Phthalocyanine nanoliposomes responsive to micro-acid and glutathione and preparation and optical diagnosis and treatment applications thereof
By designing phthalocyanine nanoliposomes responsive to microacids and glutathione, the problem of insufficient photosensitizer selectivity in photodynamic therapy was solved, achieving specific activation of tumor tissue and efficient photodynamic therapy, significantly inhibiting tumor growth, and improving the precision and safety of treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FUZHOU UNIV
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-09
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical applications of nanomaterials, specifically relating to a phthalocyanine nanoliposome responsive to microacids and glutathione, its preparation, and its application in cancer optical diagnosis and treatment. Background Technology
[0002] Malignant tumors, as one of the major diseases in today's society, seriously threaten human health and life. Clinically, most patients are diagnosed at an advanced stage, often missing the optimal treatment window. This phenomenon mainly stems from the limitations of traditional imaging methods (such as ultrasound, X-ray, CT, and MRI) in terms of specificity, sensitivity, and resolution, making it difficult to accurately detect minute lesions and early-stage tumors. On the other hand, the highly invasive pathological characteristics of malignant tumors make it difficult to precisely locate tumor boundaries, which also restricts the realization of precision treatment to some extent.
[0003] In recent years, photodynamic therapy (PDT) has emerged as a promising new treatment for tumors. The PDT reaction is based on the interaction between a photosensitizer, oxygen, and light of a suitable wavelength. The photosensitizer is excited by a suitable light source to produce cytotoxic reactive oxygen species (ROS). Typically, after the photosensitizer enters the body, the tumor site is irradiated with a light source of a specific wavelength, causing the photosensitizer to produce ROS, leading to tumor cell apoptosis or necrosis. During PDT, the photosensitizer, oxygen, and light work together, while any single component is non-toxic to the biological system. PDT's advantages, including high selectivity, low toxicity, low tolerance, and repeatability, make it a promising alternative to traditional cancer treatments such as surgery, chemotherapy, and radiotherapy.
[0004] As one of the most important components of photodynamic therapy, the photosensitizer's properties, such as light absorption capacity, singlet oxygen production rate, and tumor targeting, directly affect the efficacy and application prospects of photodynamic therapy. An ideal photosensitizer should possess characteristics such as low dark toxicity, high photoinducible toxicity, selective accumulation in tumors, rapid disappearance from healthy tissues, and high absorption in the red and near-infrared spectral range.
[0005] Phthalocyanines, with their unique molecular structure and photosensitizing properties, have become typical representatives of photosensitizers. Their good photostability and modifiability have also laid the foundation for further functional design. Against this backdrop, researchers have used phthalocyanines as a molecular backbone, introducing functional groups that regulate solubility, targeting, or environmental responsiveness through covalent modification of their peripheral rings or central metal atoms. Currently, four phthalocyanine photosensitizers have been approved for clinical research or use, including amino-axially substituted silicon phthalocyanine Pc4, sulfonated aluminum phthalocyanine Photosens, liposome-encapsulated zinc phthalocyanine, and the amphiphilic "fossilane". However, despite these advances, photodynamic therapy has not yet become a first-line mainstream treatment for cancer. A fundamental reason is that many photosensitizers still have limited selectivity for tumor tissues. Traditional photosensitizers are usually in a "normally on" state in vivo, and their non-specific accumulation and activation in normal tissues lead to potential photosensitivity toxicity, which severely restricts their clinical translation and application. Therefore, designing activatable photosensitizers based on the tumor microenvironment, which keep their photosensitivity "off" in normal tissues, and can be activated by specific disease markers in tumor tissues to achieve phototoxicity, has become a key to overcoming current difficulties. This method can effectively avoid photodamage to normal tissues and improve the precision of photodynamic therapy.
[0006] Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine linked by amide bonds. It is a major cellular thiol involved in cellular redox reactions. Studies have shown that during tumor development, cancer cells produce large amounts of reactive oxygen species (ROS) to support their rapid metabolism and malignant proliferation. Consequently, intracellular glutathione expression levels are upregulated to eliminate potential protein and DNA damage, thus protecting cancer cells from programmed cell death caused by oxidative stress. This results in higher glutathione expression levels in tumor cells compared to normal cells. The significant difference in glutathione concentration between most tumor cells and normal cells makes glutathione a promising biological target for activating photodynamic therapy. Upon irradiation with light of a specific wavelength, the photodynamic activity of glutathione-responsive photosensitizers is "off" in normal cells, but activated to an "on" state in tumor cells by high concentrations of glutathione, restoring their ability to produce ROS and further inducing cancer cell necrosis or apoptosis. Therefore, such glutathione-responsive photosensitizers have great potential for achieving precise tumor treatment.
[0007] Sulfonate groups, as classic "prodrug" masking groups, are susceptible to nucleophilic attack from the thiol groups in glutathione, resulting in irreversible aromatic nucleophilic substitution reactions and ester bond cleavage. Modifying sulfonate groups at the functional sites of phthalocyanine photosensitizers creates a molecular system with photoinduced electron transfer effects. This system remains "off" in normal tissues, but upon entering high-glutathione tumor cells, the ester bond breaks, the group leaves, and the photosensitizer activity is restored, thus achieving specific activation.
[0008] In summary, current advancements in photodynamic therapy are progressing towards improving tumor selectivity and treatment precision. Utilizing biomarkers such as glutathione, which are highly expressed in the tumor microenvironment, to design activatable photosensitizers, enabling intelligent regulation of photosensitivity activity—"off" in normal tissues and "on" in tumor tissues—is a key strategy for overcoming existing clinical translational bottlenecks and is of great significance for promoting precision cancer treatment. Summary of the Invention
[0009] The purpose of this invention is to provide phthalocyanine nanoliposomes responsive to microacids and glutathione, their preparation method, and their application in cancer phototherapy. These phthalocyanine nanoliposomes not only exhibit precise fluorescence imaging capabilities but also possess good photodynamic antitumor activity, demonstrating significant advantages as an anticancer drug.
[0010] To achieve the above objectives, the present invention adopts the following technical solution:
[0011] First, the present invention provides a phthalocyanine nanoliposome containing phospholipid-polyethylene glycol, cholesterol hemisuccinate and phthalocyanine with photosensitizing ability.
[0012] In one or more embodiments, the phospholipid-polyethylene glycol includes distearate phosphatidylethanolamine-polyethylene glycol, wherein the molecular weight of the polyethylene glycol includes, but is not limited to, 2000; and the phthalocyanine includes sulfonate-based pericyclic tetrasubstituted phthalocyanine.
[0013] Secondly, the present invention provides a method for preparing the above-mentioned phthalocyanine nanoliposomes, the method comprising the step of mixing phospholipid-polyethylene glycol, cholesterol hemisuccinate and phthalocyanine with photosensitizing ability to obtain phthalocyanine nanoliposomes.
[0014] In several embodiments, the preparation of the phthalocyanine nanoliposomes includes the following steps:
[0015] (1) Dissolve phospholipid-polyethylene glycol and cholesterol hemisuccinate in 8-16 mL of chloroform by ultrasonication to obtain a lipid solution; dissolve phthalocyanine in 1-8 mL of tetrahydrofuran by ultrasonication to obtain a phthalocyanine solution; mix the lipid solution and the phthalocyanine solution evenly under ultrasonic conditions to obtain a mixed organic phase solution. The molar ratio of phospholipid-polyethylene glycol, cholesterol hemisuccinate and phthalocyanine is 9-16:4-7:1.
[0016] (2) Remove the solvent from the mixed organic phase solution of (1) under reduced pressure by rotary evaporation at 32~41℃ and 50~130rpm to form a lipid film; add 3~10mL of deionized water to the lipid film and dissolve it by ultrasonication to obtain a liposome suspension; place the liposome suspension at room temperature for 0.25~48h to allow the liposomes to fully swell in water to obtain a liposome suspension.
[0017] (3) Transfer the liposome suspension from (2) to a centrifuge tube and break it up with an ultrasonic cell disruptor under ice-water bath conditions. The ultrasonic conditions are: 3-5s interval between disruptions, 3-5s disruption time, intensity 35%-65%, duration 2-10min, and repeat twice to finally obtain the nanoliposome solution.
[0018] (4) The nanoliposome solution of (3) is filtered and squeezed using a 0.22 μm and / or 0.45 μm aqueous and / or organic microporous membrane to remove large particles and aggregates, thereby obtaining a phthalocyanine nanoliposome solution.
[0019] In one or more embodiments, the phthalocyanine is prepared as follows:
[0020] 1) 4-Benzyloxyphthalonitrile and zinc acetate were dissolved in n-pentanol and reacted at 90°C for 0.5 h until the solution became clear. 1,8-diazabicyclo[5.4.0]undec-7-ene was added and the mixture was heated to 140°C in the dark for 8 h. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography and then by gel column chromatography. The product was dissolved in tetrahydrofuran, and n-hexane was added. The mixture was allowed to stand at 4°C to crystallize. The crystals were filtered, washed with n-hexane, and dried under vacuum at 45°C to obtain ZnPc-4Bn.
[0021] 2) ZnPc-4Bn is dissolved in toluene and trifluoroacetic acid and reacted at 90°C for 36 h under an argon atmosphere; hexane is added to the reaction solution and allowed to stand at 4°C to crystallize. The supernatant is discarded, and the crystallization operation is repeated. The solvent is then removed by vacuum evaporation to obtain ZnPc-4OH.
[0022] 3) ZnPc-4OH was dissolved in tetrahydrofuran, triethylamine was added, and the mixture was stirred and activated at 10°C for 30 min. A tetrahydrofuran solution of 2-chloro-4-nitrobenzenesulfonyl chloride was slowly added dropwise, and the reaction was carried out at 10°C for 4 h. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography and then by gel column chromatography. The product was dissolved in tetrahydrofuran, n-hexane was added, and the mixture was allowed to stand at 4°C to crystallize. The crystals were filtered, washed with n-hexane, and dried under vacuum at 45°C to obtain ZnPc-4ClNBS, i.e., phthalocyanine.
[0023] Finally, the present invention also provides the application of the phthalocyanine nanoliposomes in cancer fluorescence imaging and photodynamic therapy.
[0024] In one or more embodiments, the cancer includes, but is not limited to, any one of lung cancer, liver cancer, breast cancer, cervical cancer, bladder cancer, pancreatic cancer, lymphoma, esophageal cancer, gastric cancer, bile duct cancer, and colon cancer.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] (1) The phthalocyanine nanoliposomes described in this invention solve the hydrophobicity problem of phthalocyanine, which is beneficial to improving its bioavailability.
[0027] (2) The phthalocyanine nanoliposomes described in this invention have the ability to passively target cancer cells. Through the high-permeability and long-stay effect, they can accumulate significantly in tumor tissue and remain there for a long time.
[0028] (3) The phthalocyanine nanoliposomes described in this invention avoid non-specific activation in normal tissues and are specifically activated only at the tumor site, thereby improving the accuracy of tumor diagnosis.
[0029] (4) The phthalocyanine nanoliposomes described in this invention exhibit precise fluorescence imaging-guided photodynamic antitumor effects. In a mouse tumor model, tumors were significantly inhibited on day 14 of treatment, with an inhibition rate of 87%. This indicates that they have excellent antitumor efficacy and are a promising antitumor drug. Attached Figure Description
[0030] Figure 1 The fluorescence recovery of phthalocyanine molecules in a glutathione environment after different time intervals is shown.
[0031] Figure 2 This is a transmission electron microscope image of phthalocyanine nanoliposomes.
[0032] Figure 3 The particle size stability of phthalocyanine nanoliposomes in water.
[0033] Figure 4 This is a comparison of the fluorescence changes of phthalocyanine nanoliposomes and MB when DCFH is used as a fluorescent probe.
[0034] Figure 5 The acid response capability of phthalocyanine nanoliposomes.
[0035] Figure 6 This is an in vivo fluorescence imaging of phthalocyanine nanoliposomes.
[0036] Figure 7 This is a comparison of tumor growth curves in H22 tumor-bearing mice treated with phthalocyanine nanoliposomes over 14 days.
[0037] Figure 8 This is a graph showing the changes in body weight of tumor-bearing mice after different treatments. Detailed Implementation
[0038] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.
[0039] The 4-benzyloxyphthalonitrile (CAS No.: 86312-75-6), 1,8-diazabicyclo[5.4.0]undec-7-ene (CAS No.: 6674-22-2), distearylphosphatidylethanolamine-polyethylene glycol 2000 (CAS No.: 147867-65-0), and cholesterol hemisuccinate (CAS No.: 1510-21-0) used in the embodiments of the present invention are all commercially available.
[0040] Example 1:
[0041] Synthesis of 2-chloro-4-nitrobenzenesulfonate-substituted phthalocyanine ZnPc-4ClNBS
[0042]
[0043] (1) Compound ZnPc-4Bn ( Preparation of the crude product: 0.50 g (2.14 mmol) of 4-benzyloxyphthalonitrile and 0.10 g (0.54 mmol) of zinc acetate were added to a 100 mL reaction flask, followed by dissolution in 20 mL of n-pentanol. The reaction was carried out at 90 °C for 0.5 h until the reaction solution was basically transparent. Then, 0.5 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added, and the reaction was continued at 140 °C for 8 h in the dark. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a 100-200 mesh screen. First, dichloromethane (DCM) and petroleum ether (PE) (1:1, v / v) were used as the mobile phase to wash away the first yellow impurities. Then, ethyl acetate (EA) of DCM (30:1, v / v) was used as the mobile phase to wash away the remaining impurities. Finally, the blue-green fraction was collected using DCM and EA (10:1, v / v) as the mobile phase. The blue-green component was purified by rotary evaporation under reduced pressure to remove the solvent, and then purified once more under the silica gel column chromatography conditions described above to obtain a blue-green solid. The obtained blue-green solid was dissolved in a small amount of tetrahydrofuran (THF) and further purified by a Bio-Beads S-X1 gel column using THF as the eluent. The blue-green component was collected, and the solvent was removed by rotary evaporation under reduced pressure to obtain a blue solid. This solid was dissolved in 5 mL of THF and poured into 100 mL of n-hexane. The solution was placed in a refrigerator at 4°C and allowed to stand for 0.5 h to precipitate. The solution was then filtered and washed with a large amount of n-hexane until the filtrate was colorless. Finally, the solution was dried to constant weight in a vacuum drying oven at 45°C to obtain the blue solid product ZnPc-4Bn (115 mg, 13.4%).
[0044] Characterization data: 1 H NMR (500MHz, DMSO-d6) δ 9.04-8.87 (m, 4H), 8.76-8.54 (m,4H), 7.85-7.78 (m, 8H), 7.74-7.63 (m, 4H), 7.62-7.45 (m, 12H), 5.64 (d, J =5.3Hz, 8H). HRMS (ESI): m / z Calcd for C 60 H 41 N8O4Zn [M+H] + : 1001.2537, found1001.2540. Relative error: 0.30ppm.
[0045] (2) Compound ZnPc-4OH ( Preparation of ZnPc-4Bn: 120.00 mg (0.12 mmol) ZnPc-4Bn, 10 mL toluene, and 18 mL trifluoroacetic acid (TFA) were added to a 50 mL reaction flask. The reaction was carried out at 90 °C for 36 h under an argon atmosphere. After the reaction was completed, the reaction solution was slowly poured into 100 mL of n-hexane and placed in a refrigerator at 4 °C for 10 min to precipitate. The supernatant was then discarded. The above precipitation operation was repeated twice. The crude product was then subjected to rotary evaporation under reduced pressure to remove the solvent, yielding the intermediate ZnPc-4OH. Note: The intermediate ZnPc-4OH contains multiple hydroxyl groups and is chemically unstable. It is prone to oxidation, decomposition, and other side reactions during separation, purification, and spectroscopic characterization, making it difficult to obtain a pure product. 1 Characterized by ¹H NMR and HRMS, but due to the presence of small amounts of impurities in the crude product, its yield and output could not be accurately determined; the intermediate ZnPc-4OH obtained here is a crude product, which, without weighing or further purification, is used entirely for the synthesis of the target product ZnPc-4ClNBS in the next step. Subsequently, the structure of the target product ZnPc-4ClNBS was successfully prepared from this crude product. 1 The target product ZnPc-4ClNBS, confirmed by 1H NMR and HRMS, can indirectly corroborate the correctness of the structure of the intermediate ZnPc-4OH prepared in this step and the feasibility of this preparation process.
[0046] (3) Compound ZnPc-4ClNBS ( Preparation of the intermediate ZnPc-4OH obtained in step (2): Dissolve the intermediate ZnPc-4OH in 8 mL of THF, then add 5 mL of triethylamine (TEA), and stir at 10 °C for 30 min to obtain the activated solution. Dissolve 470 mg of 2-chloro-4-nitrobenzenesulfonyl chloride in 15 mL of THF, pour it into a constant pressure dropping funnel, adjust the dropping rate to slowly add it to the activated solution at a suitable rate, and react at 10 °C for 4 h. After the reaction is completed, remove the solvent by rotary evaporation under reduced pressure to obtain the crude product. Purify the crude product by silica gel column chromatography with 100-200 mesh, using THF and DCM (1:20, v / v) as the mobile phase, and collect the blue fraction. After removing the solvent by rotary evaporation under reduced pressure, further purify the blue fraction by passing it through a Bio-Beads S-X1 gel column with THF as the mobile phase, and collect the first blue fraction. The first blue component was removed by rotary evaporation under reduced pressure to remove the solvent, dissolved in 5 mL of THF, and then slowly poured into 30 mL of n-hexane. The mixture was placed in a refrigerator at 4 °C and allowed to stand for 0.5 h to precipitate. The precipitate was then filtered and washed with a large amount of n-hexane until it was colorless. Finally, it was dried in a vacuum drying oven at 45 °C to constant weight to obtain the blue solid product ZnPc-4ClNBS (130 mg, 76.7%).
[0047] Characterization data: 1H NMR (500MHz, DMSO-d6, ppm) δ 8.94 (t, J = 5.0 Hz, 4H), 8.77-8.46 (m, 16H), 7.77 (s, 4H). HRMS (ESI): m / z Calcd for C 57 H 25 Cl4N 12 O 22 S4Zn[M+HCOO] - : 1562.8100, found 1562.8158. Relative error: 3.71ppm.
[0048] Example 2: Fluorescence recovery ability test of ZnPc-4ClNBS in glutathione environment
[0049] Using ZnPc-4ClNBS as the research object, six glutathione (GSH) concentration gradients of 0.01mM, 0.1mM, 0.5mM, 1mM, 5mM, and 10mM were set. Under the condition that the concentration of ZnPc-4ClNBS was fixed at 5μM, its fluorescence emission spectrum was measured by fluorescence spectrometry to investigate the fluorescence recovery of different GSH concentration systems after responding to GSH at time gradients of 0h, 2h, 4h, 6h, and 12h.
[0050] The results show that ( Figure 1 The fluorescence recovery of ZnPc-4ClNBS under different GSH concentrations showed significant time and concentration dependence. At lower GSH concentrations (0.01 mM and 0.1 mM), the fluorescence intensity remained almost unchanged within 0–12 h, indicating that GSH at this concentration range had limited effect on ZnPc-4ClNBS and failed to effectively trigger its fluorescence recovery mechanism. When the GSH concentration increased to 1 mM and above, the fluorescence recovery became significant. The 1 mM group showed significant fluorescence recovery at 4 h, which then steadily increased; the 5 mM group showed higher fluorescence intensity at all time points than the 1 mM group; and the 10 mM group exhibited the strongest fluorescence recovery, leading other concentration groups at all time points. Overall, the intensity of fluorescence recovery was positively correlated with GSH concentration, increasing with time and not reaching saturation within 12 h. Furthermore, within the concentration range of 0.5–10 mM, the fluorescence recovery rate increased with increasing GSH concentration, further confirming that ZnPc-4ClNBS can accurately respond to high GSH concentrations in the tumor microenvironment.
[0051] Example 3: Preparation of ZnPc-4ClNBS phthalocyanine nanoliposomes
[0052] S1: Distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol hemisuccinate were added to a single-necked flask, followed by 15 mL of chloroform. The mixture was sonicated at 38°C and 150 W for 15 min to completely dissolve all the raw materials, yielding a lipid solution. Separately, ZnPc-4ClNBS was dissolved in 4 mL of tetrahydrofuran and sonicated at 38°C and 150 W for 15 min to completely dissolve all the raw materials, yielding a phthalocyanine solution. The phthalocyanine solution and the lipid solution were then sonicated together at 38°C and 150 W for 15 min to obtain a mixed organic phase solution. In this mixed organic phase solution, the concentration of distearylphosphatidylethanolamine-polyethylene glycol 2000 is 805.26 μM, the concentration of cholesterol hemisuccinate is 335.21 μM, and the concentration of ZnPc-4ClNBS is 67.11 μM. The molar ratio of the three components is approximately 12:5:1, and the relative molecular mass of distearylphosphatidylethanolamine-polyethylene glycol 2000 is 2800.
[0053] S2: The mixed organic phase solution of S1 was subjected to reduced pressure rotary evaporation at 37℃ and 100mbar (85r / min) to remove the solvent until a uniform lipid film formed at the bottom of the flask. Then, 5mL of deionized water was added to the lipid film, and the mixture was sonicated at 0-10℃ and 150W for 15min to fully disperse the lipid film from the flask wall into the water, forming a preliminary liposome suspension. This preliminary liposome suspension was allowed to stand at room temperature for 15min to allow the liposomes to fully swell, yielding a liposome suspension.
[0054] S3: Transfer the liposome suspension from S2 to a 10mL centrifuge tube and perform ultrasonic disruption using an ultrasonic cell disruptor under ice-water bath conditions at 0-4℃. Insert the ultrasonic probe below the liquid surface, with the probe approximately 1cm from the bottom of the tube. Set the ultrasonic program to 5s operation followed by 5s interval, with the output power intensity at 35% of the total power, and the total treatment time to 5min. Repeat the above ultrasonic disruption process twice to obtain a crude suspension of nanoliposomes.
[0055] S4: The crude suspension of nanoliposomes obtained in S3 was filtered and squeezed sequentially at room temperature using 0.45μm and 0.22μm aqueous microporous membranes to remove any potentially larger particles and aggregates, ultimately yielding a ZnPc-4ClNBS phthalocyanine nanoliposome solution.
[0056] Transmission electron microscopy images of ZnPc-4ClNBS phthalocyanine nanoliposomes are shown below. Figure 2 As can be seen from the figure, it exhibits a spherical nanoliposome structure.
[0057] The particle size of the obtained ZnPc-4ClNBS phthalocyanine nanoliposomes in aqueous solution was determined by a nanoparticle size analyzer to be between 100-200 nm.
[0058] Example 4: Drug concentration and encapsulation efficiency of ZnPc-4ClNBS phthalocyanine nanoliposomes
[0059] Standard curve preparation: Accurately weigh phthalocyanine ZnPc-4ClNBS and dissolve it in DMF to prepare a 2mM drug stock solution; then, sequentially add 1μL, 2μL, 3μL, 4μL, 5μL, 6μL, and 7μL of the liquid to 2mL of DMF and measure its electronic absorption spectrum at 670nm to obtain the standard curve of phthalocyanine ZnPc-4ClNBS in DMF as a function of concentration.
[0060] Take 20 μL of the ZnPc-4ClNBS phthalocyanine nanoliposome solution prepared in Example 3, add it to DMF and make up to 2 mL, so that the nanoliposome complex is largely destroyed by DMF, and the loaded ZnPc-4ClNBS is released into the solution. Then, measure the electronic absorption spectrum of the solution, and calculate the concentration C1 of phthalocyanine in the solution according to the standard curve at a wavelength of 670 nm. Then, calculate the encapsulation efficiency EE (%) and the concentration of phthalocyanine in the nanoliposome complex according to the following formula:
[0061] EE(%)=(M0 / M1)×100%,
[0062] C0×V0=C1×V1,
[0063] Where M0 represents the mass of phthalocyanine encapsulated in the nanoliposome complex, and M1 represents the amount of phthalocyanine added. C0 represents the concentration of phthalocyanine in the phthalocyanine nanoliposome solution, V0 is 20 μL, and C1 represents the concentration of diluted phthalocyanine, V1 is 2 mL.
[0064] The results showed that the encapsulation efficiency of ZnPc-4ClNBS phthalocyanine nanoliposomes was 88.4%, indicating that the prepared ZnPc-4ClNBS phthalocyanine nanoliposomes achieved a good encapsulation efficiency.
[0065] Example 5: Stability test of ZnPc-4ClNBS phthalocyanine nanoliposomes in water
[0066] The ZnPc-4ClNBS phthalocyanine nanoliposome solution prepared in Example 3 was diluted with deionized water or 10% fetal bovine serum (FBS) solution to a concentration of 5 μM, and its stability within 8 days was determined by a particle size analyzer.
[0067] The results show that ( Figure 3ZnPc-4ClNBS phthalocyanine nanoliposomes exhibited good stability within 8 days.
[0068] Example 6: Test of Reactive Oxygen Species (ROS) Generation Capacity of ZnPc-4ClNBS Phthalocyanine Nanoliposomes
[0069] Under room temperature and light-protected conditions, the generation of reactive oxygen species (ROS) by phthalocyanine photosensitizers was tested using a 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe method after a certain period of illumination. Methylene blue (MB) was used as a control. DCFH-DA hydrolyzes to DCFH in aqueous solution, and DCFH can be oxidized to DCF by ROS. DCF emits a fluorescent signal at 524 nm. Therefore, the ability of nano-photosensitizers to generate ROS can be studied by detecting the fluorescence intensity at 524 nm.
[0070] Taking ZnPc-4ClNBS phthalocyanine nanoliposomes as an example, the probe preparation method was as follows: A certain amount of DCFH-DA was weighed and dissolved in analytical grade methanol to obtain a 5 mM stock solution, which was then frozen and stored. 100 μL of the DCFH-DA stock solution was mixed with 400 μL of NaOH (0.1 mol / L) solution and reacted in the dark for 30 min to generate DCFH. Then, PBS (pH=7.4) buffer was added to the DCFH solution to bring its concentration to 200 μM. Next, the ZnPc-4ClNBS phthalocyanine nanoliposomes were diluted to 2 mL of water to obtain an aqueous solution, and then the prepared DCFH solution was added and mixed well (at this point, both the probe and phthalocyanine concentrations were 5 μM). The test was performed using a wavelength greater than 610 nm and a power density of 1 mW / cm². 2 The light was continuously irradiated with red light, and the fluorescence intensity at 524nm was measured every 20s, with a total irradiation and testing time of 120s.
[0071] The results showed that ( Figure 4 Under simulated normal physiological conditions (pH 7.4 and without glutathione), ZnPc-4ClNBS phthalocyanine nanoliposomes produced almost no singlet oxygen compared to MB, indicating that the photosensitizing activity of these nanoliposomes was "off" under these conditions. While this may seem unfavorable for photodynamic therapy, it can also be considered an advantage. This fluorescence quenching can reduce non-specific activation of the photosensitizer in the bloodstream and normal tissues, thereby reducing potential photosensitizing toxicity.
[0072] Example 7: Acid Response Test of ZnPc-4ClNBS Phthalocyanine Nanoliposomes
[0073] Singlet oxygen detection results showed that, compared with MB, no significant singlet oxygen generation was detected in ZnPc-4ClNBS phthalocyanine nanoliposomes, indicating that their photosensitizing activity was inhibited under these test conditions. However, successful photodynamic therapy depends on the photosensitizing activity of the photosensitizer. Therefore, further testing is needed to determine whether these liposomes can release the loaded phthalocyanine, especially whether they can target tumor tissue for release. Since cholesterol hemisuccinate is an acid-sensitive material, and the tumor microenvironment is weakly acidic, changes in particle size and morphology before and after acid response were used to evaluate its acid-responsive properties.
[0074] PBS solutions with pH values of 7.4, 6.5, and 5.5 were prepared. ZnPc-4ClNBS phthalocyanine nanoliposomes were mixed with these three pH PBS solutions at the same concentration. The mixtures were incubated in a shaker at 37°C for 12 hours, and then the particle size was measured. The ZnPc-4ClNBS phthalocyanine nanoliposomes were then uniformly dropped onto copper grids with the pH 7.4 and 5.5 solutions. The grids were dried in a fume hood at room temperature in the dark for 12 hours. The morphology before and after acid response was characterized by transmission electron microscopy the next day.
[0075] like Figure 5 As shown, the ZnPc-4ClNBS phthalocyanine nanoliposomes exhibit uniform and stable particle size at pH 7.4; however, at pH 6.5 and 5.5, the particle size is unstable, with the particle size at pH 5.5 being more irregular than that at pH 6.5. The transmission electron microscope (TEM) image of the ZnPc-4ClNBS phthalocyanine nanoliposomes at pH 7.4 shows a regular spherical shape, while the TEM image at pH 5.5 shows a fragmented shape. This may be because the cholesterol hemisuccinate, the acid-sensitive membrane material of the ZnPc-4ClNBS phthalocyanine nanoliposomes, can cleave under acidic conditions, indicating that these nanoliposomes can release phthalocyanine photosensitizers in the microacidic environment of tumors.
[0076] The results from Examples 2, 6, and 7 indicate that the responsive activation of ZnPc-4ClNBS phthalocyanine nanoliposomes relies on a dual mechanism: firstly, cholesterol hemisuccinate in the liposome membrane dissociates under the slightly acidic environment of the tumor, promoting the release of the phthalocyanine photosensitizer (Example 7); secondly, the sulfonate groups on the phthalocyanine molecule break under the influence of high concentrations of glutathione in tumor cells, restoring its ability to emit fluorescence and generate reactive oxygen species through photosensitization (Example 2). Under normal physiological conditions (pH 7.4, low GSH concentration), neither of these responses was triggered, and the photosensitizing activity remained "off" (Example 6). This responsive activation mechanism provides a functional basis for subsequent in vivo fluorescence imaging and photodynamic antitumor therapy.
[0077] Example 8: In vivo fluorescence imaging capability test of ZnPc-4ClNBS phthalocyanine nanoliposomes
[0078] Clean-grade female ICR mice (weighing approximately 20g) were purchased from Fuzhou Wu's Laboratory Animal Co., Ltd. After a period of feeding, their hair was removed, and mouse hepatocellular carcinoma cells (H22) purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences were subcutaneously implanted near their right hind limbs. After approximately seven days, the tumor size reached about 100mm. 3 In vivo imaging studies were conducted at that time.
[0079] Taking ZnPc-4ClNBS phthalocyanine nanoliposomes as an example, the specific steps were as follows: After the H22 tumor-bearing mouse model was successfully established, the tumor-bearing mice were anesthetized, and the trunks of mice containing tumor tissue were imaged using an IVISSPECTRUM small animal in vivo imaging system (610nm excitation, 690nm emission), and the fluorescence signal before drug administration was recorded. Then, 150μL of ZnPc-4ClNBS phthalocyanine nanoliposomes at a concentration of 150μM was injected via tail vein injection, and imaged using an IVISSPECTRUM small animal in vivo imaging system after a certain period of time. The selected imaging time points were 0h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h, and 120h. After in vivo imaging, the mice were sacrificed 120h after tail vein injection, and the main organs (heart, liver, spleen, lung, kidney, and tumor) were removed for imaging. During the test, ZnPc-4Bn phthalocyanine nanoliposomes without glutathione responsive groups were used as a control. The control sample was prepared according to the method in Example 3, and the molar amount of the feed was the same as that of ZnPc-4ClNBS phthalocyanine nanoliposomes.
[0080] Experimental results are as follows Figure 6As shown, after injection of ZnPc-4ClNBS phthalocyanine nanoliposomes, only a weak fluorescence signal was observed in the tumor area 2 hours later, indicating that the molecule maintained a fluorescence "off" state in the blood circulation and normal tissues. With prolonged time, the fluorescence signal gradually increased, reaching a peak at 48 hours and persisting for up to 120 hours, demonstrating excellent tumor accumulation ability and long-lasting activation characteristics. This delayed activation and continuously enhanced fluorescence phenomenon reflects the response of ZnPc-4ClNBS molecules to high concentrations of glutathione in the tumor microenvironment. The control group rapidly reached its fluorescence peak 2 hours after injection. This molecule lacks a glutathione-responsive group, and its fluorescence recovery mainly depends on the dissociation and release of the liposomes in the acidic tumor microenvironment, thus exhibiting a non-selective rapid activation characteristic and failing to achieve a precise response. Combined with tissue anatomical fluorescence images, the fluorescence signal of ZnPc-4ClNBS phthalocyanine nanoliposomes was mainly distributed in the tumor tissue. Therefore, ZnPc-4ClNBS phthalocyanine nanoliposomes can not only selectively accumulate in the tumor but also selectively recover fluorescence over time. More importantly, the ZnPc-4ClNBS phthalocyanine nanoliposomes exhibited no fluorescence recovery in isolated normal organs, achieving precise imaging with "activation only at the tumor site." This glutathione-responsive activation mechanism effectively avoids the non-specific fluorescence interference and potential phototoxicity of photosensitizers in normal tissues, providing a crucial safeguard against the in vivo side effects of activatable photosensitizers.
[0081] Example 9: In vivo antitumor activity test of ZnPc-4ClNBS phthalocyanine nanoliposomes
[0082] Clean-grade female ICR mice (weighing approximately 20g) were purchased from Fuzhou Wu's Laboratory Animal Co., Ltd. After one week of feeding, hair was removed, and mouse hepatocellular carcinoma cells (H22) purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences were subcutaneously implanted near the right hind limb. After approximately seven days, the tumor size reached about 100mm. 3 In vivo antitumor activity studies will be conducted.
[0083] H22 tumor-bearing mice were divided into four experimental groups: a group injected with PBS but not exposed to light, a group injected with PBS and given 0.1 W / cm² of light, and a group treated with PBS. 2 The group receiving 680nm laser irradiation, the group injected with ZnPc-4ClNBS phthalocyanine nanoliposomes and not receiving light irradiation, and the group injected with ZnPc-4ClNBS phthalocyanine nanoliposomes and given 0.1W / cm 2 The 680nm laser irradiation group consisted of 3 animals per group; the ZnPc-4ClNBS phthalocyanine nanoliposome injection + laser irradiation group received 100μL of a 150μM ZnPc-4ClNBS phthalocyanine nanoliposome aqueous solution via intravenous injection, followed by 0.1W / cm² treatment 48 hours after administration. 2The tumor sites were irradiated with a 680nm wavelength laser for 10 minutes each. After each group of mice was treated as required, they were continued to be fed. The mice were observed every other day, their weight was measured, and the long and short diameters of the tumors were measured with calipers. The measurements were taken for a total of 14 days.
[0084] The results are as follows Figure 7 As shown, compared with the tumor changes in mice treated with PBS, PBS + laser irradiation, and ZnPc-4ClNBS phthalocyanine nanoliposomes, tumor growth in mice treated with ZnPc-4ClNBS phthalocyanine nanoliposomes + laser irradiation was inhibited. After 14 days of treatment, tumors dissected in vitro showed a diameter of less than 2 cm. There were no significant differences in body weight among the mice during the treatment period. Figure 8 The results indicate that the systemic toxicity of ZnPc-4ClNBS phthalocyanine nanoliposomes in mice is negligible. Based on the tumor inhibition rate formula, the tumor inhibition rate of the ZnPc-4ClNBS phthalocyanine nanoliposome + laser irradiation group was 87%, indicating that ZnPc-4ClNBS phthalocyanine nanoliposomes have a good photodynamic antitumor effect.
Claims
1. A phthalocyanine nanoliposome responsive to microacids and glutathione, characterized in that: The phthalocyanine nanoliposomes contain phthalocyanine, phospholipid-polyethylene glycol, and cholesterol hemisuccinate; the phthalocyanine is a sulfonate-based pericyclic monosubstituted phthalocyanine or a sulfonate-based pericyclic tetrasubstituted phthalocyanine; wherein, the chemical structural formula of the sulfonate-based tetrasubstituted phthalocyanine is: or or or or or or .
2. The method for preparing phthalocyanine nanoliposomes as described in claim 1, characterized in that: Includes the following steps: (1) Dissolve phospholipid-polyethylene glycol and cholesterol hemisuccinate in chloroform by ultrasonication to obtain a lipid solution; dissolve phthalocyanine in tetrahydrofuran by ultrasonication to obtain a phthalocyanine solution; mix the lipid solution and the phthalocyanine solution by ultrasonication to obtain a mixed organic phase solution; (2) Remove the solvent by rotary evaporation under reduced pressure to form a lipid film; add deionized water to the lipid film and sonicate to dissolve the film and form a liposome suspension; let the liposome suspension stand at room temperature to allow the liposomes to fully expand and obtain a liposome suspension. (3) The liposome suspension was subjected to ultrasonic disruption under ice-water bath conditions to obtain a crude nanoliposome suspension; (4) The crude suspension of nanoliposomes was filtered and squeezed through a microporous membrane at room temperature to remove large particles and aggregates, thus obtaining a phthalocyanine nanoliposome solution.
3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of phospholipid-polyethylene glycol, cholesterol hemisuccinate, and phthalocyanine is 9~16:4~7:1; the volume ratio of chloroform and tetrahydrofuran is 2~6:
1.
4. The preparation method according to claim 2, characterized in that: In step (2), the conditions for ultrasound are: temperature 0~10℃, power 150W, time 15min; the resting time is 0.25~48h.
5. The preparation method according to claim 2, characterized in that: In step (3), the conditions for ultrasonic disruption are: 35%~65% output power, continuous action for 2~10 minutes with an interval of 3~5 seconds on and 3~5 seconds off, repeated twice.
6. The preparation method according to claim 2, characterized in that: In step (4), the microporous filter membrane is an aqueous and / or organic microporous filter membrane with a diameter of 0.22 μm and / or 0.45 μm.
7. The preparation method according to claim 2, characterized in that: In step (1), the preparation method of the phthalocyanine is as follows: 1) 4-Benzyloxyphthalonitrile and zinc acetate were dissolved in n-pentanol and reacted at 90°C for 0.5 h until the solution became clear. 1,8-diazabicyclo[5.4.0]undec-7-ene was added and the mixture was heated to 140°C in the dark for 8 h. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography and then by gel column chromatography. The product was dissolved in tetrahydrofuran, and n-hexane was added. The mixture was allowed to stand at 4°C to crystallize. The crystals were filtered, washed with n-hexane, and dried under vacuum at 45°C to obtain ZnPc-4Bn. 2) ZnPc-4Bn is dissolved in toluene and trifluoroacetic acid and reacted at 90°C for 36 h under an argon atmosphere; hexane is added to the reaction solution and allowed to stand at 4°C to crystallize. The supernatant is discarded, and the crystallization operation is repeated. The solvent is then removed by vacuum evaporation to obtain ZnPc-4OH. 3) ZnPc-4OH was dissolved in tetrahydrofuran, triethylamine was added, and the mixture was stirred and activated at 10°C for 30 min; a tetrahydrofuran solution of 2-chloro-4-nitrobenzenesulfonyl chloride was slowly added dropwise, and the reaction was carried out at 10°C for 4 h; the solvent was removed by vacuum distillation, the crude product was subjected to silica gel column chromatography, and then purified by gel column chromatography; the product was dissolved in tetrahydrofuran, n-hexane was added, and the mixture was allowed to stand at 4°C to crystallize, filtered, washed with n-hexane, and dried under vacuum at 45°C to obtain ZnPc-4ClNBS, i.e., phthalocyanine.
8. The use of the phthalocyanine nanoliposomes as described in claim 1 in the preparation of tumor therapeutic drugs and / or tumor imaging reagents.
9. The application according to claim 8, characterized in that: The tumor treatment drug is used for photodynamic therapy.
10. The application according to claim 8, characterized in that: The tumor imaging reagent is used for fluorescence imaging.