CN108210938B - Multifunctional targeted nano fluorescent probe and preparation and application thereof - Google Patents
Multifunctional targeted nano fluorescent probe and preparation and application thereof Download PDFInfo
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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Abstract
The invention discloses a multifunctional targeted nano fluorescent probe and preparation and application thereof. Firstly, grafting hydrophobic two-photon conjugated oligomer to hydrophilic tumor targeting biomolecule hyaluronic acid through a side chain containing a disulfide bond to obtain an amphiphilic polymer; the amphiphilic polymer can form a nano fluorescent probe through supermolecule self-assembly, and meanwhile, an anti-tumor drug is loaded; the nano fluorescent probe and the drug carrier have strong degradation resistance and higher stability during in vivo circulation, and when reaching tumor cells, the nano fluorescent probe and the drug carrier are degraded by highly expressed reductive substances glutathione and hyaluronidase to generate fluorescence enhancement response, so that targeted two-photon fluorescence imaging is realized, the rapid release of the loaded drug is triggered, and the anti-tumor drug can more efficiently play a therapeutic role. The prepared multifunctional targeted nano fluorescent probe and the drug carrier can simultaneously realize tumor targeted laser confocal two-photon fluorescence imaging and drug delivery, and can monitor the release of the drug in real time without damage.
Description
Technical Field
The invention belongs to the technical field of fluorescence imaging materials and biological drug loading, and particularly relates to a multifunctional nano fluorescence probe and a drug carrier prepared by combining two-photon conjugated oligomer and hyaluronic acid, and application of the multifunctional nano fluorescence probe and the drug carrier in fluorescence biological imaging and biological drug loading technologies.
Background
The two-photon conjugated material can absorb two photons simultaneously so as to realize fluorescence emission, so that the common two-photon excitation wavelength of the material is in a near-infrared waveband, two-photon fluorescence imaging can have better penetration depth, the interference of background stray light is reduced, and the damage to biological tissues can be reduced due to lower energy of light in the near-infrared waveband. The two-photon water-soluble conjugated material realizes two-photon absorption and has water solubility, can keep stronger fluorescence intensity in aqueous solution, and the fluorescent material with the excellent properties can be well applied to organisms as a fluorescent probe.
HA and its derivatives have wide applications in the pharmaceutical and biomedical fields, and are a natural linear polysaccharide in organisms, and have important functions in the functions of organisms, such as stabilizing and organizing extracellular matrix, regulating cell adhesion, and mediating cell proliferation and differentiation. HA receptors (CD44 and RHAMM) are over-expressed on the surfaces of various tumor cells, HA also HAs a plurality of functional groups for chemical modification, and a plurality of researches are carried out to utilize the hydrophilicity and the tumor targeting capacity of the HA for delivering anticancer drugs.
Some studies indicate that the rapid release of drugs from the nanocarriers that reach the pathological site can improve the therapeutic effect and reduce the probability of drug resistance of cancer cells. The physiological environment of the tumor tissue is greatly different from that of the normal tissue, for example, the pH value is acidic, and the tumor cells excessively express reductive Glutathione (GSH), hyaluronidase (HAase) and the like. Based on the characteristics that the reductive GSH can break a disulfide bond and HAase can degrade hyaluronic acid, the two-photon conjugated oligomer is combined with the hyaluronic acid, and the multifunctional targeted nano fluorescent probe with multiple responses and the drug carrier are prepared. The nano fluorescent probe and the drug carrier have strong anti-degradation capability and higher stability in-vivo circulation, and when reaching tumor cells, the nano particles generate fluorescence enhancement response due to degradation by GSH and HAase, so that targeted two-photon fluorescence imaging is realized, the rapid release of the loaded drug is initiated, and the anti-tumor drug can more efficiently play a therapeutic role.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a multifunctional targeted nano fluorescent probe, preparation and application thereof, and application thereof in the aspects of fluorescent biological imaging and biological drug loading technology. The multifunctional targeted nano fluorescent probe can not only deliver drugs in a targeted manner, but also realize the rapid release of the drugs through reductive response or enzymatic degradation, and also has the function of targeted two-photon fluorescence imaging, so that the multifunctional targeted nano fluorescent probe has potential wide application prospects in the aspects of fluorescence imaging and biological drug loading.
The technical scheme is as follows: according to the multifunctional targeted nano fluorescent probe, a hydrophobic two-photon conjugated oligomer is grafted to a hydrophilic tumor targeted biomolecule hyaluronic acid HA through a side chain containing a disulfide bond to obtain an amphiphilic polymer; the amphiphilic polymer forms a nano fluorescent probe through supermolecule self-assembly, and simultaneously loads an anti-tumor drug; the nano fluorescent probe can generate fluorescence enhanced response to high-expression reducing substances Glutathione (GSH) and hyaluronidase (HAase) in tumor cells, so that fluorescent imaging is realized and drugs are released.
Wherein the content of the first and second substances,
the amphiphilic polymer has the following molecular formula:
wherein, the 9-substituted fluorenyl is an electron donor, referred to as D for short, the olefinic bond is a conjugate bridge, the aromatic heterocyclic ring Ar with a narrow band gap is an electron acceptor, referred to as A for short, a conjugated structure of D-pi-A-pi-D is formed, and the two-photon absorption property is realized; r is selected from the following groups: r1CONH, where R1Is one of the following groups: alkyl, alkoxy; r' is selected from the following groups R2COOH，R2Is one of the following groups: alkyl, alkoxy; ar is one of 4, 7-bis (benzene-2-yl) -2,1, 3-benzothiadiazole group or 4, 7-bis (thiophene-2-yl) -2,1, 3-benzothiadiazole group, and HA is hyaluronic acid.
The amphiphilic polymer is characterized in that the amphiphilic polymer,
a. when Ar is a 4, 7-bis (phen-2-yl) -2,1, 3-benzothiadiazole group, one of the typical structures is as follows: r has the structure R1CONH, R' having the structure R2COOH，R1And R2Same, are all alkyl; the structural formula is as follows:
b. when Ar is a 4, 7-bis (thien-2-yl) -2,1, 3-benzothiadiazole group, one of the typical structures is as follows: r has the structure R1CONH, R' having the structure R2COOH，R1And R2And the alkyl groups have the following structural formula:
the preparation method of the multifunctional targeted nano fluorescent probe provided by the invention comprises the following steps:
dissolving an amphiphilic polymer in water, dissolving a hydrophobic antitumor drug in a polar organic solvent dimethyl sulfoxide (DMSO) which can be mixed with water, then dropwise adding the DMSO solution into a polymer aqueous solution in an ultrasonic state, carrying out intermittent ultrasonic treatment in an ice water bath by using a tip type ultrasonic instrument, stirring at room temperature, then transferring into a dialysis bag, and firstly, respectively carrying out volume ratio: and (3) dialyzing the mixed solution of methanol and water in a ratio of 2:1 and 1:1 for 12 to 15 hours, dialyzing the mixed solution in deionized water for 24 to 26 hours to remove the unencapsulated drugs and form stably dispersed nanoparticles, filtering the nanoparticles by using a filter membrane, and freeze-drying the filtered nanoparticles to obtain the targeted nano fluorescent probe.
The preparation method of the amphiphilic polymer comprises the following steps:
1) adopting organic metal catalyzed Heck coupling reaction to couple single bromofluorene derivative monomers on two sides of a divinyl aromatic heterocyclic monomer with a narrow band gap, or coupling vinyl fluorene derivative monomers on two sides of a dibromo aromatic heterocyclic monomer with a narrow band gap to obtain a D-pi-A-pi-D conjugated structure, and then preparing a two-photon conjugated oligomer with a side chain containing carboxyl through side chain carboxylation reaction;
2) grafting the two-photon conjugated oligomer prepared in the step 1) to HA modified by cystamine under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS to obtain the amphiphilic polymer.
The application of the multifunctional targeted nano fluorescent probe provided by the invention is applied to tumor targeted two-photon fluorescent cell imaging and drug delivery, and comprises the following steps:
1) grafting a two-photon conjugated oligomer onto cystamine modified HA to prepare an amphiphilic polymer;
2) mixing the amphiphilic polymer with a hydrophobic anti-tumor drug, and preparing a nano fluorescent probe with a hydrophobic cavity wrapped with the drug by self-assembly through a dialysis method;
3) the targeted nano fluorescent probe is applied to tumor cells, so that the fluorescence enhancement response to GSH and HAase is realized, and the tumor targeted laser confocal two-photon fluorescence imaging and the drug delivery are realized.
Has the advantages that: in the invention, the multifunctional nano fluorescent probe and the drug carrier are prepared by supermolecule self-assembly. After the nano-drug carrier is formed by self-assembly, the fluorescence of the two-photon conjugated oligomer is weakened due to aggregation; the multifunctional nano fluorescent probe and the nano drug carrier can be stably conveyed to a tumor part, are selectively taken up by tumor cells through the endocytosis mediated by HA receptors, and can be degraded by reducing GSH and HAase once internalized into the tumor cells, so that the release effect of intracellular drugs is improved, and the anti-tumor effect is improved; and the fluorescence of the two-photon conjugated oligomer is enhanced, so that the aim of cooperatively performing the tumor targeted two-photon fluorescence cell imaging and the drug release monitoring process is fulfilled, and the nano material with multiple functions has potential wide application prospect in the aspects of fluorescence imaging and biological drug loading.
Drawings
FIG. 1 is a schematic structural diagram of a nano fluorescent probe HA-TPO/CPT NP carrying camptothecin CPT as an antitumor drug prepared by the invention.
FIG. 2 shows fluorescence emission spectra (excitation wavelength: 425nm) of two-photon conjugated oligomer TPO, amphiphilic polymer HA-TPO, targeted nanoparticle HA-TPO NP and CPT-loaded nano fluorescent probe HA-TPO/CPT NP prepared by the invention in water, wherein TPO concentration in each material is 5 × 10-6mol/L。
FIG. 3 shows the cell imaging of the targeting nanoparticle HA-TPO NP prepared by the present invention under the laser confocal fluorescence microscope two-photon mode in tumor cell HeLa of overexpression GSH and HAase (excitation wavelength: 720nm, collection wavelength range: 500nm-700 nm).
FIG. 4 shows the targeted nanoparticle HA-TPO NP prepared by the invention in the environment containing different concentrations of reducing Dithiothreitol (DTT): the particle size of 10mM DTT (simulating the reducing environment of tumor cells), 10uM DTT (simulating the reducing environment in human plasma), and Dynamic Light Scattering (DLS) in the absence of DTT were varied with time.
Detailed Description
In order to better understand the contents of the present invention, the following examples further illustrate the technical solutions of the present invention, but should not be construed as limiting the scope of the present invention.
Example (b):
1) the monomers (1) and (2) were prepared by charging (1) and (2) in a mass ratio of 1:2Material, then in Pd (OAc)2/P(o-tolyl)3Coupling the two through Heck reaction under catalysis, and obtaining neutral two-photon conjugated oligomer after 24 hours of reaction; and further using trifluoroacetic acid to carry out carboxylation reaction for 4 hours to obtain the two-photon conjugated oligomer TPO.
2) Hyaluronic acid-Cystamine (HA-Cystamine) was prepared by the following method, TPO was dissolved in DMSO, catalysts EDC and NHS were dissolved in Phosphate Buffered Saline (PBS), respectively, added to the DMSO solution on TPO, reacted for 2 hours, then 2-mercaptoethanol was added to quench the reactivity of EDC, and after 5 minutes of reaction, a certain amount of HA-Cystamine was added, followed by stirring at room temperature overnight. The solution in the reaction flask was transferred into a dialysis bag with a molecular weight cut-off (MWCO) of 3500Da, and unreacted EDC, NHS, 2-mercaptoethanol, unreacted TPO and other intermediates were removed by dialysis, followed by freeze-drying for 36 hours to obtain an amphiphilic polymer HA-TPO.
3) Dissolving the HA-TPO prepared in the step 2) in a mixed solution of DMSO and water (DMSO: H)2O ═ 1:1), then sonicated with a tip-type sonicator for 10 minutes in an ice-water bath, stirred for 2 hours, transferred into a dialysis bag with MWCO of 3500Da, dialyzed in deionized water for 24 hours, during which water was changed 3 times, then filtered with a 0.45 μm filter and freeze-dried to give targeted nanoparticle HA-TPO NPs.
4) Dissolving an antitumor drug CPT in DMSO, dropwise adding the antitumor drug CPT into the aqueous solution dissolved with the HA-TPO prepared in the step 2) under the ultrasonic state, then carrying out ultrasonic treatment for 10 minutes in an ice water bath by using a tip type ultrasonic instrument, stirring for 2 hours, transferring the mixture into a dialysis bag with the MWCO of 3500Da, firstly dialyzing the mixture in a mixed solution of methanol and water of 2:1 and 1:1(V: V) for 12 hours, then dialyzing the mixture in deionized water for 24 hours, then filtering the mixture by using a 0.45 mu m filter membrane, and freeze-drying the mixture to finally obtain the CPT-loaded targeted nanoprobe HA-TPO/CPT NP.
5) Incubating tumor cells HeLa excessively expressing GSH and HAase by using a culture medium containing a targeting nano probe HA-TPO/CPT NP, placing the cells under a laser confocal fluorescence microscope after 4 hours, exciting by using 720nm laser in a two-photon mode, collecting fluorescence between 500 and 700nm, and taking a picture to obtain a cell imaging result.
Claims (4)
1. A multifunctional targeted nano fluorescent probe is characterized in that a hydrophobic two-photon conjugated oligomer is grafted to a hydrophilic tumor targeted biomolecule hyaluronic acid HA through a side chain containing a disulfide bond to obtain an amphiphilic polymer; the amphiphilic polymer forms a nano fluorescent probe through supermolecule self-assembly, and simultaneously loads an anti-tumor drug; the nano fluorescent probe can generate fluorescence enhanced response to high-expression reducing substances Glutathione (GSH) and hyaluronidase (HAase) in tumor cells, so that fluorescent imaging is realized and drugs are released;
the amphiphilic polymer has the following molecular formula:
wherein, the 9-substituted fluorenyl is an electron donor, referred to as D for short, the olefinic bond is a conjugate bridge, the aromatic heterocyclic ring Ar with a narrow band gap is an electron acceptor, referred to as A for short, a conjugated structure of D-pi-A-pi-D is formed, and the two-photon absorption property is realized; r is selected from the following groups: r1CONH, where R1Is one of the following groups: alkylene, alkyleneoxy; r' is selected from the following groups R2COOH，R2Is one of the following groups: alkylene, alkyleneoxy; ar is one of 4, 7-bis (benzene-2-yl) -2,1, 3-benzothiadiazole group or 4, 7-bis (thiophene-2-yl) -2,1, 3-benzothiadiazole group, and HA is hyaluronic acid.
2. The multifunctional targeted nano fluorescent probe according to claim 1, characterized in that the amphiphilic polymer is:
a. when Ar is 4, 7-bis (benzene-2-yl) -2,1, 3-benzothiadiazole group, the structural formula is as follows:
or:
b. when Ar is 4, 7-bi (thiophene-2 group) -2,1, 3-benzothiadiazole group, the structural formula is as follows:
3. the method for preparing the multifunctional targeted nano fluorescent probe as claimed in claim 1, which is characterized in that the preparation method comprises the following steps:
dissolving an amphiphilic polymer in water, dissolving a hydrophobic antitumor drug in a polar organic solvent dimethyl sulfoxide (DMSO) which can be mixed with water, then dropwise adding the DMSO solution into a polymer aqueous solution in an ultrasonic state, carrying out intermittent ultrasonic treatment in an ice water bath by using a tip type ultrasonic instrument, stirring at room temperature, then transferring into a dialysis bag, and firstly, respectively carrying out volume ratio: and (3) dialyzing the mixed solution of methanol and water in a ratio of 2:1 and 1:1 for 12 to 15 hours, dialyzing the mixed solution in deionized water for 24 to 26 hours to remove the unencapsulated drugs and form stably dispersed nanoparticles, filtering the nanoparticles by using a filter membrane, and freeze-drying the filtered nanoparticles to obtain the targeted nano fluorescent probe.
4. The preparation method of the multifunctional targeted nano fluorescent probe according to claim 3, characterized in that the preparation method of the amphiphilic polymer comprises the following steps:
1) adopting organic metal catalyzed Heck coupling reaction to couple single bromofluorene derivative monomers on two sides of a divinyl aromatic heterocyclic monomer with a narrow band gap, or coupling vinyl fluorene derivative monomers on two sides of a dibromo aromatic heterocyclic monomer with a narrow band gap to obtain a D-pi-A-pi-D conjugated structure, and then preparing a two-photon conjugated oligomer with a side chain containing carboxyl through side chain carboxylation reaction;
2) grafting the two-photon conjugated oligomer prepared in the step 1) to HA modified by cystamine under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS to obtain the amphiphilic polymer.
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