阅读说明：本技术 一种高度生物相容性纳米级超声造影剂及其制备方法与应用 (A kind of high degree of biocompatibility nanoscale ultrasound contrast agents and the preparation method and application thereof ) 是由 李�杰 周晓莹 时丹丹 郭鲁 商蒙蒙 孙霄 孟冬 刘欣欣 赵亚丁 于 2019-10-11 设计创作，主要内容包括：本发明涉及一种高度生物相容性纳米级超声造影剂及其制备方法与应用。本发明中的纳米级超声造影剂是以全氟己烷为核心,以壳聚糖、棕榈酸、卵磷脂为壳膜材料,全氟己烷作为核心可显著提高造影剂的产出效率,并使超声造影剂的结构更加稳定。所述的高度生物相容性纳米级超声造影剂的粒径为300～900nm,平均粒径为519.6±72.66nm,在纳米级范围,能穿过肿瘤组织血管壁间隙,对于肿瘤的治疗具有靶向性和高效性；该造影剂具有较强的体内增强显像能力,较高的药物包封率及载药量,生物安全性高。(The present invention relates to a kind of high degree of biocompatibility nanoscale ultrasound contrast agents and the preparation method and application thereof.Nanoscale ultrasound contrast agents in the present invention are using perflexane as core, and using chitosan, palmitinic acid, lecithin as Shell membrane materials, perflexane is remarkably improved the output efficiency of contrast agent as core, and keeps the structure of acoustic contrast agent more stable.The partial size of the high degree of biocompatibility nanoscale ultrasound contrast agents is 300~900nm, and average grain diameter is 519.6 ± 72.66nm, in nano-scale range, can pass through tumor blood vessels wall gap, has targeting and high efficiency for the treatment of tumour；The contrast agent has stronger internal enhancing visualization capabilities, higher entrapment efficiency and drugloading rate, and biological safety is high.)

1. a kind of high degree of biocompatibility nanoscale ultrasound contrast agents, which is characterized in that the nanoscale ultrasound contrast agents are with shell Glycan, palmitinic acid, lecithin are shell membrane, are enclosed with perflexane inside shell membrane, the partial size of the nanoscale ultrasound contrast agents is 300~900nm.

2. high degree of biocompatibility nanoscale ultrasound contrast agents as described in claim 1, which is characterized in that the palmitinic acid and The mass ratio of lecithin is 1:4, and the mass ratio of the lecithin and chitosan is (1~2): 4.

3. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents described in claim 1, which is characterized in that step It is as follows:

(1) palmitinic acid is dissolved by heating into ultrapure water to obtain palm acid solution, palm acid solution is mixed in lecithin ultrapure In water, the chitosan solution for being dissolved in ultrapure water is added, is vortexed mixes at room temperature, obtain mixed solution；

(2) perflexane is added into mixed solution made from step (1), is vortexed mixes at room temperature, emulsify (1~3) min, obtain Emulsification product；

(3) by ultrafiltration centrifugation after the dilution of emulsification product made from step (2) to get high-biocompatibility nanoscale ultrasonic contrast Agent.

4. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly the mass ratio of palmitinic acid described in (1) and lecithin is 1:4, and the concentration of palmitinic acid is 0.1~0.2g/ in the mixed solution L, wherein palmitinic acid purity >=99%.

5. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly the mass ratio of lecithin described in (1) and chitosan is (1~2): 4, wherein the molecular weight of chitosan is 100~300kD.

6. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly percent by volume of the perflexane described in (2) in mixed solution is 0.2~0.4%.

7. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly diluted multiple described in (3) is 5~10 times.

8. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly the molecular cut off for the ultra-filtration centrifuge tube that the centrifugation of ultrafiltration described in (3) uses is 30KD.

9. the preparation method of high degree of biocompatibility nanoscale ultrasound contrast agents as claimed in claim 3, which is characterized in that step Suddenly the centrifugation of ultrafiltration described in (3) is that 500-1000rpm is centrifuged 1-5min.

10. high degree of biocompatibility nanoscale ultrasound contrast agents described in claim 1 prepare antineoplastic carrying adriamycin Application in object.

Technical field

The present invention relates to a kind of high degree of biocompatibility nanoscale ultrasound contrast agents and the preparation method and application thereof, belong to super Sound molecular imaging technical field.

Background technique

With swift and violent 0 development of ultrasound molecular imaging technique and bio-nanotechnology, nanoscale ultrasound contrast agents development is fast Speed more and more has been proven that their potential importances in cancer treatment about medicament-carried nano systematic research.It receives The advantages of rice drug delivery system, is that the drug that can will collect middle dosage is transported to specific region using in-vivo tumour tissue as target. Administration nano-drug administration system not only increases local action of the drug to tumour, and reduces chemotherapeutics during blood circulation Into the side effect of its hetero-organization.However, most of Nano medication slow-released systems still face some ask in clinical practice application Topic, for example, how to increase after Nano medication slow-released system is assembled and is adhered to tumour cell to intracellular release, Wu Fashi When observe and control therapeutic process.Therefore, therapeutic scheme further can not be evaluated and is improved over the course for the treatment of. Ultrasound guidance drug delivery system can destroy the enhancing reversible permeability of cytoplasma membrane by ultrasound targeted microbubble, there is drug Effect enters tumour cell.The observation therapeutic process of real-time and precise can be imaged in this method by external supersonic simultaneously.

Ultrasonic guidance drug delivery system is just becoming one of the hot spot of targeted therapy research.In order to be easier to penetrate new green blood The partial size in endothelial cell gap, acoustic contrast agent gradually decreases to nanometer by micron.With grinding for nanoscale ultrasound contrast agents Study carefully and deepen continuously with clinical application, safety causes the extensive concern of people, injection contrast agent is before clinical application It must assure that biological safety.In all materials for nanoscale ultrasound contrast agents, protein material may cause allergy Reaction.Many fixatives or surfactant such as glutaraldehyde and tween are harmful to the human body, and high molecular material structure safe to the human body At potential risk.Therefore, there is an urgent need to prepare nanoscale ultrasound contrast agents highly-safe, curative for effect.Chitosan is nature Common a kind of natural polysaccharide in boundary.Largely studies have shown that chitosan has good biocompatibility and biological degradability, And there is antibacterial and antitumor characteristic.Chinese patent literature CN106139174A (application number 201610693711.0) is provided A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents for wrapping up liquid fluorocarbon, is by acylation reaction pair Carboxymethyl chitosan is modified, and synthesis has amphiphilic positive hexanoyl carboxymethyl chitosan, and liquid fluorine is added on this basis The nanoscale ultrasonic contrast being made of liquid fluorocarbon kernel and chitosan derivatives shell is made using ultrasonic emulsification method for carbon Agent.The nanoscale ultrasound contrast agents that the invention is prepared are negatively charged, and the positive hexanoyl carboxymethyl chitosan of synthesis has used shell Glycan enlivens amino, is unfavorable for contrast agent carrying medicaments, and the material n-caproic anhydride and methanol used during the preparation process It is all noxious material, its biological safety can not ensure if it can not completely remove.(the Shen Chinese patent literature CN109260480A Number please 201811219930.0) disclose a kind of chitosan nano meter level acoustic contrast agent for carrying adriamycin and preparation method thereof with Using the nanoscale ultrasound contrast agents are using chitosan as shell membrane, and adriamycin (DOX) and perfluoropropane gas are wrapped in chitosan Inside shell membrane, there are stronger enhancing visualization capabilities, the form of the nanoscale ultrasound contrast agents of invention preparation is nanometer vacuole, Perfluoropropane nanometer vacuole output capacity is low, and stability is poor.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of high degree of biocompatibility nanoscale ultrasound contrast agents and its Preparation method and application, the present invention in nanoscale ultrasound contrast agents be using perflexane as core, with chitosan, palmitinic acid, Lecithin is Shell membrane materials, and perflexane is remarkably improved the output efficiency of contrast agent as core, and makes acoustic contrast agent Structure is more stable.The contrast agent has stronger internal enhancing visualization capabilities, higher entrapment efficiency and drugloading rate, biology It is highly-safe.

The present invention also provides above-mentioned nanoscale ultrasound contrast agents in load anti-tumor drug and interior therapeutic tumour Using.

Term explanation:

Room temperature: have well known to a person skilled in the art meanings, generally refer to 25 ± 2 DEG C.

Technical scheme is as follows:

A kind of high degree of biocompatibility nanoscale ultrasound contrast agents, the nanoscale ultrasound contrast agents are with chitosan, palm Acid, lecithin are shell membrane, are enclosed with perflexane inside shell membrane, the partial size of the nanoscale ultrasound contrast agents is 300~ 900nm。

Preferred according to the present invention, the mass ratio of the palmitinic acid and lecithin is 1:4, the lecithin and chitosan Mass ratio is (1~2): 4.

The preparation method of above-mentioned high degree of biocompatibility nanoscale ultrasound contrast agents, steps are as follows:

(1) palmitinic acid is dissolved by heating and obtains palm acid solution in ultrapure water, palm acid solution is mixed in lecithin In ultrapure water, the chitosan solution for being dissolved in ultrapure water is added, is vortexed mixes at room temperature, obtain mixed solution；

(2) perflexane is added into mixed solution made from step (1), is vortexed mixes at room temperature, emulsify (1~3) Min obtains emulsification product；

(3) ultrafiltration centrifugation after the dilution of emulsification product made from step (2) is made to get high-biocompatibility nanoscale ultrasound Shadow agent.

Preferred according to the present invention, the mass ratio of palmitinic acid described in step (1) and lecithin is 1:4, and the mixing is molten The concentration of palmitinic acid is 0.1~0.2g/L in liquid, wherein palmitinic acid purity >=99%, is purchased from sigma, product number 200-312- 9；Lecithin is purchased from sigma, product number 232-715-0.

Preferred according to the present invention, the mass ratio of lecithin described in step (1) and chitosan is (1~2): 4, wherein shell The molecular weight of glycan is 100~300kD, is purchased from sigma, product number MFCD00161512.

It is preferred according to the present invention, percent by volume of the perflexane in mixed solution described in step (2) be 0.2~ 0.4%.

Preferred according to the present invention, diluted multiple described in step (3) is 5~10 times.

Preferred according to the present invention, the molecular cut off for the ultra-filtration centrifuge tube that the centrifugation of ultrafiltration described in step (3) uses is 30KD。

Preferred according to the present invention, the centrifugation of ultrafiltration described in step (3) is that 500-1000rpm is centrifuged 1-5min.

Above-mentioned high degree of biocompatibility nanoscale ultrasound contrast agents prepare application in anti-tumor drug carrying adriamycin.

The above-mentioned experimental procedure not elaborated is carried out according to the art routine operation.

Technical characterstic of the invention:

The present invention obtains emulsification product using chitosan, palmitinic acid and lecithin as Shell membrane materials, by kernel of perflexane, Obtain that partial size is smaller and uniform nanoscale ultrasound contrast agents after ultrafiltration centrifugation.Chitosan, palmitinic acid and lecithin all have good Good biocompatibility and biological degradability, is the good material for being used to prepare acoustic contrast agent, in addition, palmitinic acid and lecithin Rouge all has negative electrical charge, attracts to form the shell of nanometer drop by positive and negative charge with positively charged chitosan.

Lecithin is also surfactant (existing hydrophobic grouping also has hydrophilic radical) simultaneously, in external hydrophilic easy to form The nano-liquid droplet structure in portion hydrophobic (kernel perflexane is not soluble in water).

The utility model has the advantages that

1, high degree of biocompatibility nanoscale ultrasound contrast agents prepared by the present invention are with chitosan, palmitinic acid, lecithin For shell membrane, perflexane is wrapped in the nano-liquid droplet inside shell membrane, and the partial size of the acoustic contrast agent is 300~900nm, average Partial size is 519.6 ± 72.66nm, in nano-scale range, can pass through tumor blood vessels wall gap, have for the treatment of tumour Targeting and high efficiency.

2, high degree of biocompatibility nanoscale ultrasound contrast agents prepared by the present invention are using perflexane liquid as core, perfluor Hexane can significantly improve the output efficiency of contrast agent, and blebbing rate is high, and keeps the structure of acoustic contrast agent more stable, and 25 DEG C put After setting 6 hours, still without apparent modal variation.

3. nanoscale ultrasound contrast agents vivo biodistribution compatibility prepared by the present invention is high, biological safety is high, 80mg/kg is small Tail vein injection is without overt toxicity.

4, there is high degree of biocompatibility nanoscale ultrasound contrast agents prepared by the present invention stronger internal enhancing to image energy Power, and can maintain to develop in a longer period of time, entrapment efficiency and drugloading rate also with higher.

5, present invention high degree of biocompatibility nanoscale ultrasound prepared by the method by the way of ultrasound wave irradiation is made Tumour is treated in shadow agent, and nanoscale ultrasound contrast agents development on the one hand can be enhanced, on the other hand can be by ultrasonic spoke Promote carrying medicaments or small molecule more to enter in tumour cell according to the acoustic horn effect of generation, realizes diagnosis and treatment integration.

6, high degree of biocompatibility nanoscale ultrasound contrast agents carrying medicaments prepared by the present invention enter internal combining ultrasonic The extraction that can significantly improve drug is treated, the treatment of tumor disease is conducive to.

Detailed description of the invention

Fig. 1 is the BCNDs optical microscope photograph that embodiment 1 is prepared；In figure, A is untreated BCNDs optics Microscope photo；B is the BCNDs optical microscope photograph after placing 6 hours at 25 DEG C；

Fig. 2 is the BCNDs partial size and potential curve figure that embodiment 1 is prepared；In figure, the particle size that A is BCNDs is divided Butut, B are the potential image of BCNDs；

Fig. 3 is the HE stained photographs of mice organs；In figure, A-1 is the HE stained photographs of control group mice heart, and A-2 is The HE stained photographs of control group mice liver, A-3 are the HE stained photographs of control group mice spleen, and A-4 is control group mice lung HE stained photographs, A-5 is the HE stained photographs of control group mice kidney；B-1 is the HE stained photographs of experimental mice heart, B-2 is the HE stained photographs of experimental mice liver, and B-3 is the HE stained photographs of experimental mice spleen, and B-4 is that experimental group is small The HE stained photographs of mouse lung, B-5 are the HE stained photographs of experimental mice kidney；

Fig. 4 is different the encapsulation rate and drugloading rate curve graph for the DOX-BCNDs that doxorubicin concentration is prepared；In figure, A For the drugloading rate curve graph of DOX-BCNDs, abscissa is doxorubicin concentration, and ordinate is drugloading rate；B is the packet of DOX-BCNDs Envelope rate curve graph, abscissa are doxorubicin concentration, and ordinate is encapsulation rate；

Fig. 5 is the ultrasonic development image of DOX-BCNDs in vitro and in vivo；In figure, A-1 is external grayscale visualising image, A-2 To enhance visualising image in vitro；B-1 is internal grayscale visualising image, and B-2 is to enhance visualising image in vivo；

Fig. 6 is the fluorescence imaging result of tumor-bearing mice and histoorgan；In figure, A is DOX-BCNDs group and DOX group mouse In the living imaging result of different time；B be mouse tissue organ fluorescence imaging as a result, in figure histoorgan from left to right according to Secondary is tumor tissues, heart, spleen, lung, liver, kidney；

Fig. 7 is different disposal group mouse tumor volume change figure；In figure, A is gross tumor volume picture after mouse dissection, and B is Mouse tumor volume change histogram；

Fig. 8 is serum creatinine (CREA) in different disposal group mouse blood, urea nitrogen (BUN), lactic dehydrogenase (LDH) and flesh Acid phosphoric acid kinases (CK) contents level histogram；In figure abscissa be from left to right followed successively by Normal group, control group, DOX group, DOX ultrasound group, DOX-BCNDs ultrasound group, doubling dosage DOX-BCNDs ultrasound group；

Fig. 9 is the microscope photo of different disposal group mouse tumor tissue paraffin section de HE dyeing；

Figure 10 is the fluorescent microscopy images of different disposal group mouse tumor histocyte apoptosis situation；

Figure 11 is the fluorescent microscopy images of different disposal group mouse tumor tissue cell proliferation situation；

Figure 12 is different disposal group mouse tumor tissue cell proliferation situation histogram, in figure, abscissa from left to right according to Secondary is control group, DOX group, DOX ultrasound group, DOX-BCNDs ultrasound group, doubling dosage DOX-BCNDs ultrasound group, and abscissa is Ki67 average optical density.

Specific embodiment

The present invention will be further explained with reference to the examples below, but protection scope of the present invention is not limited to that.

Palmitinic acid is purchased from sigma, product number 200-312-9；

Lecithin is purchased from sigma, is Epikuron200, product number 232-715-0；

Chitosan is purchased from sigma, and molecular weight is 100~300kD, product number MFCD00161512；

Adriamycin is purchased from sigma, product number 246-818-3.

The present embodiment is related to drug and reagent unless otherwise specified, is ordinary commercial products.