CN109966513B

CN109966513B - Preparation method and application of multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy

Info

Publication number: CN109966513B
Application number: CN201711455843.0A
Authority: CN
Inventors: 戴志飞; 陈敏; 梁晓龙; 岳秀丽
Original assignee: Peking University; Harbin Institute of Technology
Current assignee: Peking University; Harbin Institute of Technology
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2022-05-17
Anticipated expiration: 2037-12-28
Also published as: CN109966513A

Abstract

The invention relates to a multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy, and relates to a preparation method of the multifunctional microbubble and application of the multifunctional microbubble in tumor diagnosis and treatment. The structural schematic diagram of the multifunctional microbubble integrating ultrasonic/fluorescence bimodal imaging and photodynamic therapy/chemotherapy is shown in the figure, the membrane components of the multifunctional microbubble comprise lipid containing photosensitizer functional groups for photodynamic therapy, amphiphilic drug conjugate for chemotherapy and conventional phospholipid, the proportion of the photosensitization drug and the chemotherapeutic drug is regulated and controlled according to the requirements, and the drug-loading rate is greatly improved. Under the action of ultrasound, the multifunctional microvesicle can realize fixed-point targeted blasting on a tumor part to be converted into nano particles, thereby greatly improving the enrichment and uptake of the medicine on the tumor part and effectively improving the effect of inhibiting the tumor growth by combining photodynamic/chemotherapy.

Description

Preparation method and application of multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy, and application thereof in tumor diagnosis and treatment.

Background

Noninvasive ultrasound imaging has been widely used for early diagnosis of cancer because of its incomparable advantages such as convenience, low price, and real-time imaging. The ultrasound microbubble contrast agent improves the imaging effect by introducing materials such as gas with different acoustic characteristics from tissues, can display the morphological information of tumors with high quality and can also effectively reflect the biological characteristics of the tumors, thereby obviously improving the accuracy of diagnosis. Ultrasonic imaging receives acoustic signals, and compared with optical imaging, imaging depth is greatly increased. The near infrared fluorescence/ultrasound bimodal imaging can make up for the deficiencies of the above materials, and provides a new method for diagnosing tumors with depth consideration.

Fluorescence imaging is an extremely important tool for molecular biology and medical research. The light absorption of the biomolecules in the near infrared region (with the wavelength of 600-900 nm) is the lowest, the autofluorescence is the lowest, and a large amount of infrared light can pass through tissues and skin to be detected. Thus, its wavelength range is considered to be the "diagnostic window" of the optical imaging. The unique advantages are as follows: the sensitivity is high; secondly, the targeted imaging of various tumors can be realized through the design of different fluorescent probes; and the real-time dynamic tumor living body imaging can be provided. However, near infrared dyes are limited in imaging depth (no more than 1cm), thereby affecting their use in visualizing deep tumors.

The light therapy (photothermal) of tumor is a new effective means for tumor treatment following surgery, radiotherapy and chemotherapy due to its low treatment cost, small tissue trauma, small side effects and high efficiency. Photodynamic therapy (PDT) is a non-invasive treatment developed in recent years and has been widely used clinically. PDT refers to delivery of photosensitizer drugs to tumor cells, and generation of "highly active" singlet oxygen and free Radicals (ROS) by illumination of specific wavelengths, and ROS can destroy lipid molecules, DNA, proteins, etc. of cancer cells, thereby inducing apoptosis, etc. of cancer cells.

Although PDT and chemotherapy alone have made significant progress for the treatment of tumors, nanomaterials that can combine both chemotherapy and PDT treatment are still relatively rare. If the combination of chemotherapy and PDT can be realized by the micro-nano technology, the treatment effect can be expected to be further improved. Therefore, there is a need to develop new materials to achieve synergistic anticancer effects in combination with photodynamic and chemotherapy. In addition, ideal light therapy should kill tumor tissue while minimizing damage to surrounding normal tissue to ensure efficacy and safety of the treatment. For light therapy, a photosensitizer is a core element of light therapy, and the photosensitizer can only generate light therapy effect at the position where the photosensitizer exists to damage cells; because the laser energy used in the treatment is generally low, the pure laser irradiation has no killing effect on cells under the condition of lacking the photosensitizer. Tumor targeting for light therapy therefore relies on good accumulation of the photosensitizer at the target area. How to achieve the accurate accumulation of photosensitizer chemotherapeutic drugs at the tumor site is a big problem in the light treatment.

With the rapid development of the ultrasonic microbubble preparation technology, the ultrasonic microbubble contrast agent can be used as an excellent ultrasonic imaging contrast enhancer, plays an important role in tumor diagnosis, and has huge application potential in tumor treatment. In the field of treatment, the ultrasonic microvesicles can be used as controlled release carriers of other therapeutic substances such as drugs, genes, nano materials and the like to achieve the aim of targeted delivery. However, as a drug carrier, the problem of low drug loading of ultrasound microbubbles needs to be overcome first.

Based on the consideration, the multifunctional ultrasonic microvesicle integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy is characterized in that lipid with photosensitizer functional groups and amphiphilic drug conjugate with chemotherapy effect are assembled into a membrane component of an ultrasonic contrast agent, the microvesicle can be broken at a tumor site under the guidance of ultrasound to be converted into nano particles, and the nano particles are more taken up by tumor cells under the action of ultrasonic cavitation. And then under the guidance of fluorescence imaging, performing photodynamic combined treatment on the tumor part at the optimal enrichment time to achieve the synergistic treatment effect of chemotherapy and photodynamic treatment.

Disclosure of Invention

The invention aims to provide a multifunctional ultrasonic microbubble contrast agent integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy and a preparation method of the microbubble contrast agent.

The invention also aims to provide the application of the multifunctional ultrasonic microbubble contrast agent integrating ultrasonic/fluorescence bimodal imaging and photodynamic therapy/chemotherapy in tumor diagnosis and treatment.

The structure of the multifunctional ultrasonic microbubble contrast agent integrating ultrasonic/fluorescence bimodal imaging and photodynamic therapy/chemotherapy is shown in figure 1.

The multifunctional microbubble contrast agent is characterized in that the shell layer of the microbubble is composed of a lipid monomolecular layer, and the multifunctional microbubble contrast agent simultaneously comprises the following components: lipid containing photosensitive functional groups for photodynamic therapy, amphiphilic drug conjugate for chemotherapy and various conventional phospholipids, wherein inert gas or liquid is loaded in the microbubbles, and the amphiphilic drug conjugate and the lipid containing the photosensitive functional groups can be self-assembled together with the conventional phospholipids in aqueous solution to form the microbubbles.

Wherein the drug conjugate is characterized in that the chemotherapeutic agent is selected from the group consisting of paclitaxel, camptothecin, pentoxifyllide, doxorubicin, ifosfamide, vincristine, vinblastine, etoposide, vinblastine, carboplatin, cisplatin, mitomycin, vinblastine amide, epirubicin, vinblastine, and methotrexate, and has the general structure:

wherein A represents various hydrophobic chemotherapeutic drug molecules, B represents various hydrophilic chemotherapeutic drug molecules, X and Y represent various connecting groups, and X and Y can be the same or different; a is 2 or 3; b is 2 or 3, and a and b may be the same or different. The drug conjugate can self-assemble in aqueous solution to form liposome after sol-gel process.

The lipid containing the photosensitizer functional group generally means that the photosensitizer functional group is covalently linked to the lipid, and the structure of the lipid is generally as follows:

wherein R1, R2 ═ C6-18 alkyl; a, b ═ 2 or 3; x ═ N or O, i.e. the photosensitizer and the lipid are linked by an ester or amide bond; the lipid containing the photosensitive functional group can be self-assembled in aqueous solution to form the liposome after the sol-gel process. The photosensitive functional group includes hematoporphyrin, protoporphyrin, tetraphenylporphyrin, pyropheophorbide, bacteriochlorophyll, chlorophyll a, benzoporphyrin derivative, tetrahydrophenylchlorin, benzochlorin, naphthochlorin, phthalocyanine or naphthalocyanine, etc.

The invention relates to a preparation method of multifunctional microvesicle integrating ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy, which comprises the following steps:

1) dissolving required phospholipid in ethanol, respectively dissolving lipid containing photosensitizer functional groups in tetrahydrofuran, dissolving drug conjugates in dimethyl sulfoxide, and uniformly mixing and dissolving the drugs and the conjugates according to a certain proportion (the proportion of the lipid containing the photosensitizer functional groups is 0-30%, and the proportion of the drug conjugates is 0-50%).

2) And (3) dripping the uniformly mixed system into physiological saline at the temperature of 40-60 ℃ by adopting an ethanol injection method, and carrying out water bath ultrasound for 15-30 minutes.

3) Dialyzing the obtained system 2) in physiological saline at room temperature for 2-4 h by using a 8000-14000Da dialysis bag.

4) And transferring the obtained system into a penicillin bottle, adding propylene glycol and glycerol serving as stabilizing agents, and uniformly mixing.

5) Filling inert inner packing materials into a penicillin bottle, sealing the penicillin bottle, then violently oscillating the penicillin bottle for 45s by using a silver mercury mixer, and separating and purifying the penicillin bottle to obtain the multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy.

In step l), the phospholipid contains a carbon chain length of 12 to 24 carbons and includes phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylglycerol, such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG5000), and the like.

The inert inner inclusion substance in the step 5) comprises air, nitrogen, carbon dioxide and fluorocarbon gas, and the liquid is selected from C₅-C₁₂A fluorocarbon.

The invention integrates ultrasonic/fluorescence bimodal imaging and photodynamic therapy/chemotherapy into a whole, the lipid with photosensitizer functional groups, the amphiphilic drug conjugate and the phospholipid are formed into a membrane, the proportion of the photosensitive drug and the chemotherapeutic drug is respectively regulated and controlled according to the requirement, and the drug loading rate is greatly improved; meanwhile, the device can integrate two imaging modes of fluorescence and ultrasound, and accurately position the tumor position; moreover, the targeted blasting of the microbubbles is realized under the action of the ultrasound, and the enrichment of the photosensitizer/chemotherapeutic drug at the tumor part is increased; the photodynamic therapy and chemotherapy are combined to treat the tumor, the effect of the photodynamic therapy or chemotherapy is better than that of the independent photodynamic therapy or chemotherapy, and the curative effect is effectively improved.

Drawings

FIG. 1 is a structural diagram of a multifunctional microbubble contrast agent described in the present invention, wherein 1 represents a lipid containing a photosensitizer functional group, 2 represents an amphiphilic drug conjugate, and 3 represents an encapsulated substance; FIG. 2 is a graph showing the distribution of the particle size of the multifunctional microbubbles prepared in example 1; FIG. 3 is the singlet oxygen generation rate of the multifunctional microbubble contrast agent under near infrared light irradiation in different concentrations in the specific example 5; FIG. 4 is a confocal laser image of the uptake of microbubble contrast agent by cancer cells in example 6; FIG. 5 is an ultrasonic image of the microbubble contrast agent at the tumor tissue of the animal in the embodiment 7; FIG. 6 is fluorescence imaging of the microbubble contrast agent of the embodiment example 8 before and 24 hours after injection in tumor model mice; FIG. 7 is a fluorescence distribution diagram of major tissues and organs of an animal with or without the microbubble contrast agent being destroyed by ultrasound at tumor tissue of the animal in the embodiment 8; FIG. 8 is a graph of tumor growth in animals treated with the combination photodynamic therapy/chemotherapy of the microbubble contrast agent of example 9.

Detailed Description

The following detailed description will help to understand the present invention, but does not limit the contents of the present invention.

Example 1

Mixing Distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), cholesterol (chol), porphyrin lipid (PGL) and drug Conjugate (CF) according to a certain molar ratio (30%: 10%: 10%: 40%), and then injecting the mixture into 0.8ml of water by ethanol injection under the water bath ultrasonic condition at 50 ℃; putting the obtained solution into a dialysis bag with the cut-off molecular weight of 8000-14000Da, dialyzing for 2-4 h, taking out, adding 100 mu L of glycerol and 100 mu L of propylene glycol respectively, and uniformly mixing. The mixed solution is filled into a 3.5mL penicillin bottle and is filled with enough perfluoropropane (C)₃F₈) Oscillating the gas with an oscillator for 45s, separating and purifying to obtain the multifunctional microbubble (PCF-MBs) integrating ultrasonic/fluorescence bimodal imaging and photodynamic therapy/chemotherapy. The distribution of the microbubble particle size is shown in figure 2.

Example 2

Mixing Distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), cholesterol (chol), porphyrin lipid (PGL) and drug Conjugate (CF) according to a certain molar ratio (30%: 10%: 10%: 40%), and then injecting the mixture into 0.8ml of water by ethanol injection under the water bath ultrasonic condition at 50 ℃; putting the obtained solution into a dialysis bag with the cut-off molecular weight of 8000-14000Da, dialyzing for 2-4 h, taking out, adding 100 mu L of glycerol and 100 mu L of propylene glycol respectively, and uniformly mixing. And filling the mixed solution into a 3.5mL penicillin bottle, filling sufficient perfluorobutane gas, oscillating for 45s by an oscillator, separating and purifying to obtain the multifunctional microbubble (PCF-MBs) integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy.

Example 3

Distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), cholesterol (chol), porphyrin lipid (PGL) and drug Conjugate (CF) are mixed according to a certain molar ratio (30%: 10%: 10%: 10%: 40%), and then the mixture is injected into 0.8ml of water by an ethanol injection method under the condition of water bath ultrasound at 50 ℃; putting the obtained solution into a dialysis bag with the cut-off molecular weight of 8000-14000Da, dialyzing for 2-4 h, taking out, adding 100 mu L of glycerol and 100 mu L of propylene glycol respectively, and uniformly mixing. And (3) filling the mixed solution into a 3.5mL penicillin bottle, adding sufficient perfluorooctyl bromide, oscillating for 45s by an oscillator, separating and purifying to obtain the multifunctional microbubble (PCF-MBs) integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy.

Example 4

To evaluate the ability of the multifunctional microbubbles obtained in examples 1-3 to generate singlet oxygen, microbubbles of different concentrations were converted to nanoparticles after ultrasonic irradiation, and irradiated with 650nm laser (0.2W/cm)²And 10min), and simultaneously detecting the generation amount of singlet oxygen in the process by using a singlet oxygen probe ADPA. The results are shown in FIG. 3, and the decreasing speed of ADPA absorption peak increases gradually as the concentration increases from 2.5uM to 10uM, which can be used to characterize the singlet oxygen generation rate at different concentrations.

Example 5

To trace the uptake of PCF-MBs by tumor cells, 10uM PCF-MBs were co-cultured with the cells, and after 4 hours, the medium was removed, stained with Acridine Orange (AO), fixed with 4% paraformaldehyde, and observed with a confocal laser microscope. The specific result is shown in FIG. 4, using 405nm excitation light, collecting 600-750nm red fluorescence, the collected signal represents the distribution of PGL; using 405nm excitation, fluorescence near 450nm shows the blue fluorescence of Camptothecin (CPT) in CF. It can be seen that the fluorescence of PGL is enriched only in cytoplasm, while the fluorescence of CF can penetrate into nucleus in addition to cytoplasm.

Example 6

The multifunctional microvesicles obtained in examples 1 to 3, which integrate ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy, were injected into HT-29 tumor-bearing model nude mice via tail vein, using an ultrasound diagnostic apparatus trace mode, MI:0.04 (mechanical index), probe frequency: 3-12MHz, and observing the ultrasonic contrast effect in the microvesicle body. After injecting 200ul PCF-MBs, the microbubble gradually enhances the ultrasonic signal near the tumor along with the blood flow (figure 5), after the tumor part is broken by using low-intensity focused ultrasound, the ultrasonic signal is greatly weakened, then the microbubble is again perfused into the tumor blood vessel for several seconds, the enhanced ultrasonic contrast signal is gradually enhanced, and then after injecting about 3min, the ultrasonic enhanced signal is gradually attenuated to the level before injecting the microbubble.

Example 7

To evaluate the ability of the multifunctional microvesicles obtained in examples 1-3 to fluorescently image tumors in vivo, nude mice inoculated with subcutaneous HT-29 tumors were fluorescently imaged. The concentration of microbubbles is 10⁸A solution of 1mL/kg was injected into mice via tail vein, followed by 100. mu.L of physiological saline. The experimental group carries out ultrasonic irradiation on tumor parts under the guidance of ultrasonic imaging to smash micro-bubbles (1.03MHz, 50% duty, 1W/cm)²3min), then the mice are subjected to near infrared fluorescence imaging for 0.5h, 3h, 12h and 24h respectively (figure 6), and the contrast group is not subjected to ultrasonic irradiation. In vivo fluorescence imaging images are shown in figure 7, after microbubbles are broken by ultrasonic waves at a tumor part, a mouse is dissected after 24 hours, heart, liver, spleen, lung, kidney and tumor tissues are collected for fluorescence imaging, quantitative analysis shows that the fluorescence of a control group is mainly distributed in the liver, the enrichment amount of the liver of an experimental group is reduced by half under the mediation of the breaking of the ultrasonic targeted microbubbles, and the enrichment amount of the tumor tissues is improved by 5 times, so that the prepared multifunctional microbubbles can effectively improve the enrichment amount of the tumor part under the directional ultrasonic breaking.

Example 8

In vivo photodynamic therapy/chemotherapy combination therapy experiments investigated whether the multifunctional microvesicles obtained in examples 1-3 could effectively inhibit tumor growth. Nude mice bearing HT-29 subcutaneous tumors were randomly divided into 6 groups and treated with PBS, PCF-MBs, PCF-MBs + Light, pMBs + US + Light, PCF-MBs + US and PCF-MBs + US + Light, respectively, wherein the administration was 200ul by tail vein injection. After treatment of each group of mice, changes in tumor volume and body weight were recorded daily (tumor volume: 1/2 × length × width)². The tumor volume growth rate of the combined treatment group of the photodynamic therapy and the chemotherapy is far less than that of the pure chemotherapy or the pure photodynamic group, while the single photodynamic (pMBs + US + Light) group has better inhibition effect and slow growth in the previous week, but then the phenomenon of rapid growth appears, which shows that the single photodynamic group only kills most tumor cells in the treatment process, still has a small amount of tumor cells to survive, and rapidly divides and grows in the later period, so that the effect of the combined treatment is better than that of the pure chemotherapy or the photodynamic therapy; compared with the single microbubble group (PCF-MBs), the microbubble combined ultrasonic group (PCF-MBs + US) has the advantage that the tumor inhibition efficiency is doubled, because the microbubble combined ultrasonic tumor site targeted disruption technology can enable more therapeutic agents to be taken up by tumor cells and play a more effective therapeutic role. The results are shown in FIG. 8.

Claims

1. A multifunctional microbubble for integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy is characterized in that a shell layer of the microbubble is composed of a lipid monomolecular layer, and the microbubble comprises the following components: lipid containing photosensitizer functional groups for photodynamic therapy, amphiphilic drug conjugate for chemotherapy and various conventional phospholipids, wherein inert gas or liquid is loaded in the microbubbles, and the lipid containing the photosensitizer functional groups, the amphiphilic drug conjugate for chemotherapy and the conventional phospholipids are self-assembled together in aqueous solution to form the microbubbles, wherein the amphiphilic drug conjugate for chemotherapy has the following structure:

the lipid containing photosensitizer functional groups has the following structure:

2. the multifunctional microbubble integrating ultrasound/fluorescence dual-mode imaging and photodynamic therapy/chemotherapy as claimed in claim 1, wherein the microbubble can be converted into nanoparticles under the action of ultrasound, and the particle size of the nanoparticles is in the range of 20nm to 700 nm.

3. The multifunctional microbubble integrating ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy as claimed in claim 1, wherein the multifunctional microbubble is formed by a gas or liquid encapsulated by a film forming material, and the particle size of the multifunctional microbubble ranges from 200nm to 8 μm.

4. The multifunctional microbubble integrating ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy as claimed in claim 1, wherein the microbubble can be used for diagnosis and treatment of tumor.

5. The method of claim 1, wherein the method comprises the steps of:

1) dissolving and uniformly mixing a certain proportion of phospholipid, lipid containing photosensitizer functional groups and amphiphilic drug conjugate in a hydrophilic organic solvent, wherein the proportion of the lipid containing photosensitizer functional groups is 0-30%, and the proportion of the drug conjugate is 0-50%;

2) dripping the mixed system into physiological saline at 40-60 ℃ by adopting an injection method, and carrying out water bath ultrasound for 15-30 minutes;

3) dialyzing the obtained system 2) in physiological saline at room temperature for 2-4 h by using a 8000-14000Da dialysis bag;

4) transferring the obtained system into a penicillin bottle, adding propylene glycol and glycerol as stabilizers, and uniformly mixing;

5) filling inert gas or liquid into a penicillin bottle, sealing the penicillin bottle, then using a silver mercury mixer to violently oscillate for 45s, separating and purifying to obtain the multifunctional microbubble integrating ultrasonic/fluorescent bimodal imaging and photodynamic therapy/chemotherapy.

6. The method according to claim 5, wherein the multifunctional microbubble is a mixture of ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy, the phospholipid comprises a carbon chain length of 12-24 carbons and comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylglycerol, and is specifically selected from 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and distearoylphosphatidylethanolamine-polyethylene glycol 5000(DSPE-PEG 5000).

7. The method for preparing multifunctional microbubbles integrating ultrasound/fluorescence bimodal imaging and photodynamic therapy/chemotherapy as claimed in claim 5, wherein the inert gas in step 5) is fluorocarbon gas, and the liquid is selected from C₅-C₁₂A fluorocarbon.