CN108421040B - Conjugated polymer nanometer photosensitive material with two-photon imaging and photodynamic curative effects, and preparation and application thereof - Google Patents
Conjugated polymer nanometer photosensitive material with two-photon imaging and photodynamic curative effects, and preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of antitumor drugs, and discloses a conjugated polymer nanometer photosensitive material with two-photon imaging and photodynamic curative effects, and preparation and application thereof. The nanometer photosensitive material is water-soluble nanometer particles, and the interior of the nanometer photosensitive material contains conjugated high molecules, a photosensitizer and a red light luminescent compound, wherein the conjugated high molecules are energy donor materials, and the photosensitizer and the red light luminescent compound are energy acceptor materials; the shell layer of the nano-particle is formed by a surfactant, and the surface of the nano-particle is modified by a modifier containing a functional group with a targeting recognition effect on tumor cells. The nanometer photosensitive material has good biocompatibility, is easy to enter cells, and has a high-efficiency two-photon imaging function and a two-photon photodynamic curative effect; and can be selectively combined with cancer cells, can treat tumors by adopting a photodynamic therapy method of synchronous two-photon imaging, and has high treatment efficiency and less side effects. The preparation method is simple, mild in synthesis conditions and easy to implement.
Description
Technical Field
The invention belongs to the technical field of antitumor drugs, and particularly relates to a conjugated polymer nanometer photosensitive material, a preparation method thereof, application of the conjugated polymer nanometer photosensitive material in two-photon imaging in cancer cells and application of the conjugated polymer nanometer photosensitive material as a tumor photodynamic therapy photosensitizer.
Background
Cancer is one of the most important diseases threatening human health and life. According to the worldwide cancer report issued by the world health organization in 2014, the global cancer morbidity is severe, and the morbidity and the mortality are in a continuously rising trend. The incidence of cancer in China accounts for almost half of the whole world, and thus, the Chinese cancer is one of major diseases which must be overcome independently in China. Photodynamic therapy is a new technology for treating tumors which begins to develop in the end of the seventies of the 20 th century, and clinical trials have been conducted in some countries. To date, photodynamic therapy has been used in more than 3000 patients with cancer, including skin, esophagus, lung, bladder, ovary, head and neck, in many countries around the world. Photodynamic therapy is considered as an effective cancer treatment mode due to the advantages of high spatial precision, small wound, repeatability and the like in clinical research and practice of cancer treatment, is rapidly researched and developed and becomes one of the most active research fields in the tumor prevention and treatment science in the world.
The photodynamic therapy mainly utilizes the photosensitizer which is enriched in cancer cell tissues to be activated under illumination to generate singlet oxygen, and the singlet oxygen reacts with organelle proteins and nucleic acid to cause cancer cells to be necrotic or apoptotic.
The working wavelength of the photosensitizer used in the traditional photodynamic therapy is mainly concentrated in visible light and red light, and the photodynamic killing depth to human tissues is usually only a few millimeters, so that the photosensitizer has a limited clinical application range, is only suitable for treating superficial tumors of skin or the inner wall of an organ cavity, and has difficult good curative effect on tumors in deep tissues.
Two-photon photodynamic therapy is a new generation photodynamic therapy using two-photon excitation technology. This emerging technology utilizes femtosecond lasers with wavelengths in the near infrared region to activate the photosensitizer by two-photon absorption. Since the near infrared light is located in the optical window of the biological tissue (700-1200nm), the optical fiber has larger biological tissue permeability (up to several centimeters). Two-photon photodynamic therapy can thus greatly increase the depth of photodynamic killing.
In order to simultaneously realize high-brightness two-photon fluorescence imaging and high-efficiency photodynamic therapy effect so as to be better applied to a biological system, the two-photon photodynamic therapy technology needs to solve the following key problems: (1) in order to achieve a high photodynamic therapeutic effect, the photosensitizer material needs to have a high two-photon absorption cross section. However, the two-photon absorption capacity of conventional photosensitizer small molecules is low, and the two-photon absorption cross section of the conventional photosensitizer small molecules is only 1-200GM (1GM is 10)-50cm4s photon-1) Very powerful femtosecond lasers are required for effective excitation. The use of high-intensity femtosecond laser can cause potential damage to biological tissues and cannot fully exert the advantages of two-photon photodynamic therapy. (2) In order to realize high-brightness two-photon imaging, the material is required to have higher fluorescence quantum efficiency in a red light region. However, the main function of photosensitizers is to generate singlet oxygen, requiring efficient intersystem crossing (S) from singlet excited to triplet state1→T1) Inevitably, photosensitizers with very low fluorescence quantum efficiency cannot be used for simultaneous biological imaging.
Disclosure of Invention
In order to overcome the defects of the conventional photosensitizer material, the invention aims to provide a conjugated polymer-based nano photosensitive material which can be selectively combined with cancer cells and has a two-photon imaging function and a two-photon photodynamic curative effect.
Another object of the present invention is to provide a method for preparing the conjugated polymer based photosensitive nanomaterial.
The present invention also aims at providing the application of the nanometer photosensitive material of the conjugated polymer. The conjugated high-molecular nanometer photosensitive material is applied to two-photon imaging and a photosensitizer for photodynamic therapy, in particular to the application of cell imaging excited by near infrared two photons and the photosensitizer for two-photon photodynamic therapy of tumors. The invention enhances the singlet oxygen generation amount of a small molecular photosensitizer (energy receptor) and the fluorescence intensity of a red light luminophore (energy receptor) molecule in the nano material through the fluorescence resonance energy transfer effect, and has better effect in near-infrared two-photon excited cell imaging and photodynamic curative effect. The tumor cells are preferably KB cells.
The purpose of the invention is realized by the following technical scheme:
a nanometer photosensitive material of conjugated polymer with two-photon imaging and photodynamic curative effect is a water-soluble nanoparticle, wherein the inside of the water-soluble nanoparticle contains the conjugated polymer, a photosensitizer and a red light luminescent compound, the conjugated polymer is an energy donor material, and the photosensitizer and the red light luminescent compound are energy acceptor materials;
the conjugated polymer has a structural formula as follows:
the molar ratio of the conjugated polymer to the photosensitizer to the red light-emitting compound is 1 (0-0.1): (0-0.1), the dosage of the photosensitizer to the red light-emitting compound is not 0, and the dosage of the photosensitizer to the red light-emitting compound is preferably 1: (0.01-0.05): (0.01-0.05).
The absorption spectrum of the photosensitizer is overlapped with the emission spectrum of the conjugated polymer; the absorption spectrum of the red light-emitting compound is overlapped with the emission spectrum of the conjugated polymer.
The photosensitizer is tetraphenylporphyrin TPP, zinc tetraphenylporphyrin (ZnTPP) or meso-tetra (4-carboxyphenyl) porphin (T790), preferably tetraphenylporphyrin TPP;
the structural formula of the tetraphenylporphyrin TPP is as follows:
the red light emitting compound is preferably TPD (4, 4' - ([1,2,5] thiadiazole [3,4-c ] pyridine-4, 7-substituted) -bis (N, N-diphenylamine)), and the structural formula is as follows:
the shell layer of the water-soluble nanoparticles is formed by a surfactant such as (styrene-co-maleic anhydride) or
One or two of F-127 is used as a surfactant. The surfactant can form nanoparticles with the conjugated polymer, the photosensitizer, and the red light emitting compound and the nanoparticles are water soluble.
The surface of the water-soluble nano-particle is modified by a modifier, wherein the modifier is preferably a modifier containing a functional group with a targeting recognition effect on tumor cells, and is more preferably a folic acid functional group; the modifier DSPE-PEG (2000) Folate and DSPE-PEG (2000), preferably DSPE-PEG (2000) Folate.
The structural formula of poly (styrene-co-maleic anhydride) is as follows:
the nano photosensitive material is stored and used in the form of aqueous solution.
The preparation method of the conjugated polymer nanometer photosensitive material with the two-photon imaging and the photodynamic curative effect comprises the following steps:
(1) preparing a conjugated polymer, a photosensitizer, a red light luminescent compound, a surfactant and a modifier into a mixed solution by adopting an organic solvent;
(2) and mixing the mixed solution with water, carrying out ultrasonic treatment, and removing the organic solvent to obtain an aqueous solution of water-soluble nanoparticles, wherein the nanoparticles in the aqueous solution are the nano photosensitive material.
The molar ratio of the conjugated polymer to the surfactant is 1 (0.1-0.3), preferably 1 (0.1-0.2).
The molar ratio of the conjugated polymer to the photosensitizer to the red light-emitting compound is 1 (0-0.1): (0-0.1), the dosage of the photosensitizer to the red light-emitting compound is not 0, and the dosage of the photosensitizer to the red light-emitting compound is preferably 1: (0.01-0.05): (0.01-0.05).
The molar mass ratio of the conjugated polymer to the modifier is 1 mu mol: (0.002-0.4) mg.
The organic solvent is preferably tetrahydrofuran.
The concentration of the conjugated polymer in the mixed solution is (30-50) mu mol/L, and preferably 40 mu mol/L.
The volume ratio of the water to the mixed solution is (3-40): 1, preferably 4: 1.
The ultrasonic time is 30-60 s.
The size of the nano particles in the photosensitive material is mainly influenced by the dosage of the matrix raw material. The content of the acceptor is low relative to the energy donor, and the energy transfer is insufficient, so that the energy donor cannot be sufficiently utilized. In order to maximize the use of energy donors, a sufficient number of energy acceptor molecules are required, while avoiding a too high content of acceptor molecules leading to self-quenching. Thus, the amount of acceptor molecule added to the energy donor directly affects the efficiency of energy transfer. The invention optimizes the concentration of small acceptor molecules (TPP and TPD) through single-photon and two-photon excitation fluorescence spectrum analysis (the concentration is relative to the energy donor and accounts for the percentage of the energy donor), thereby achieving the optimal energy transfer effect.
The photosensitive material of the invention is a water-soluble nano-particle, and the nano-particle photosensitive material has the following advantages: (1) water-solubility and good biocompatibility. (2) The size of the nanospheres is 40-55 nm, and the nanospheres can well enter cells within 100 nm. (3) The two-photon absorption cross section is large.
The nanometer photosensitive material based on conjugated polymer is prepared with conjugated polymer with high two-photon absorption section as matrix and introduced photosensitizer (e.g. Tetraphenylporphyrin (TPP)) and red luminophore (e.g. TPD) as energy acceptor. The singlet oxygen generation amount of the photosensitizer and the fluorescence intensity of the red luminophor (the two-photon excitation efficiency of the red luminophor or the photosensitizer can be enhanced to reach the level ofSeveral hundred times). Poly (styrene-co-maleic anhydride) or
F-127 acts as a surfactant for better nanoparticle formation. And the nano photosensitive material is subjected to surface modification by a modifier (a compound containing folic acid groups) so that the nano particles can selectively act on cancer cells. The photosensitive material can be selectively combined with cancer cells, has a high-efficiency two-photon imaging function and a two-photon photodynamic curative effect, and is high in treatment efficiency and less in side effect.
Compared with the prior art, the invention has the beneficial effects that:
1. the conjugated polymer nanometer photosensitive material has the characteristics of no toxicity to cells in a dark environment and high damage effect to the cells under the near-infrared illumination condition; the cell is a cancer cell, particularly a KB cell;
2. the nanometer photosensitive material of the conjugated polymer is water-soluble and has good biocompatibility; has nanometer size and is easy to enter cells; under the excitation of two photons, the singlet oxygen generation amount of the photosensitizer and the fluorescence intensity of a red luminophore can be enhanced through fluorescence resonance energy transfer (the singlet oxygen generation amount of the photosensitizer is enhanced by 161 times, and the fluorescence intensity of the red luminophore TPD is enhanced by 23 times), so that the photosensitive material has a high-efficiency two-photon imaging function and a two-photon photodynamic curative effect;
3. the photosensitive material can be selectively combined with cancer cells, can treat tumors by adopting a synchronous two-photon imaging photodynamic therapy method, and has high treatment efficiency and less side effect;
4. the conjugated polymer nanometer photosensitive material has strong two-photon excited fluorescence emission property, good light stability, strong application value and capability of utilizing the photodynamic treatment process of synchronous two-photon imaging;
5. the nanometer photosensitive material of the conjugated polymer has the advantages of easily available raw materials, mild synthesis conditions and simple preparation method.
It should be emphasized that in the prior art, no document reports the conjugated polymer nano photosensitive material of the present invention. The material is used as a high-efficiency nanometer photosensitive material with a two-photon imaging function and a two-photon photodynamic curative effect by simultaneously introducing two receptor molecules and respectively applying the two receptor molecules to singlet oxygen generation and cell imaging.
Drawings
FIG. 1 shows normalized ultraviolet-visible absorption spectrogram (PPBF-Abs, TPP-Abs, TPD-Abs) and normalized fluorescence emission spectrogram (PPBF-PL, TPP-PL, TPD-PL) of conjugated polymer nano photosensitive material prepared by the present invention in tetrahydrofuran;
FIG. 2 is a graph showing a particle size distribution of a nano photosensitive material (FA-PPBF/TPP (2%)/TPD (2%) NPs) of a conjugated polymer prepared in example 1;
FIG. 3 is a transmission electron microscope image of the nano photosensitive material of the conjugated polymer prepared in example 1;
FIG. 4 shows fluorescence spectra of the nanoparticles and the conjugated polymers prepared in examples 2 to 5; wherein (A) fluorescence spectra of nanoparticle materials containing different amounts of photosensitizer TPP in example 2 and TPP (2%) NPs in example 4 under single photon excitation (400 nm); (B) fluorescence spectra under two-photon excitation (750nm) of the nanoparticle material of example 2 containing different amounts of photosensitizer TPP and of the TPP (2%) NPs of example 4, the TPP (2%) NPs x10 representing a 10-fold amplification of the spectral signal of the TPP (2%) NPs; (C) fluorescence spectra of the nano-photosensitive materials containing different contents of the red light-emitting compound TPD in example 3, the PPBF NPs and the PPBF/TPP (2%) NPs in example 2, the TPP (2%) NPs in example 4 and the TPD (2%) NPs in example 5 under single photon excitation (400 nm); (D) fluorescence spectra under two-photon excitation (750nm) of the nano-photosensitive materials containing different contents of red light-emitting compounds TPD in example 3, PPBF NPs and PPBF/TPP (2%) NPs in example 2, TPP (2%) NPs in example 4 and TPD (2%) NPs in example 5, wherein TPP (2%) NPs x10 represents that the spectral signal of TPP (2%) NPs is amplified by 10 times;
FIG. 5 is a graph of the fluorescence enhancement times under single and two-photon excitation of the nano-photosensitive material under different TPD contents in example 3; FIG. A is the fluorescence enhancement factor of PPBF to TPP under single-and two-photon excitation in NPs of nanometer photosensitive material PPBF/TPP (2%)/TPD (1%, 2%, 3%, 5%) with the addition of TPD; panel B shows the fluorescence enhancement factor of PPBF on TPD under single and two-photon excitation with the addition of TPD in PPBF/TPP (2%)/TPD (1%, 2%, 3%, 5%) NPs; A. the ordinate corresponding to 400nm in the graph B is the single photon enhancement multiple, and 750nm corresponds to the two-photon enhancement multiple;
FIG. 6 shows the nano-sized photosensitive materials (PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 3, TPP (20%) NPs and H in example 52The change curve of the singlet oxygen yield of O, namely the change curve of the absorbance value along with the time;
FIG. 7 is a bar graph of cell viability of the nano-photosensitive material FA-PPBF/TPP (2%)/TPD (2%) NPs of the conjugated polymer prepared in example 1, the nano-photosensitive material PPBF/TPP (2%)/TPD (2%) NPs prepared in example 3, and the FA-TPD (2%) NPs prepared in example 5 with respect to photodynamic therapy effect of KB cells under femtosecond laser 750nm illumination;
FIG. 8 is a graph showing the effect of the conjugated polymer nano-photosensitive material (FA-PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 1 on the survival rate of KB cells at various concentrations ((0-4. mu. mol/L));
FIG. 9 is an image of the conjugated polymer nano-photosensitive material (FA-PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 1, the PPBF/TPP (2%)/TPD (2%) NPs prepared in example 3, and the FA-TPD (2%) NPs prepared in example 5 in KB cell under the excitation condition of femtosecond laser 750 nm; panel A is FA-PPBF/TPP (2%)/TPD (2%) NPs, panel B is PPBF/TPP (2%)/TPD (2%) NPs, and panel C is FA-TPD (2%) NPs; A. b, C, the left side is the bright field image of the cell, and the middle is the image of the two-photon excited fluorescence in the cell; the right side is a superimposed image of bright field and two-photon excitation fluorescence imaging of the cells;
FIG. 10 is a graph showing the two-photon absorption cross-sectional values of the conjugated polymers PPBF, TPP, TPD, which are the raw materials for preparing the conjugated polymer nano photosensitive material according to the present invention, change with wavelength.
Detailed Description
The present invention will be described in further detail with reference to specific examples and drawings, but the present invention is not limited thereto. References referred to the synthesis of conjugated polymer PPBF in the examples: liu, p.; li, S.; jin, y.; qian, l.; gao, n.; yao, s.q.; huang, f.; xu, q. -h.; cao, Y, Red-Emitting DPSB-Based Conjugated Polymer Nanoparticles with High Two-Photon luminance for Cell Membrane imaging ACS.Appl.Mater.Interfaces2015,7(12), 6754-6763.
References to the synthesis of the red-emitting compound TPD: jiang, j.; hu, d.; hanif, m.; li, X.; su, s.; xie, z.; liu, l.; zhang, s.; yang, B.; ma, Y., Twist Angle and Rotation free Effects on luminescence Donor-Acceptor Materials, Crystal Structures, Photophysical Properties, and OLED application, adv, Opt, Mater, 2016,4(12), 2109-.
DSPE-PEG (2000) template refers to 1, 2-distaroy-sn-glycerol-3-phosphoethanomine-N- [ Folate (polyethylene glycol) -2000] (ammonium salt) (abbreviated as DSPE-PEG (2000) -FA) and DSPE-PEG (2000) are both available from Avanti Polar Lipids.
Poly (styrene-co-maleic anhydride) (abbreviated as PSMA) used in the present invention was purchased from Sigma Aldrich.
Example 1
A preparation method of a conjugated polymer nanometer photosensitive material with two-photon imaging and photodynamic curative effects comprises the following steps:
(1) respectively dissolving conjugated polymer (PPBF), Tetraphenylporphyrin (TPP), red light luminescent compound (TPD) and poly (styrene-co-maleic anhydride) (PSMA), and DSPE-PEG (2000) -FA in a tetrahydrofuran solvent, mixing the conjugated polymer (PPBF), the Tetraphenylporphyrin (TPP) and the red light luminescent compound (TPD) with a solution of poly (styrene-co-maleic anhydride) (PSMA), adding a tetrahydrofuran solution of DSPE-PEG (2000) -FA, and uniformly mixing to obtain 1mL of a mixed solution; the concentration of conjugated polymer (PPBF) in the mixed solution is 40 mu mol/L, the concentration of Tetraphenylporphyrin (TPP) is 0.8 mu mol/L, the concentration of red-light luminophore (TPD) is 0.8 mu mol/L, the concentration of poly (styrene-co-maleic anhydride) (PSMA) is 8 mu mol/L, and the concentration of DSPE-PEG (2000) -FA is 0.004 mg/mL;
(2) and (3) quickly injecting the mixed solution into 4mL of deionized water, carrying out ultrasonic treatment for 30s, and carrying out rotary evaporation to remove tetrahydrofuran in the solution to obtain a conjugated polymer nano photosensitive material (marked as FA-PPBF/TPP (2%)/TPD (2%) NPs), wherein the nano photosensitive material is stored in the form of an aqueous solution which is a clear bright yellow aqueous solution. The concentration of the prepared conjugated polymer nanometer photosensitive material is 10 mu mol/L according to the concentration of the conjugated polymer PPBF. The particle size distribution diagram of the conjugated polymer nano photosensitive material prepared in this example is shown in fig. 2. As can be seen from FIG. 2, the particle size of the nano photosensitive material prepared by the invention is between 40 and 55nm, and the average diameter is about 45 nm.
The transmission electron microscope image of the conjugated polymer nano photosensitive material prepared in this example is shown in fig. 3.
Example 2
Preparation of several nanoparticle materials:
1. preparation of nanoparticle PPBF NPs: dissolving and mixing conjugated polymer (PPBF), poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) by adopting tetrahydrofuran to obtain 1mL of mixed solution, wherein the concentration of the conjugated polymer (PPBF) in the mixed solution is 40 mu mol/L, the concentration of the PSMA is 8 mu mol/L, and the concentration of the DSPE-PEG (2000) is 0.004 mg/mL; quickly injecting the mixed solution into 4mL of deionized water, performing ultrasonic treatment for 30s, and performing rotary evaporation to remove tetrahydrofuran in the solution to obtain a nanoparticle product PPBF NPs;
2. preparation of nanoparticle PPBF/TPP (1%, 2%, 3%) NPs: dissolving and mixing a conjugated polymer (PPBF), Tetraphenylporphyrin (TPP) with different contents, poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) by adopting tetrahydrofuran to respectively obtain 1mL of mixed solution with different TPP contents, wherein the concentration of the PPBF in the mixed solution is 40 mu mol/L, the concentration of the TPP in the mixed solution is 0.4 mu mol/L, 0.8 mu mol/L and 1.2 mu mol/L, the concentration of the PSMA in the mixed solution is 8 mu mol/L, and the concentration of the DSPE-PEG (2000) in the mixed solution is 0.004 mg/mL; respectively and rapidly injecting mixed solutions with different TPP contents into 4mL of deionized water, carrying out ultrasonic treatment for 30s, and carrying out rotary evaporation to remove tetrahydrofuran in the solutions to respectively obtain nanoparticle products PPBF/TPP (1%) NPs, PPBF/TPP (2%) NPs and PPBF/TPP (3%) NPs.
Example 3
Through the analysis of the nanoparticle material (nanoparticles prepared in example 2) prepared by adding photosensitizer TPP with different proportions to the conjugated polymer, the addition amount of TPP is optimal when the addition amount is 2% of the molar content of the conjugated polymer, so that red-light luminophores TPD with different proportions are added on the basis, and the energy of PPBF is fully utilized.
Preparing a nano photosensitive material:
preparation of nanoparticle PPBF/TPP (2%)/TPD (1%, 2%, 3%, 5%) NPs:
dissolving and mixing conjugated polymer (PPBF), Tetraphenylporphyrin (TPP), red light-emitting compounds (TPD) with different contents, poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) by adopting tetrahydrofuran to obtain 1mL of mixed solution with different TPD contents, wherein the concentration of the conjugated polymer PPBF in the mixed solution is 40 mu mol/L, the concentration of the TPP is 0.8 mu mol/L, the concentrations of the red light-emitting compounds TPD are respectively 0.4 mu mol/L, 0.8 mu mol/L, 1.2 mu mol/L and 2.0 mu mol/L, the concentration of the PSMA is 8 mu mol/L, and the concentration of the DSPE-PEG (2000) is 0.004 mg/mL; the mixed solution is rapidly injected into 4mL of deionized water, ultrasonic treatment is carried out for 30s, tetrahydrofuran in the solution is removed through rotary evaporation, and nanometer photosensitive materials PPBF/TPP (2%)/TPD (1%) NPs, PPBF/TPP (2%)/TPD (2%) NPs, PPBF/TPP (2%)/TPD (3%) NPs and PPBF/TPP (2%)/TPD (5%) NPs are respectively obtained.
Example 4
Preparation of TPP (20%) NPs and TPP (2%) NPs: dissolving and mixing Tetraphenylporphyrin (TPP), poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) with different contents by adopting tetrahydrofuran to obtain 1mL of mixed solution with different TPP contents, wherein the concentrations of TPP in the mixed solution are respectively 0.8 mu mol/L and 8 mu mol/L, the concentration of PSMA is 8 mu mol/L, and the concentration of DSPE-PEG (2000) is 0.004 mg/mL; the mixed solution with different TPP contents is respectively and rapidly injected into 4mL of deionized water, ultrasonic treatment is carried out for 30s, and rotary evaporation is carried out to remove tetrahydrofuran in the solution, so that products TPP (20%) NPs and TPP (2%) NPs are respectively obtained.
Example 5
Preparation of TPD (2%) NPs: dissolving and mixing a red light luminescent compound (TPD), poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) by adopting tetrahydrofuran to obtain 1mL of mixed solution, wherein the concentration of TPD in the mixed solution is 0.8 mu mol/L, the concentration of PSMA is 8 mu mol/L, and the concentration of DSPE-PEG (2000) is 0.004 mg/mL; the mixed solution was rapidly injected into 4mL of deionized water, sonicated for 30s, and rotary evaporated to remove tetrahydrofuran from the solution to give the product TPD (2%) NPs.
Preparation of FA-TPD (2%) NPs: dissolving and mixing a red light luminescent compound (TPD), poly (styrene-co-maleic anhydride) (PSMA) and DSPE-PEG (2000) by adopting tetrahydrofuran to obtain 1mL of mixed solution, wherein the concentration of TPD in the mixed solution is 0.8 mu mol/L, the concentration of PSMA is 8 mu mol/L, and the concentration of DSPE-PEG (2000) -FA is 0.004 mg/mL; the mixed solution was rapidly injected into 4mL of deionized water, sonicated for 30s, and rotary evaporated to remove tetrahydrofuran from the solution to give the product FA-TPD (2%) NPs.
And (3) performance testing:
1. the normalized ultraviolet absorption spectrogram and the normalized fluorescence emission spectrogram of the raw materials of the conjugated polymer nanometer photosensitive material (PPBF), the photosensitizer (TPP) and the red light luminescent compound (TPD) in tetrahydrofuran are shown in figure 1. As can be seen from FIG. 1, the emission spectrum of PPBF and the absorption spectra of TPP and TPD have good overlap, which indicates that the effective fluorescence resonance energy transfer can be realized between donor and acceptor molecules in the nanomaterial.
The two-photon absorption cross section value of the raw materials PPBF, TPP and TPD of the conjugated polymer nano photosensitive material prepared by the invention changes with the wavelength in a curve chart as shown in figure 10.
The test condition is that the two-photon absorption cross section is calculated according to the following formula by taking fluorescein as a reference:
wherein the subscript s refers to the sample being measured and r refers to the reference; phis,ΦrIs the fluorescence quantum yield; ss,SrIs the integral area of the two-photon excitation fluorescence spectrum; cs,CrAre respectively the measured samplesAnd the mass concentration of the reference substance.
2. FIG. 4 shows fluorescence spectra of the nanoparticles and the conjugated polymers prepared in examples 2 to 5; wherein (A) fluorescence spectra of nanoparticle materials containing different amounts of photosensitizer TPP in example 2 and TPP (2%) NPs in example 4 under single photon excitation (400 nm); (B) fluorescence spectra under two-photon excitation (750nm) of the nanoparticle material containing different amounts of photosensitizer TPP in example 2 and of TPP (2%) NPs in example 4, TPP (2%) NPs × 10 representing 10-fold amplification of the spectral signal of TPP (2%) NPs; (C) fluorescence spectra of the nano-photosensitive materials containing different contents of the red light-emitting compound TPD in example 3, the PPBF NPs and the PPBF/TPP (2%) NPs in example 2, the TPP (2%) NPs in example 4 and the TPD (2%) NPs in example 5 under single photon excitation (400 nm); (D) fluorescence spectra of the nano-photosensitive materials containing different contents of the red light emitting compound TPD in example 3, the PPBF NPs, the PPBF/TPP (2%) NPs in example 2, the TPP (2%) NPs in example 4, and the TPD (2%) NPs in example 5 under two-photon excitation (750nm), where TPP (2%) NPs × 10 represents 10-fold amplification of the spectral signal of the TPP (2%) NPs.
The testing steps are as follows: performing spectrum test on the nanoparticles (PPBF/TPP NPs), analyzing the energy transfer efficiency of the PPBF to the TPP in the PPBF/TPP NPs, selecting the concentration of the TPP when the energy transfer of the PPBF to the TPP is about 50%, and generating no self-quenching phenomenon of the TPP under the concentration. As shown in fig. 4 a and B, when 2% of TPP is added to the conjugated polymer nanoparticle system, the energy transfer efficiency of two-photon excited PPBF to TPP is about 50%. And no self-quenching phenomenon of TPP occurs at the concentration. In order to fully utilize the residual energy of the conjugated polymer PPBF, TPD with different proportions is further added on the basis, and the energy transfer efficiency of the PPBF to the TPD in the PPBF/TPP/TPDNPs is analyzed. The energy transfer efficiency of PPBF to TPP and TPD is considered together to select an optimum concentration of TPP and TPD. As can be seen from C and D in FIG. 4, the single-photon and two-photon fluorescence spectra trends of PPBF/TPP/TPD NPs are similar to that of PPBF/TPP NPs. With the increase of the concentration of the TPD of the receptor molecules, the emission peak intensity of the PPBF is further weakened, and the emission peak intensity of the TPD is gradually enhanced; when the substance ratio of TPD/PPBF is 0-2%, the fluorescence peak intensity of TPD increases in proportion to the concentration of TPD. When the substance ratio of TPD/PPBF is more than 2%, the peak at the wavelength of 650nm is weakened as the concentration of TPD is further increased (when the substance ratio of TPD/PPBF is 2% -5%), and the energy transfer of TPP by PPBF is inhibited because the molecular concentration of TPD as an acceptor is too high. Therefore, in order to obtain high two-photon induced fluorescence and high two-photon induced singlet oxygen yield simultaneously, the concentrations of TPP and TPD are fully considered, and finally, a nano material (PPBF/TPP (2%)/TPD (2%) NPs) containing 2% of TPP and 2% of TPD concentration is optimally prepared by taking the concentration of conjugated polymer PPBF as a reference standard.
3. FIG. 5 is a graph of the fluorescence enhancement times under single and two-photon excitation of the nano-photosensitive material under different TPD contents in example 3; FIG. A is the fluorescence enhancement factor of PPBF to TPP under single-and two-photon excitation in NPs of nanometer photosensitive material PPBF/TPP (2%)/TPD (1%, 2%, 3%, 5%) with the addition of TPD; panel B shows the fluorescence enhancement factor of PPBF on TPD under single and two-photon excitation with the addition of TPD in PPBF/TPP (2%)/TPD (1%, 2%, 3%, 5%) NPs; A. the ordinate of the graph B corresponding to 400nm is the single photon enhancement factor, and the ordinate of the graph B corresponding to 750nm is the two-photon enhancement factor.
According to the calculation of the enhancement times, under the two-photon excitation, the sample PPBF/TPP (2%)/TPD (2%) NPs can enhance the singlet oxygen generation amount of the photosensitizer TPP by 161 times through fluorescence resonance energy transfer, and enhance the fluorescence intensity of the red luminophore TPD by 23 times.
4. FIG. 6 shows the nano-sized photosensitive materials (PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 3, TPP (20%) NPs and H in example 42The singlet oxygen yield of O is a curve in which the absorption value changes with time.
FIG. 6 is a diagram showing the spectral method for detecting the amount of singlet oxygen generated from the nano-sized photosensitive material prepared according to the present invention. In photodynamic therapy, singlet oxygen, a major reactive oxygen species, kills cancer cells. Once singlet oxygen exists, 9, 10-anthryl-bis (methylene) diacrylic acid (abbe number is ABDA) is oxidized and degraded to reduce the absorption value, so that ABDA can be used as a chemical probe to indirectly determine the position of the nano photosensitive materialThe amount of singlet oxygen produced. The amount of singlet oxygen generated by the nano photosensitive material is determined by detecting the change in ABDA absorption. In the detection of singlet oxygen, 0.5mL of 10. mu. mol/L aqueous solution of nano photosensitive material (PPBF/TPP (2%)/TPD (2%) NPs) (or aqueous solution of TPP (20%) NPs, or H2O) was added to the reaction solution so that the concentration of ABDA was 0.01 mol/L. Placing the aqueous solution of the mixture of the nanometer photosensitive material and ABDA at an optical power density of 0.91Wcm-2The change of the maximum absorption value of ABDA at 260nm with the irradiation time was detected under a femtosecond laser of 750 nm. The change of absorption spectrum of ABDA in 22min was continuously detected at 2min intervals. Samples PPBF/TPP (2%)/TPD (2%) NPs, TPP (20%) NPs and H were compared from FIG. 62The oxidation effect of ABDA induced by two-photon excitation of O is known as follows:
the efficiency of the composite nano material for generating singlet oxygen under two-photon excitation (750nm) is enhanced by 149 times compared with that of the pure TPP nano material. This shows that in the nanoparticle PPBF/TPP (2%)/TPD (2%) NPs, the amount of singlet oxygen generated is significantly enhanced through the fluorescence resonance energy transfer process of the conjugated polymer to the small molecule photosensitizer.
5. FIG. 7 is a bar graph of cell viability of the nano-photosensitive material FA-PPBF/TPP (2%)/TPD (2%) NPs of the conjugated polymer prepared in example 1, the nano-photosensitive material PPBF/TPP (2%)/TPD (2%) NPs prepared in example 3, and the FA-TPD (2%) NPs prepared in example 5 on photodynamic therapy effect of KB cells under femtosecond laser 750nm illumination. Under the condition of 750nm femtosecond laser illumination for 15min, the cell survival conditions after the treatment of FA-PPBF/TPP (2%)/TPD (2%) NPs, PPBF/TPP (2%)/TPD (2%) NPs and FA-TPD (2%) NPs are detected, obvious two-photon red light signals can be observed in the cells added with the samples of FA-PPBF/TPP (2%)/TPD (2%) NPs, while no obvious two-photon fluorescence signals can be observed in the cells of the samples without the target (PPBF/TPP (2%)/TPD (2%) NPs) and the samples only containing the micromolecular red luminophore (FA-TPD (2%)), which indicates that the samples modified with the targeting FA (FA-PPBF/TPP (2%)/TPD (2%) NPs) can well enter into the cells and the nanoparticles with the conjugated polymer PPBF has the effect of the energy transfer of the PPBF on the micromolecular D, thereby allowing significant enhancement of the fluorescence of the TPD. The FA-PPBF/TPP (2%)/TPD (2%) NPs under two-photon excitation can selectively act on cancer cells, and have stronger two-photon photodynamic curative effect.
6. Cytotoxicity test: cytotoxicity test of FA-PPBF/TPP (2%)/TPD (2%) NPs. KB cells were seeded in 96-well confocal Petri dishes with glass bottom and 5% CO at 37 ℃2The cells were cultured in the incubator for 24 hours until the cells reached 20-30% saturation. After the conjugated polymer nanometer photosensitive materials with different concentrations (0-4 mu mol/L) are added into the cells, the cells are continuously cultured in an incubator for 24 hours, and the cell survival rate under each treatment condition is detected according to an MTT method. The test results are shown in fig. 8.
FIG. 8 is a graph showing the effect of the conjugated polymer nano-photosensitive material (FA-PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 1 on the survival rate of KB cells at various concentrations ((0-4. mu. mol/L)). The cell activity of KB cell in FA-PPBF/TPP (2%)/TPD (2%) NPs of up to 4 mu mol/L still reaches 85%, which shows that the nano photosensitive material FA-PPBF/TPP (2%)/TPD (2%) NPs have good non-toxicity, and can be better applied to organisms.
7. And (3) testing the photodynamic treatment effect: KB cells were seeded in confocal dishes at 37 ℃ with 5% CO2The cells were cultured in the incubator for 24 hours until the cells reached 70-80% saturation. After cells were added with FA-PPBF/TPP (2%)/TPD (2%) NPs, PPBF/TPP (2%)/TPD (2%) NPs and an equal amount of FA-TPD (2%) NPs at a concentration of 1. mu. mol/L and further cultured in an incubator for 12 hours, the cells were observed with a two-photon confocal scanning microscope (OLYMPUS IX73) and had an acceptance range of spectrum of 610-700nm and an objective lens of 100 times oil-scope used for cell imaging.
FIG. 9 is an image of the conjugated polymer nano-photosensitive material (FA-PPBF/TPP (2%)/TPD (2%) NPs) prepared in example 1, the PPBF/TPP (2%)/TPD (2%) NPs prepared in example 3, and the FA-TPD (2%) NPs prepared in example 5 in KB cell under the excitation condition of femtosecond laser 750 nm; panel A is FA-PPBF/TPP (2%)/TPD (2%) NPs, panel B is PPBF/TPP (2%)/TPD (2%) NPs, and panel C is FA-TPD (2%) NPs; A. b, C, the left side is the bright field image of the cell, and the middle is the image of the two-photon excited fluorescence in the cell; the right is the overlay of the bright field and two-photon excited fluorescence imaging of the cells. According to a cellular fluorescence imaging graph under 750nm two-photon excitation (as shown in fig. 9), a significant two-photon red light signal can be observed in cells added with a sample of FA-PPBF/TPP (2%)/TPD (2%) NPs, while no significant two-photon fluorescence signal can be observed in cells with no target on the surface (PPBF/TPP (2%)/TPD (2%) NPs) and only a small-molecule red luminophore sample (FA-TPD (2%) NPs), indicating that the sample modified with targeting FA (FA-PPBF/TPP (2%)/TPD (2%) NPs) can well enter KB cells and in nanoparticles with conjugated high molecular PPBF, the fluorescence of TPD is significantly enhanced due to the energy transfer effect of PPBF as an energy donor on the small-molecule TPD. This also corresponds to the data of the fluorescence spectrum (FIG. 4). The FA-PPBF/TPP (2%)/TPD (2%) NPs can selectively act on cancer cells under two-photon excitation, and have stronger cell fluorescence imaging effect compared with a pure red-light luminophore TPD nano material.
The conclusions for the photosensitive materials in FIGS. 4-6 are still applicable to the photosensitive materials prepared by DSPE-PEG (2000) -FA instead of DSPE-PEG (2000).
In summary, compared with the existing photosensitizer, the nanometer photosensitive material containing the conjugated polymer, the red luminophore and the photosensitizer provided by the invention can fully exert the respective advantages of the donor conjugated polymer PPBF and the two acceptor molecules TPP and TPD while ensuring the biocompatibility. FA-PPBF/TPP/TPD NPs can enhance the generation of photosensitizer singlet oxygen and the fluorescence intensity of red luminophores under the action of fluorescence resonance energy under the near-infrared two-photon excitation, and realize high-efficiency synchronous two-photon fluorescence imaging and photodynamic curative effect. Two-photon excitation has significant advantages: (1) the near infrared light at the long wave position is less influenced by scattering than the short wavelength light, so that the biological tissue permeability is higher, and the photodynamic therapy of deeper tissues can be carried out; (2) the focusing point is better, and the signal to noise ratio is improved; (3) near infrared light at long wavelengths is less cytotoxic than light at short wavelengths.
The nanometer photosensitive material has a very high two-photon absorption cross section, can greatly enhance the effects of two-photon imaging and photodynamic curative effect through the fluorescence resonance energy transfer process, overcomes the defects that the traditional photosensitizer has a small two-photon cross section and can not synchronously carry out cell imaging, and has no toxicity to cancer cells in a dark environment. And the utilization of two-photon excitation has significant advantages: (1) the near-infrared light with long wavelength is less influenced by scattering than the light with short wavelength, so that the biological tissue permeability can be higher, and the photodynamic therapy of deeper tissues can be carried out; (2) the focusing point is better, and the signal to noise ratio is improved; (3) near infrared light at long wavelengths is less cytotoxic than light at short wavelengths.
The above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.
Claims (5)
1. The conjugated polymer nanometer photosensitive material with the two-photon imaging and photodynamic curative effects is characterized by being prepared by the following steps:
(1) preparing a conjugated polymer, a photosensitizer, a red light luminescent compound, a surfactant and a modifier into a mixed solution by adopting an organic solvent;
(2) mixing the mixed solution with water, performing ultrasonic treatment, and removing the organic solvent to obtain an aqueous solution of water-soluble nanoparticles, wherein the water-soluble nanoparticles in the aqueous solution are nano photosensitive materials;
the nanometer photosensitive material is water-soluble nanometer particles, conjugated high molecules, a photosensitizer and a red light luminescent compound are contained in the water-soluble nanometer particles, the conjugated high molecules are energy donor materials, and the photosensitizer and the red light luminescent compound are energy acceptor materials; the conjugated polymer has a structural formula as follows:
the absorption spectrum of the photosensitizer is overlapped with the emission spectrum of the conjugated polymer; the absorption spectrum of the red light-emitting compound is overlapped with the emission spectrum of the conjugated polymer;
the molar ratio of the conjugated polymer to the photosensitizer to the red light-emitting compound is 1 (0-0.1): 0-0.1, and the dosages of the photosensitizer and the red light-emitting compound are not 0;
the photosensitizer is tetraphenylporphyrin, zinc tetraphenylporphyrin or meso-tetra (4-carboxyphenyl) porphin;
the red light-emitting compound is TPD, and the structural formula is as follows:
the nanometer photosensitive material is stored and used in the form of aqueous solution;
the shell layer of the water-soluble nano-particle is formed by a surfactant, and one or two of (styrene-co-maleic anhydride) or F-127 is/are used as the surfactant;
the surface of the water-soluble nano-particle is modified by a modifier, and the modifier is a modifier containing a functional group with a targeting recognition effect on tumor cells.
2. The conjugated polymer based nano photosensitive material with two-photon imaging and photodynamic therapy effects as claimed in claim 1, wherein: the functional group is a folate functional group.
3. The conjugated polymer based nano photosensitive material with two-photon imaging and photodynamic therapy effects as claimed in claim 2, wherein: the modifier is DSPE-PEG (2000) -FA.
4. The conjugated polymer based nano photosensitive material with two-photon imaging and photodynamic therapy effects as claimed in claim 1, wherein: the molar ratio of the conjugated polymer to the surfactant is 1 (0.1-0.3);
the molar mass ratio of the conjugated polymer to the modifier is 1 mu mol: (0.002-0.4) mg;
the concentration of the conjugated polymer in the mixed solution is (30-50) mu mol/L;
the ultrasonic time is 30-60 s.
5. The use of the conjugated polymer based nano photosensitive material according to any one of claims 1 to 4, which has two-photon imaging and photodynamic therapy effects, in the preparation of two-photon imaging agents and photodynamic therapy photosensitizers.
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| "Highly Efficient, Conjugated-Polymer-Based Nano-Photosensitizers for Selectively Targeted Two-Photon Photodynamic Therapy and Imaging of Cancer Cells";Xiaoqin Shen等;《Chem. Eur. J.》;20141202(第21期);摘要，第2215页，第2219页右栏，Figure 1 * |
| "Red-Emitting DPSB-Based Conjugated Polymer Nanoparticles with High Two-Photon Brightness for Cell Membrane Imaging";Peng Liu等;《ACS Appl. Mater. Interfaces》;20150312(第7期);摘要，第6759页右栏，第6760页，Scheme 1-2 * |
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