CN111529704B - Aggregation luminescence photosensitizer/antibacterial drug multifunctional nano micelle and preparation method and application thereof - Google Patents
Aggregation luminescence photosensitizer/antibacterial drug multifunctional nano micelle and preparation method and application thereof Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention belongs to the technical field of targeted imaging and treatment, and discloses an aggregated luminescent photosensitizer/antibacterial drug multifunctional nano-micelle and a preparation method and application thereof, wherein the nano-micelle is of a core-shell structure, the core of the nano-micelle is composed of an aggregated luminescent photosensitizer TTD and an antituberculosis drug, the shell is a monolayer lipid membrane, and the diameter of the multifunctional nano-micelle is 100-120 nm; the antituberculous drug is rifampicin. The aggregation luminescence photosensitizer TTD in the nano micelle can generate a large amount of ROS oxygen free radicals for inducing the photodynamic killing of tubercle bacillus, and the antitubercular drug rifampicin slowly released in the micelle can further inhibit the growth of the tubercle bacillus, so that the photodynamic and drug synergistic treatment function is exerted. The multifunctional nano micelle can be used as a contrast agent for locating tuberculosis focus and can also be used as a therapeutic agent for synergistically killing tubercle bacillus.
Description
Technical Field
The invention relates to the technical field of targeted imaging and treatment, in particular to an aggregated luminescent photosensitizer/antibacterial drug multifunctional nano micelle and a preparation method and application thereof.
Background
Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium Tuberculosis (m.tb), which seriously threatens human health. According to the statistics of the 2018 worldwide tuberculosis report, about 1000 ten thousand of new tuberculosis cases are discovered globally in 2017, and the number of deaths per year reaches over 160 ten thousand. More feared, over 23% of the world's population (about 17 million people) is now a latent infection of tuberculosis, of which about 10% will develop tuberculosis throughout its lifetime. As the only tuberculosis treatment method on the market at present, the antibiotic treatment has the defects and disadvantages of long administration time, obvious toxic and side effects, poor patient compliance and the like. Moreover, the occurrence of a large amount of multi-drug resistant tubercle bacillus (MDR-m.tb) and pan-drug resistant tubercle bacillus (XDR-m.tb) significantly reduces the efficacy of the antibiotic. Therefore, the development of a novel tuberculosis treatment method has important significance for reducing the death rate of tuberculosis and improving the life quality of tuberculosis patients.
Tubercle nodules, a characteristic focus of tuberculosis, consist of a close packing of immune cells, surrounded by a mass of dead or non-dead tubercle bacilli. Initially, tubercles were thought to sequester tubercle bacillus inside and inhibit their growth and proliferation. However, evidence suggests that early nodules can promote the spread of tubercle bacillus; the dense structure of tubercle bacillus limits the access of antibiotics; tubercle bacillus in a resting state inside the nodule is itself insensitive to antibiotics. This all renders tuberculosis a chronic and persistent infectious disease. Therefore, the nano-material mediated photodynamic therapy is used in combination with chemotherapy, so that the inherent defects of anti-simple hormone treatment can be overcome, and the nano-material mediated photodynamic therapy can be a new tuberculosis treatment strategy.
Disclosure of Invention
Aiming at the defects in the prior art, the invention firstly provides the multifunctional nano-micelle for tuberculosis targeted imaging and multi-modal therapy.
The second purpose of the invention is to provide a preparation method of the multifunctional nano-micelle.
The third purpose of the invention is to provide the application of the multifunctional nano-micelle.
The purpose of the invention is realized by the following technical scheme:
the multifunctional nano-micelle is used for targeted imaging of tuberculosis and multi-modal therapy, and is characterized in that the multifunctional nano-micelle is of a core-shell structure, the core of the multifunctional nano-micelle is composed of an aggregated luminescent photosensitizer TTD and an anti-tuberculosis drug, the shell is a monolayer lipid membrane, and the diameter of the multifunctional nano-micelle is 100-120 nm; the anti-tuberculosis drug is rifampicin.
The invention takes aggregation luminescent photosensitive molecules TTD and antituberculosis drugs RIF as co-carriers to form the core of the nano-micelle, uses a liposome membrane to embed TTD and RIF according to a unique design scheme, and designs and synthesizes TTD/RIF @ lipid-PEG nano-micelle to obtain the multifunctional nano-micelle which has aggregation luminescent imaging and visible light excitation and simultaneously plays the photodynamic and drug functions of tubercle bacillus. The TTD/RIF @ lipid-PEG nanomicelles have a diameter of 100-120nm, which is equivalent to the size of some proteins and bio-organic compounds present in the human and animal body, thereby facilitating the delivery and absorption of contrast agents in the region of interest.
In the multifunctional nano-micelle, the aggregation luminescent photosensitizer TTD is an aggregation luminescent material with a symmetrical structure and has the characteristic of aggregation induced luminescence; compared with the traditional fluorescent molecules (such as pyrene) with aggregation-induced quenching phenomenon, the aggregation luminescent agent can emit strong fluorescence in an aggregation state, so that the method is more suitable for fluorescence imaging in the field of biology; furthermore, in the invention, TTD is wrapped in the nano-micelle core through hydrophobic interaction and is in a stronger aggregation state, so that stronger fluorescence can be emitted. The TTD molecule also has the characteristics of a photosensitizer, can be excited by visible light and reacts with molecular oxygen to generate oxygen free Radicals (ROS) for photodynamic killing of tubercle bacillus. The antibacterial medicament RIF is an antituberculosis first-line medicament, and can inhibit the generation of bacterial cell walls and the growth and the reproduction of tubercle bacillus.
The invention also provides a preparation method of the multifunctional nano micelle, which comprises the following steps:
(1) dissolving rifampicin in THF solvent, and adding aggregated luminescent photosensitizer TTD and amphiphilic micelle molecule polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine to form THF mixed solution; wherein the mass ratio of the aggregated luminescent photosensitizer TTD to rifampicin to the amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine is 1-2: 12-30: 2-4.
(2) Injecting the THF mixed solution into ultrapure water, performing ultrasonic and photophobic magnetic stirring, and evaporating the THF solvent to dryness;
(3) and filtering for multiple times, and washing away the unbound rifampicin and amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine to obtain the TTD/RIF @ lipid-PEG multifunctional nano-micelle.
In the multifunctional nano micelle synthesis raw materials, polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) has the amphiphilic characteristic, the aggregated luminescent photosensitive molecule TTD has hydrophobicity, and the antituberculous drug RIF has slightly water solubility. Therefore, under the action of probe ultrasound, DSPE-PEG2000The hydrophobic end DSPE of (a) will self-assemble,and coating hydrophobic substance TTD and slightly water-soluble RIF to form nano micelle core, DSPE-PEG2000Hydrophobic end PEG of2000Will distribute to the surface of the nano micelle and maintain the stability of the structure.
The TTD/RIF @ lipid-PEG multifunctional nano micelle constructed by the method has the characteristics of high biocompatibility, high illumination stability and the like. In order to improve the circulation time in vivo of the nano-micelle and reduce the phagocytosis of Reticuloendothelial system (Reticuloendothelial system), the invention adopts DSPE-PEG approved by FDA and used for clinic2000(polyethylene glycol-distearoylphosphatidylethanolamine) liposome membrane, which is effective in increasing systemic circulation time of drugs.
Preferably, the mass ratio of the aggregated luminescent photosensitizer TTD, rifampicin and amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine in the step (1) is 1: 20: 2.
preferably, the ultrasonic condition of the step (2) is 60-80W, 20-25kHz and the time is 1-5 min.
Preferably, the multiple filtration in step (3) is performed by filtering through a 0.22 μm-pore-size syringe filter and then performing ultrafiltration through 10kDa ultrafiltration.
The invention also provides a preparation for treating tuberculosis, which comprises the multifunctional nano-micelle. The multifunctional nano micelle has a fluorescence imaging function on tubercle nodules of tubercle foci, realizes the targeting of the tubercle foci, and has the functions of photodynamic killing and drug killing synergistic treatment on tubercle bacillus.
The invention also provides application of the multifunctional nano-micelle in preparing a contrast agent for imaging tuberculosis lesions.
Or, the invention also provides the application of the multifunctional nano-micelle in the preparation of the photodynamic medicament synergistic killing agent for tubercle bacillus. The TTD/RIF @ lipid-PEG multifunctional nano micelle can be used for fluorescence imaging of tuberculosis nodules; and because of the improvement of the sensitivity, the dosage of the contrast agent can be reduced, and the toxic and side effects are further reduced. Under the irradiation of visible light, the TTD embedded inside gathers luminescent photosensitive molecules, can effectively convert light energy into chemical energy, generate a large amount of ROS oxygen free radicals, and further kill mycobacterium tuberculosis; meanwhile, the anti-tuberculosis drug RIF contained in the drug can be slowly released, and the growth of tubercle bacillus is inhibited.
The TTD/RIF @ lipid-PEG multifunctional nano-micelle constructed by the invention can be administered by any known delivery method: systemic delivery (intravenous), intra-arterial, parenteral, intrapulmonary or regional delivery forms of local administration. If the medicine is administrated by intravenous injection, the target imaging of the focus part and the imaging-mediated photodynamic and medicinal treatment can be realized.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes aggregation luminescent photosensitive molecules TTD and antituberculosis drugs RIF as co-carriers to form the core of the nano-micelle, uses a liposome membrane to embed TTD and RIF according to a unique design scheme, and designs and synthesizes TTD/RIF @ lipid-PEG nano-micelle to obtain the multifunctional nano-micelle which has aggregation luminescent imaging and visible light excitation and simultaneously plays the photodynamic and drug functions of tubercle bacillus. The TTD/RIF @ lipid-PEG nanomicelles have a diameter of 100-120nm, which is equivalent to the size of some proteins and bio-organic compounds present in the human and animal body, thereby facilitating the delivery and absorption of contrast agents in the region of interest.
In the multifunctional nano-micelle, the aggregation luminescent photosensitizer TTD is an aggregation luminescent material with a symmetrical structure and has the characteristic of aggregation induced luminescence; compared with the traditional fluorescent molecules (such as pyrene) with aggregation-induced quenching phenomenon, the aggregation luminescent agent can emit strong fluorescence in an aggregation state, so that the method is more suitable for fluorescence imaging in the field of biology; furthermore, in the invention, TTD is wrapped in the nano-micelle core through hydrophobic interaction and is in a stronger aggregation state, so that stronger fluorescence can be emitted. The TTD molecule also has the characteristics of a photosensitizer, can be excited by visible light and reacts with molecular oxygen to generate oxygen free Radicals (ROS) for photodynamic killing of tubercle bacillus. The prepared multifunctional nano micelle can be used for target imaging of tuberculosis focus and can also be used for photodynamic and drug synergistic treatment of tubercle bacillus. The invention can also overcome the defects of poor curative effect, long administration time, obvious toxic and side effects, poor patient compliance and the like in the traditional tuberculosis antibiotic treatment scheme, and becomes a new tuberculosis focus targeting and tuberculosis treatment strategy.
Drawings
FIG. 1 is a schematic diagram of a tuberculosis targeted imaging and treatment method based on TTD/RIF @ lipid-PEG multifunctional nano-micelle;
FIG. 2 is a chemical synthesis route for the aggregated luminescent photosensitizer TTD;
FIG. 3 is a transmission electron microscope image (FIG. 3A) and a hydrated particle size and zeta potential diagram (FIG. 3B) of TTD/RIF @ lipid-PEG multifunctional nano-micelle;
FIG. 4 is a graph of an ultraviolet-visible absorption spectrum and an emission spectrum of the TTD/RIF @ lipid-PEG multifunctional nano-micelle;
FIG. 5 is a graph of ROS oxygen radical generation efficiency by in vitro illumination of TTD/RIF @ lipid-PEG multifunctional nano-micelles (ABDA is ROS generation indicator);
FIG. 6 is a target imaging diagram of a nodule in a Mycobacterium marinum infected zebrafish embryo local tubercle model by TTD/RIF @ lipid-PEG multifunctional nano-micelles;
FIG. 7 is a target imaging graph of TTD/RIF @ lipid-PEG multifunctional nano-micelles on nodules in a Mycobacterium marinum infected mouse tail nodule model;
FIG. 8 is a CFU coated plate (left panel) and a CFU counting statistical chart (right panel) showing the killing effect of TTD/RIF @ lipid-PEG multifunctional nano-micelles on tubercle bacillus.
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Synthesis of aggregated luminescent photosensitizer TTD
The synthesis procedure of TTD is described in the prior art, and the specific synthetic route is shown in FIG. 2.
Example 2 preparation of TTD/RIF @ lipid-PEG multifunctional nanomicelle
20.0mg of Rifampicin (Rifamicin, RIF, Sigma, USA) as an antituberculous drug is dissolved in 1mL of Tetrahydrofuran (THF) solution, then 1.0mg of aggregated luminescent photosensitizer TTD and 2.0mg of amphiphilic micelle molecule polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG2000, Avanti, USA) are added thereto, and after being mixed uniformly, the mixture is rapidly injected into 9mL of Milli-Q ultrapure water (Millipore, USA), and is subjected to ultrasonication for 2min by using an ultrasonic probe of 12W and 25,000Hz, and then the mixture is poured into a 50mL flask, and then a magneton is added thereto to perform stirring at 500rpm at room temperature and in the absence of light overnight to remove the THF solvent in the mixture. And removing large particles by using a needle filter with the pore size of 0.22 mu m, then carrying out ultrafiltration by using a 10KDa ultrafiltration tube and washing for multiple times, and washing away all unassembled free RIF and DSPE-PEG2000 to finally obtain the TTD/RIF @ lipid-PEG multifunctional nano micelle.
The prepared TTD/RIF @ lipid-PEG multifunctional nano micelle is characterized as follows:
(1) transmission Electron Microscope (TEM): the transmission electron microscope image shows the morphology and the nanometer size of the TTD/RIF @ lipid-PEG nano-micelle, and the result is shown in FIG. 3A, and the transmission electron microscope image shows that the TTD/RIF @ lipid-PEG nano-micelle is a spherical structure with uniform size under a scale of 100 nm.
(2) Zetasizer Nano particle size/zeta potential detector: the Zetasizer Nano particle size/zeta potential detector diagram shows the hydrated particle size distribution and zeta potential value of TTD/RIF @ lipid-PEG Nano micelle, the Nano particle size result (figure 3B) shows that the hydrated particle size of the TTD/RIF @ lipid-PEG Nano micelle is about 100-120nm, and the result is consistent with the electron microscope result; as a result of the Zeta potential, the surface was negatively charged to about-60 mV, and the solution was maintained in a uniform and stable state.
(3) Ultraviolet-visible spectrophotometer (UV-Vis): and the ultraviolet-visible absorption spectrum represents the characteristic absorption spectrum of the TTD/RIF @ lipid-PEG nano-micelle. Results referring to FIG. 4, TTD/RIF @ lipid-PEG nanomicelles have 2 characteristic absorption spectra, 350nm and 490nm, respectively.
(4) Fluorescence spectrophotometer: and (3) a fluorescence spectroscopy spectrum which represents the characteristic emission spectrum of the TTD/RIF @ lipid-PEG nano-micelle. Results referring to FIG. 4, TTD/RIF @ lipid-PEG nanomicelles were excited with 488nm excitation light so that they emitted strong red fluorescence with a maximum emission wavelength of 635 nm.
(5) ABDA in vitro ROS detection kit: ABDA (sigma, usa) possesses 3 characteristic absorption peaks, the peak of which is proportional to the concentration. When ROS free radicals are present in the solution, ABDA is rapidly degraded and its 3 absorption peaks are reduced. Referring to FIG. 5, after an aqueous ABDA solution containing TTD/RIF @ lipid-PEG nanomicelles is irradiated by LED white light, 3 characteristic absorption peaks of ABDA decrease along with time gradient, which indicates that the TTD/RIF @ lipid-PEG nanomicelles can generate a large amount of ROS oxygen free radicals under illumination.
(6) Tuberculosis nodule in vivo targeted imaging capability: after the TTD/RIF @ lipid-PEG nano micelle aqueous solution is intravenously injected into an animal body which is provided with a zebra fish local nodule model caused by Mycobacterium marinum (M.m) infection and a mouse tail nodule model caused by Mycobacterium marinum infection, after a period of in vivo circulation, the TTD/RIF @ lipid-PEG nano micelle is used for observing the target imaging effect of the TTD/RIF @ lipid-PEG nano micelle on nodules and the distribution condition of each organ in the body by a fluorescence microscope and a small animal fluorescence imaging system. The result is shown in figure 6, after the TTD/RIF @ lipid-PEG nano-micelle is injected intravenously and circulates for 24 hours in a zebra fish embryo body successfully constructing local nodules, and the nano-micelle can be successfully co-localized with the nodules through observation of an inverted fluorescence microscope, so that the in-vivo nodule targeting imaging capability of the nano-micelle is shown. The result is shown in figure 7, after the TTD/RIF @ lipid-PEG nano-micelle is injected intravenously in a mouse body with successfully constructed tail nodules and circulates for 12 hours, and the nano-micelle can be successfully co-localized with the nodules through observation of a small animal fluorescence imaging system, so that the in-vivo nodule targeting imaging capability of the nano-micelle is shown; in addition, the TTD/RIF @ lipid-PEG nano micelle is mainly aggregated in the liver, and has no aggregation phenomenon in other organs, which indicates that the TTD/RIF @ lipid-PEG nano micelle is mainly metabolized by the liver.
(7) Killing ability of tubercle bacillus
TTD/RIF @ lipid-PEG nano micelles with different concentrations are used for co-incubation with tubercle bacillus, and after white light irradiation is carried out for 30min, CFU plate coating counting is used for judging the sterilization and bacteriostasis capability of the nano micelle. With reference to fig. 8, under the irradiation of LED white light, the CFU of tubercle bacillus is significantly decreased with the increase of the concentration of TTD/RIF @ lipid-PEG nano-micelle, compared to the unirradiated group, which indicates that TTD/RIF @ lipid-PEG nano-micelle has a photodynamic killing effect on tubercle bacillus. And with the increase of the concentration of the TTD/RIF @ lipid-PEG nano micelle, the CFU of the tubercle bacillus in the non-irradiated group is also reduced in a gradient manner, which shows that the anti-tuberculosis drug RIF wrapped inside the TTD/RIF @ lipid-PEG nano micelle also plays a role in bacteriostasis. The results prove that the TTD/RIF @ lipid-PEG nano-micelle can kill tubercle bacillus and inhibit the proliferation of tubercle bacillus more effectively through photodynamic and drug combination treatment.
Claims (8)
1. The multifunctional nano-micelle is used for targeted imaging of tuberculosis and multi-modal therapy, and is characterized in that the multifunctional nano-micelle is of a core-shell structure, the core of the multifunctional nano-micelle is composed of an aggregated luminescent photosensitizer TTD and an anti-tuberculosis drug, the shell is a monolayer lipid membrane, and the diameter of the multifunctional nano-micelle is 100-120 nm; the anti-tuberculosis drug is rifampicin.
2. The multifunctional nanomicelle for tuberculosis targeted imaging and multimodal therapy according to claim 1, characterized in that the unilamellar lipid membrane is aggregated from amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine.
3. The method for preparing multifunctional nanomicelle according to claim 2, comprising the steps of:
(1) dissolving rifampicin in THF solvent, and adding aggregated luminescent photosensitizer TTD and amphiphilic micelle molecule polyethylene glycol 2000-distearoyl phosphatidyl ethanolamine to form THF mixed solution; wherein the mass ratio of the aggregated luminescent photosensitizer TTD to rifampicin to the amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine is 1-2: 12-30: 2-4;
(2) injecting the THF mixed solution into ultrapure water, performing ultrasonic and photophobic magnetic stirring, and evaporating the THF solvent to dryness;
(3) and filtering for multiple times, and washing away the unbound rifampicin and amphiphilic micelle molecule polyethylene glycol 2000-distearoylphosphatidylethanolamine to obtain the TTD/RIF @ lipid-PEG multifunctional nano-micelle.
4. The preparation method of multifunctional nano-micelle of claim 3, wherein the conditions of the ultrasound in the step (2) are 60-80W, 20-25kHz, and the time is 1-5 min.
5. The method for preparing multifunctional nanomicelle according to claim 3, wherein the multiple filtration of step (3) is performed by a needle filter with a pore size of 0.22 μm and then by ultrafiltration with ultrafiltration of 10 kDa.
6. A formulation for use in the treatment of tuberculosis, characterized in that it comprises the multifunctional nanomicelle according to any one of claims 1 to 2.
7. Use of the multifunctional nanomicelle according to any one of claims 1 to 2 for the preparation of a contrast agent for imaging of tuberculosis lesions.
8. Use of the multifunctional nanomicelle according to any one of claims 1 to 2 for the preparation of a photodynamic, drug co-biocide for tubercle bacillus.
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