CN115737812A - Preparation method and application of near-infrared photosensitizer nano preparation - Google Patents

Abstract

The invention relates to a preparation method and application of a near-infrared photosensitizer nano preparation. The nano preparation is prepared by respectively adopting traditional medicine liposome and amphiphilic block polymer as medicine carriers to load near infrared double-reduction fluorine-containing porphyrin photosensitizer (FBC). In contrast to the commercially available photosensitizer Hematoporphyrin (HP)The prepared nano preparation shows high-efficiency singlet oxygen: ( ¹ O ₂ ) The generating capacity is enhanced, the killing effect on the tumor is enhanced, and a good photodynamic treatment effect is shown. Meanwhile, the nano preparation has long blood circulation time in vivo and less residue, and shows higher bioavailability and biocompatibility. The nano preparation provided by the invention has a good application prospect in clinical photodynamic therapy, and provides a new choice for photodynamic tumor therapy.

Description

Preparation method and application of near-infrared photosensitizer nano preparation

Technical Field

The invention relates to the field of nano biomedical materials, in particular to a design of a medicament dosage form loaded with a near infrared FBC photosensitizer, pharmacokinetics research of the medicament and pharmacodynamics research on a cell and animal level.

Background

In the past decades, chemotherapy and the like have played a very important role in cancer treatment. However, traditional tumor treatment methods such as chemotherapy and radiotherapy have the obvious defects of low selectivity, large toxic and side effects and the like, and the treatment effect is poor. In recent years, photodynamic therapy has become an effective and promising mode of cancer therapy due to its non-invasive, high spatial accuracy and controllability, minimal drug resistance, low biological toxicity and immune-stimulatory benefits. Although photodynamic therapy is expected to be an effective method for treating tumors, its wide clinical application is limited due to the limitations of conventional photosensitizers. Most of the existing photosensitizers are hydrophobic photosensitizers, and are easy to accumulate in normal tissues or blood vessels, so that only a small amount of photosensitizers can reach tumor parts, and the treatment effect is influenced. In addition, conventional photosensitizers have poor selectivity and poor tissue penetration ability, which is a fatal weakness in the photodynamic treatment of deep tumors.

The development of nano-drug carriers in recent years effectively improves the problem of poor solubility of anticancer drugs, and brings great hope for tumor treatment. The nano-drug carrier increases the solubility and stability of the anticancer drug by encapsulating the hydrophobic anticancer drug in a lipid soluble region, thereby prolonging the blood circulation time of the anticancer drug in vivo. In addition, the drug-loaded nanoparticles can be passively targeted to tumor parts through high permeability and retention Effect (EPR), so that accumulation in tumor tissues is facilitated, and the bioavailability of the anticancer drug is improved.

The absorption wavelength of the near infrared photosensitizer lies in the red region of the spectrum between 700 and 900 nm, and light in this band enables good tissue penetration and photosensitizer activation, representing an optimal therapeutic window. In recent years, the inventor has developed a near infrared photosensitizer (FBC) with a maximum absorption wavelength of 750 nm and strong molar absorption, so that the photosensitizer drug achieves the possibility of photodynamic therapy of deep tumors under the irradiation of more penetrating near infrared light. In order to better realize the photodynamic therapy effect of the photosensitizer, the photosensitizer is coated by liposome or amphiphilic block polymer to prepare a nano-drug preparation, so that the utilization efficiency of the near-infrared photosensitizer can be effectively improved.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation method and application of a near-infrared photosensitizer nano preparation.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

the invention provides a near-infrared photosensitizer nano preparation, which specifically comprises a drug carrier and a hydrophobic photosensitizer drug FBC, wherein the FBC has the following structure:

。

the drug carriers are respectively liposome and amphiphilic polyethylene oxide-poly-D-lactic acid block copolymer (PEO-PDLA) so as to improve the utilization efficiency of the photosensitizer.

The invention provides a preparation method of a liposome drug loaded with FBC photosensitizer, which comprises the following steps: mixing phospholipid, cholesterol and FBC in a certain proportion in a certain amount of anhydrous ethanol by adopting an ethanol injection method, performing ultrasonic mixing uniformly, dropwise adding into a constant-temperature water phase by using a syringe, emulsifying for 1 h, removing the anhydrous ethanol by dialysis, and then passing the green suspension through a 0.22-micron water phase membrane to obtain a uniform liposome nano solution.

The mass ratio of the phospholipid to the cholesterol is (3-5): 1 (preferably 3: (5-20) (preferably 1.

The dialysis process employs dialysis bags with a molecular weight cut-off of 2000-5000 (preferably 3500).

Also, according to the method for preparing the liposome drug, an HP-Lips liposome solution can be prepared.

The invention also provides a preparation method of the polymer nano-particle loaded with double-reduction fluorine-containing porphyrin photosensitizer (FBC), which comprises the following steps: mixing PEO-PDLA and FBC in a certain amount of tetrahydrofuran according to a certain proportion, ultrasonically mixing uniformly, dropwise adding the mixture into the water phase by using a syringe, stirring for 12 h, and dialyzing to remove the organic solvent to obtain the PEO-PDLA @ FBC nanoparticles with uniform particle size.

The mass ratio of the FBC photosensitizer medicine to the amphiphilic block polymer is 1: (5-20) (preferably 1.

The dialysis process employs a dialysis bag with a molecular weight cut-off of 7000-14000 (preferably 8000-14000). Likewise, according to the preparation method of the polymer nanoparticles, PEO-PDLA @ HP nanoparticles can be prepared.

The invention provides the analysis of the singlet oxygen generation performance of two types of nanoparticles, and experimental results show that the rate of singlet oxygen generation of PEO-PDLA @ FBC nanoparticles is obviously higher than that of PEO-PDLA @ HP nanoparticles, and the nanoparticles have high-efficiency singlet oxygen generation capacity.

The invention provides pharmacokinetic research of PEO-PDLA @ FBC nano particles, and experimental results show that the PEO-PDLA @ FBC nano particles are rapidly distributed in vivo and slowly cleared, so that the blood circulation time of a photosensitizer in vivo is prolonged, and the bioavailability is improved.

The invention provides a tissue distribution research of PEO-PDLA @ FBC nanoparticles, and experimental results show that the PEO-PDLA @ FBC nanoparticles are slowly cleared in normal tissues but have less residues, and have almost no toxic effect on the tissues.

The invention provides an in vitro cytotoxicity experiment of liposome drugs and nanoparticles, and the experimental result shows that the biocompatibility and the cancer cell growth inhibition rate of two FBC-loaded nano preparations are superior to those of HP-loaded nano preparations.

The invention provides in-vivo anti-tumor experiments of two types of nanoparticles in different injection modes, and the experimental results show that compared with the PEO-PDLA @ HP nanoparticles, the PEO-PDLA @ FBC nanoparticles have better in-vivo anti-tumor effect.

The invention has the beneficial effects that: FBC has the following properties compared to common photosensitizers to overcome the challenges faced by conventional photosensitizers: 1) The perfluoroporphyrin structure is clear and very stable; 2) The skin has low phototoxicity and does not need to be strictly protected from light; 3) The treatment wavelength is 750 nm, the tissue penetration depth is ideal, and the PDT effect can be obviously improved. The FBC is wrapped by the drug carrier to form a near-infrared nano preparation, so that the utilization efficiency of the FBC is effectively improved. Meanwhile, the nano preparation has the characteristics of simple synthesis process, less residue, low toxic and side effects, obvious phototoxicity, obvious tumor inhibition effect and the like, thereby having potential application in photodynamic therapy.

Drawings

FIG. 1 shows UV-visible absorption spectra of FBC-Lips.

FIG. 2 shows UV-visible absorption spectra of HP-Lips.

FIG. 3 is the UV-VIS absorption spectrum of PEO-PDLA @ FBC.

FIG. 4 is the UV-visible absorption spectrum of PEO-PDLA @ HP.

FIG. 5 is a graph of PEO-PDLA @ FBC production under light conditions ¹ O ₂ Influence on ultraviolet-visible light absorption of the capture agent DPBF.

FIG. 6 is a graph of the production of PEO-PDLA @ HP under light conditions ¹ O ₂ Influence on ultraviolet-visible light absorption of the capture agent DPBF.

FIG. 7 is fluorescence emission spectra of PEO-PDLA @ FBC plasma samples and blank plasma.

FIG. 8 is a pharmacokinetic profile of tail vein injection of PEO-PDLA @ FBC in Kunming mice.

FIG. 9 is the biological tissue distribution of tail vein injection of PEO-PDLA @ FBC in Kunming mice.

FIG. 10 shows cytotoxicity of FBC-Lips and HP-Lips in different cell lines measured by MTT method.

FIG. 11 shows the cytotoxicity of FBC-Lips and HP-Lips in different cell lines measured by MTT method.

FIG. 12 shows cytotoxicity of PEO-PDLA @ FBC and PEO-PDLA @ HP in different cell lines measured by MTT method.

FIG. 13 shows the cytotoxicity of PEO-PDLA @ FBC and PEO-PDLA @ HP in different cell lines measured by MTT method.

FIG. 14 is the tumor growth curve of BALB/c nude mice treated differently when the drug is injected into tail vein.

FIG. 15 shows the tumor growth curves of BALB/c nude mice treated differently when drugs are injected intratumorally.

Detailed Description

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, which are included to demonstrate that the invention may be practiced, and to provide those skilled in the art with a full description that enables the technical disclosure to be more clearly and easily understood. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 preparation of FBC-Lips Liposomal drugs

In this example, a photosensitizer drug is encapsulated in a nanoliposome formulation using a phospholipid, cholesterol, or other material. The preparation method is an ethanol injection method, and the weight ratio of phospholipid: dissolving cholesterol in absolute ethyl alcohol according to the proportion of 3. Fig. 1 shows the uv-vis absorption spectrum of FBC liposomes, demonstrating the successful loading of FBC by liposomes. The instrument model is Evolution 220UV-Vis, and the spectrum scanning range is from 300 nm to 800 nm.

HP-Lips liposome drug was prepared according to the liposome drug preparation method of example 1, as shown in FIG. 2.

Example 2 preparation method of PEO-PDLA @ FBC nanoparticles

In the embodiment, a photosensitizer FBC is wrapped by a PEO-PDLA block copolymer to form nanoparticles, so that the problem of lipid solubility of the FBC is solved. Dissolving a polymer and a photosensitizer in a ratio of 10:1 in 1 mL of tetrahydrofuran, uniformly dissolving the mixture by using ultrasound, dropwise adding an organic solvent into 5 mL of water phase by using a syringe, and stirring for 12 hours to form a uniform nano solution. The solution was filled into dialysis bags (MWCO = 8000-14000) and dialyzed against deionized water to remove the organic solution. In this way, PEO-PDLA @ FBC nanoparticles were obtained. FIG. 3 shows the UV-visible absorption spectrum of PEO-PDLA @ FBC, demonstrating that PEO-PDLA @ FBC nanoparticles were successfully prepared. The instrument model is Evolution 220UV-Vis, and the spectrum scanning range is from 300 nm to 800 nm.

PEO-PDLA @ HP nanoparticles were prepared according to the nanoparticle method of example 2, as shown in FIG. 4.

Example 3 singlet oxygen Generation ability in nanoparticles

In this example, 1, 3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen scavenger to verify whether the nanoparticles can generate singlet oxygen after being irradiated with light. A solution containing a fixed concentration of PEO-PDLA @ FBC nanoparticles and DPBF was added to a quartz tube and irradiated with a 750 nm laser for 15 s. Because the generation of the singlet oxygen of the nanoparticles is directly related to the reduction of DPBF absorbance in an ultraviolet visible spectrum, the absorbance of DPBF at 420 nm is measured every 3 s, and the generation efficiency of the singlet oxygen of the prepared PEO-PDLA @ FBC nanoparticles in the aqueous solution can be evaluated.

Similarly, a set of absorbance decrease curves was obtained by irradiating an aqueous DPBF solution containing PEO-PDLA @ HP nanoparticles with 650 nm laser for 15 s and measuring the absorbance of the aqueous DPBF solution at 420 nm every 3 s. As shown in FIGS. 5 and 6, the decrease of the absorbance of the PEO-PDLA @ FBC nanoparticles is greater than that of the PEO-PDLA @ HP nanoparticles, which indicates that the rate of singlet oxygen generation of the PEO-PDLA @ FBC nanoparticles is higher than that of the PEO-PDLA @ HP nanoparticles, and the tumor killing effect can be effectively enhanced.

Example 4 validation of the fluorescence method

This example shows the feasibility of fluorescence detection of the FBC content of a photosensitizer in a biological sample. Blank plasma samples and plasma samples containing PEO-pdla @ fbc nanoparticles were scanned using a lumine fluorometer at room temperature. The excitation wavelength is 510 nm, the emission wavelength is 600-900 nm, and the excitation and emission slits are 20 nm. FIG. 7 is a fluorescence spectrum showing that plasma has no effect on the fluorescence measurement of PEO-PDLA @ FBC nanoparticles.

Example 5 pharmacokinetic Studies of PEO-PDLA @ FBC nanoparticles

In this example, three different doses of PEO-PDLA @ FBC nanoparticles were injected into mice via caudal vein, and the amount of the drug in vivo was detected by fluorescence at different time points after injection, indicating that the drug distribution speed in vivo is fast and the clearance is slow.

In Kunming mice, high (5 mg/kg), medium (2 mg/kg) and low dose (1 mg/kg) PEO-PDLA @ FBC nano solutions are respectively administered to tail veins, blood is taken from eye sockets of the mice after intravenous injection administration for 0.083, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours, the blood is placed in a centrifugal tube containing 1.5 mL of heparin sodium and is uniformly mixed, the centrifugal tube is centrifuged at low temperature for 15 min under the condition of 3000 r/min, 0.2 mL of upper plasma samples are taken in a centrifugal tube containing 1.8 mL of physiological saline, and fluorescence is measured by uniformly mixing. The sample was excited at 510 nm and the fluorescence spectrum was recorded in the range 600-900 nm. The measured fluorescence curves are used to calculate the drug content, and pharmacokinetic curves under three drug doses are obtained. As shown in fig. 8, the drug is rapidly distributed in vivo and slowly cleared, and the blood circulation time of the photosensitizer in vivo is prolonged, thereby improving bioavailability.

Example 6 biological tissue distribution analysis of PEO-PDLA @ FBC nanoparticles

In this example, 5 mg/kg dose of PEO-PDLA @ FBC nanoparticles was injected into mice tail vein, mice were sacrificed at different time points after injection, mouse tissues were dissected out, and the content of drug in different tissues was detected by fluorescence.

Injecting PEO-PDLA @ FBC drug-loaded nano solution of 5 mg/kg into tail vein, killing mice at 0.083, 0.5, 1, 2, 4, 8, 12, 24 and 48 h after intravenous injection, quickly dissecting and taking out heart, liver, spleen, lung and kidney tissues, washing off stains with normal saline, weighing after filter paper is sucked dry, and adding 20 times of normal saline to carry out tissue homogenate. The tissue homogenate was centrifuged at 3000 r/min for 15 min at 4 ℃ and the supernatant was taken for fluorescence measurement. The measured fluorescence curves were used to calculate the drug content to study the time-dependent changes in the distribution of the drug in different tissues. As shown in FIG. 9, PEO-PDLA @ FBC nanoparticles cleared slowly but remained less in normal tissues with little toxic effect on tissues.

Example 7 in vitro cell experiments

In order to further study the use of the porphyrin photosensitizer in photodynamic therapy, the inventors also performed in vitro cell experiments. The cytotoxicity is tested by adopting a MTT method and the absorbance is measured by a spectrophotometric enzyme-labeling instrument by taking various cancer cell strains as research objects.

Cytotoxicity of FBC-Lips and HP-Lips under no light and light conditions was measured by MTT method using various cancer cells as the study targets, and the cytotoxicity data graphs of cytotoxicity and dark toxicity of fig. 10 and 11 were obtained. Experiments show that: under the condition of no illumination, the biocompatibility of FBC-Lips to cells is better than that of HP-Lips, which indicates that the photosensitizer FBC has less toxicity than hematoporphyrin HP, and basically has no killing effect on cells at low concentration. Under the condition of illumination, the cell activity is gradually reduced along with the increase of the concentration of porphyrin, namely, the phototoxicity is gradually enhanced. And the toxicity of the FBC-Lips to cells is higher than that of the HP-Lips, which shows that after laser irradiation, the FBC can generate enough singlet oxygen to effectively kill tumor cells.

Similarly, cytotoxicity of PEO-PDLA @ FBC and PEO-PDLA @ HP in the absence of light and in the presence of light was measured by MTT assay using various cancer cells as the study subjects, and the cytotoxicity and dark toxicity data of FIG. 12 and FIG. 13 were obtained, and the results of the experiment were in agreement with the study of liposome drugs.

Example 8 PDT of mouse Breast cancer by Tail intravenous injection of PEO-PDLA @ FBC nanoparticles

This example demonstrates that PEO-pdla @ fbc nanoparticles produce tumor regression/necrosis when exposed to light of the appropriate wavelength.

The tumor model was 4T1 mouse breast cancer cultured in RPMI medium containing 5% fetal bovine serum and supplemented with antibiotics. Cells at 37 ℃ and containing 5% CO ₂ In a humid atmosphere. The cancer cells (1X 10) ⁶ ) Taken in phosphate buffered saline and transplanted subcutaneously into BALB/c miceThe right flank. The tumor volume is up to 120-150 mm ³ The treatment is started at the left and right.

BALB/c nude mice were divided into the following experimental groups:

(1) 4 animals received 2 mg/kg of PEO-PDLA @ HP via tail vein injection;

(2) 4 animals received tail vein injection of 2 mg/kg PEO-PDLA @ FBC;

(3) 4 animals received tail vein injection of 2 mg/kg PEO-PDLA @ HP, light at 650 nm for 7 min;

(4) 4 animals received 2 mg/kg PEO-PDLA @ FBC via tail vein injection and 5 min of 750 nm illumination;

(5) 4 animals received tail vein injection of PBS as a control group.

Tumor length (L) and width (W) were measured with a vernier caliper on the day of injection, day 0. Tumor size in mice was measured postero-septal with formula V = 0.5 xl × W ² The volume is calculated and recorded. The tumor growth curve was then plotted over 14 days using the formula for relative tumor volume. Relative Tumor Volume (RTV) = V/V ₀ （V ₀ Initial tumor volume).

FIG. 14 is a graph showing the relative tumor volume increase in mice after 14 days of tail vein injection. The tumor sizes of the animals in the light treatment group are smaller than those in the untreated group, which shows that the FBC and the HP can generate singlet oxygen to kill cancer cells under the light, but the treatment effect of the FBC is better than that of the HP, and the FBC shows higher tumor inhibition effect after treatment.

Example 9 intratumoral injection of PEO-PDLA @ FBC nanoparticles for PDT of mouse Breast cancer

This example shows that PEO-pdla @ fbc nanoparticles produce tumor regression/necrosis when exposed to light of the appropriate wavelength.

The tumor model was 4T1 mouse breast cancer, cultured in RPMI medium containing 5% fetal bovine serum and supplemented with antibiotics. Cells at 37 ℃ and contain 5% CO ₂ In a humid atmosphere. The cancer cells (1X 10) ⁶ ) Taken up in phosphate buffered saline and implanted subcutaneously in the right flank of BALB/c mice. When the tumor volume is up to 120-150 mm ³ The treatment is started at the left and right.

BALB/c nude mice were divided into the following experimental groups:

(1) 4 animals received intratumoral injection of 2 mg/kg PEO-PDLA @ HP;

(2) 4 animals received intratumoral injection of 2 mg/kg PEO-PDLA @ FBC;

(3) 4 animals received intratumoral injection of 2 mg/kg PEO-PDLA @ HP under 650 nm light for 7 min;

(4) 4 animals received 2 mg/kg PEO-PDLA @ FBC intratumorally, 5 min 750 nm light;

(5) 4 animals received intratumoral injection of PBS as a control group.

Tumor length (L) and width (W) were measured with a vernier caliper on the day of injection, day 0. Tumor size in mice was measured postero-septal with formula V = 0.5 xl × W ² The volume is calculated and recorded. The tumor growth curve was then plotted over 14 days using the formula for relative tumor volume. Relative Tumor Volume (RTV) = V/V ₀ （V ₀ Initial tumor volume).

FIG. 15 is a graph showing the relative tumor volume increase in mice after the intratumoral injection of the drug for 14 days, the experimental results being consistent with the tail vein injection study. But compared with tail vein injection, the PDT effect of intratumoral injection is better.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (11)

1. A near-infrared photosensitizer nanoformulation comprising a drug carrier and a near-infrared hydrophobic photosensitizer drug (FBC) having a maximum absorption wavelength of 750 nm, said FBC having the structure:

。

2. the near-infrared photosensitizer nano-formulation according to claim 1, wherein the drug carriers are respectively liposome and amphiphilic polyethylene oxide-poly-d-lactic acid block copolymer (PEO-PDLA).

3. The preparation method of the liposome drug loaded with the FBC photosensitizer is characterized by comprising the following steps:

(1) Dissolving FBC photosensitizer medicine, phospholipid and cholesterol in a certain proportion in absolute ethyl alcohol, and performing ultrasonic treatment to uniformly dissolve the FBC photosensitizer medicine, the phospholipid and the cholesterol;

(2) Slowly dripping the uniformly mixed organic phase solution into a water phase which is stirred at a high speed and is kept at a constant temperature of 45 ℃ by using an injector, gradually changing the solution from colorless transparency to green suspension, and continuously emulsifying for one hour at the constant temperature;

(3) Dialyzing the emulsified nanosuspension, and passing through a 220 nm aqueous phase membrane to obtain an opalescent FBC-liposome solution (FBC-Lips).

4. The method for preparing a liposomal drug according to claim 3, wherein the mass ratio of the phospholipid to the cholesterol in the step (1) is (3-5): 1, the mass ratio of the FBC photosensitizer medicine to the liposome is 1: (5-20).

5. The method for preparing a liposomal pharmaceutical agent as claimed in claim 3, wherein in step (3), the dialysis process uses a dialysis bag with a molecular weight cut-off of 2000-5000.

6. The method for preparing a liposome drug according to claim 3, wherein a Hematoporphyrin (HP) photosensitizer-loaded liposome solution (HP-Lips) is also prepared.

7. The preparation method of the polymer nano particles loaded with the FBC photosensitizer is characterized by comprising the following steps of:

(1) Dissolving a certain amount of near-infrared FBC photosensitizer and PEO-PDLA block copolymer in tetrahydrofuran, and performing ultrasonic treatment to uniformly mix the solution;

(2) Dropwise and slowly adding the uniformly mixed polymer solution into a water phase under a stirring condition, wherein the water solution gradually changes from colorless and transparent to green clear solution;

(3) And dialyzing the nano solution to finally obtain the successfully-coated polymer nano solution (PEO-PDLA @ FBC).

8. The method for preparing nanoparticles according to claim 7, wherein in the step (1), the mass ratio of the FBC photosensitizer drug to the amphiphilic block polymer is 1: (5-20).

9. The method for preparing nanoparticles according to claim 7, wherein in step (3), the dialysis process is performed by using a dialysis bag with a molecular weight cut-off of 7000-14000.

10. The method for preparing nanoparticles according to claim 7, wherein polymer nanoparticles loaded with Hematoporphyrin (HP) photosensitizer (PEO-PDLA @ HP) are prepared in the same way.

11. Use of the liposomal drug and the polymeric nanoparticle as defined in any one of claims 1 to 10 for the preparation of an antitumor drug.

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