CN111617246B - Self-assembled nanoparticles of pure photosensitizer and preparation and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to a pure photosensitizer self-assembly nanoparticle, which has the effects of high drug loading, good stability, low toxic and side effects, specific disintegration of tumor parts, alleviation of aggregation-induced fluorescence quenching (ACQ) effect and improvement of antitumor activity. The invention provides a pure photosensitizer self-assembly nanoparticle, which is formed by independent self-assembly of a photosensitizer or core-shell matching self-assembly of the photosensitizer and a PEG (polyethylene glycol) modifier. The photosensitizer is one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, pyropheophorbide a hexyl ether and chlorin e 6. The PEG modifier is amphiphilic PEG modifier and amphiphilic polymer of PEG and photosensitizer. The weight ratio of the photosensitizer to the PEG modifier is as follows: 10:0.5-10:3. the invention provides a new strategy and more choices for developing a pure drug self-assembly delivery system, and meets the urgent need of high-efficiency chemotherapeutic preparations in clinic.

Description

A kind of self-assembled nanoparticles of pure photosensitizer and its preparation and application

technical field

The invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, relates to a pure photosensitizer self-assembled nanoparticle, in particular to the construction of a pure photosensitizer (pyropheophorbide a, PPa) self-assembled nanoparticle, and its Applications in drug delivery.

Background technique

Cancer is still considered one of the most serious diseases threatening human health. Currently, surgery is the most common and effective cancer treatment, especially in the early stages of non-metastatic solid tumors. For solid tumors, various other treatment strategies, such as chemotherapy and phototherapy, have also been used clinically. Among them, chemotherapy is still the main treatment option for clinical treatment of cancer, especially for those patients with inoperable and metastatic tumors. However, severe toxicity may result due to the narrow therapeutic window of most chemotherapeutic agents and off-target effects in vivo. Therefore, it is a very good option to control and treat local diseases through site-specific local treatment regimens.

Photodynamic therapy (PDT) has been extensively studied as a non-invasive cancer treatment compared to systemic chemotherapy. Under the local laser irradiation of the tumor, the photosensitizer can induce the apoptosis and necrosis of tumor cells through the generation of a large number of reactive oxygen species (ROS). Cytotoxic ROS produced by photosensitizers can destroy cell membranes and oxidize intracellular macromolecules, thereby affecting the normal physiological functions of tumor cells. Notably, without laser treatment, the photosensitizer had little cytotoxicity. Therefore, phototherapy is considered as a promising therapeutic approach for tumor-targeted non-invasive cancer treatment. Furthermore, due to the rapid development of novel photosensitizers and optical fibers, the clinical application of phototherapy has been extended to the treatment of deep visceral tumors. However, the therapeutic efficiency of phototherapy is still hampered by insufficient accumulation of photosensitizers in tumors. Therefore, the rational design of highly efficient drug delivery systems (DDSs) for photosensitizers is crucial for effective phototherapy.

With the rapid development of biomedical nanotechnology, various nano-drug delivery systems (nano-DDS) have been developed to enhance the delivery efficacy of anticancer drugs, including chemotherapeutics and photosensitizers. Most photosensitizers are non-covalently encapsulated in organic or inorganic nanocarriers for delivery. However, noncovalent drug loading methods have long been criticized for their low drug loading efficiency, poor stability, premature drug leakage, and potential toxicity associated with carrier materials. Recently, nanodelivery systems self-assembled from carrier-free small molecule drugs or prodrugs have emerged as promising nanoplatforms for efficient drug delivery. Moreover, studies have found that certain hydrophobic drugs can self-assemble into nanoparticles. However, pure drug-driven nanodelivery systems usually suffer from unsatisfactory colloidal stability due to relatively weak intermolecular interactions between small molecules. Furthermore, how to trigger pure drug nanodelivery systems to release drugs specifically at tumor sites remains challenging.

To address these challenges, we constructed a pure photosensitizer-driven nanoself-assembly system with core-shell-matched PEGylation for efficient photodynamic therapy.

Contents of the invention

The technical problem solved by the present invention is that PPa has poor hydrophobicity, insoluble in water, low drug loading due to entrapment in polymers, poor drug leakage and poor toxicity related to excipients, etc., and a pure PPa with core-shell matching is designed. Self-assembled nanoparticles, so as to achieve high drug loading, good stability, low toxicity and side effects, and the effect of targeted disintegration of tumor sites, thereby improving anti-tumor activity. At the same time, using PCL-PEG _2K modification as a control, the differences in the anti-tumor activity of different PEGylated nanoparticles were investigated, as well as the stability of PPa self-assembled nanoparticles, drug release, cytotoxicity, pharmacokinetics, Tissue distribution and pharmacodynamic effects.

The purpose of the present invention is to design pure PPa self-assembled nanoparticles (PPa/PPa-PEG _2K nanoparticles), including self-assembled photosensitizers alone, or self-assembled nanoparticles formed by photosensitizers and PEG modifiers. Prepare PPa self-assembled nano-drug delivery system, and investigate the stability, in vitro singlet oxygen production, cellular uptake, intracellular reactive oxygen production, cytotoxicity, pharmacokinetics, and tissue of different PEGylated pure PPa self-assembled nanoparticles Distribution and pharmacodynamics, comprehensively screen out the most effective preparations, provide new strategies and more options for the development of carrier-free pure drug nano-delivery systems, and meet the urgent needs of high-efficiency chemotherapy preparations in clinical practice.

The present invention realizes above-mentioned object through following technical scheme:

The invention provides a self-assembled nanoparticle of a pure photosensitizer. The nanoparticle is self-assembled by a photosensitizer alone, or self-assembled by a photosensitizer and a PEG modifier through core-shell matching.

The photosensitizer is one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, pyropheophorbide a hexyl ether, and chlorin e6.

The PEG modifier is an amphiphilic PEG modifier, an amphiphilic polymer of PEG and a photosensitizer, and the molecular weight of PEG is 2000-20000, preferably 2000-5000.

The PEG modifier is preferably PPa-PEG _2K , PCL ₅₀₀ -PEG _2K .

The weight ratio of the photosensitizer to the PEG modifier is: 10:0.5-10:3.

Further, in the present invention, self-assembled nanoparticles of pyropheophorbide a and PEG modifier are preferred, more preferably nanoparticles self-assembled of pyropheophorbide a and PPa-PEG _2K or PCL-PEG _2K .

The invention provides the preparation method of the series of pure PPa self-assembled nanoparticles, and the pure PPa nanoparticles can be non-PEGylated PPa nanoparticles and PEG-modified PPa nanoparticles.

The preparation method of the PPa self-assembled nanoparticles provided by the invention is as follows:

Dissolve a certain amount of PPa or a mixture of PPa and PEG modifier into an appropriate amount of mixed solvent of ethanol and tetrahydrofuran, and slowly add the solution dropwise into water under stirring to spontaneously form uniform nanoparticles. Finally, ethanol and tetrahydrofuran in the preparation are removed by dialysis to obtain a nano-colloid solution without any organic solvent. The PEG modifiers are PPa-PEG _2K and PCL-PEG _2K .

Wherein, the volume ratio of ethanol to tetrahydrofuran is 3:2-3:4.

The molar ratio of PPa to PEG modifier is: 10:0.5-10:3.

specifically,

(1) The preparation method of non-PEGylated PPa self-assembled nanoparticles: Dissolve a certain amount of PPa in an appropriate amount of ethanol and tetrahydrofuran (3:2), and slowly add the solution dropwise to water under stirring, and the PPa spontaneously Form uniform nanoparticles. Ethanol and tetrahydrofuran in the preparation are removed by dialysis to obtain a nano colloid solution without any organic solvent.

(2) The preparation method of PEG-modified PPa self-assembled nanoparticles: a certain amount of PEG modifier (PPa-PEG _2K or PCL-PEG _2K ) and PPa are dissolved in an appropriate amount of ethanol and tetrahydrofuran, and the solution is stirred Slowly added dropwise to water, PPa spontaneously formed uniform nanoparticles. Ethanol and tetrahydrofuran in the preparation are removed by dialysis to obtain a nano colloid solution without any organic solvent.

The present invention has the following beneficial effects: (1) PPa self-assembled nanoparticles (PPa/PPa-PEG _2K nanoparticles) and PCL-PEG _2K modified _PPa self-assembled nanoparticles ( PPa/PCL-PEG _2K nanoparticles). (2) Prepared uniform PPa self-assembled nanoparticles, the preparation method is simple and easy, and the stability is good, and the efficient entrapment of PPa is realized; (3) The stability of PPa self-assembled nanoparticles by different PEGylation was investigated, and the in vitro single-line Oxygen production, cellular uptake, intracellular ROS production, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamics. The prescription with the best effect is comprehensively screened out, which provides new strategies and more options for the development of carrier-free self-assembled nano-drug delivery system, and meets the urgent demand for high-efficiency chemotherapy preparations in clinic.

In the present invention, we discovered a unique self-assembly phenomenon of a common photosensitizer (pyropheophorbide a, PPa) used for PDT, which can self-assemble into nanoparticles alone. In addition, the amphiphilic polymer (PPa-PEG _2K ) achieves core-shell matched PEGylation on PPa nanoparticles through the hydrophobic and π-π stacking interactions between PPa and PPa-PEG _2K . The self-assembly mechanism and core-shell matching interaction were investigated by computer molecular simulations. In addition, under laser irradiation, the outer PPa-PEG _2K was destroyed, the stability of PPa/PPa-PEG _2K nanoparticles decreased, and specific disintegration occurred. This core-shell matched nanoparticle has multiple drug delivery advantages, including ultra-high drug loading efficiency (74.8%, w/w), high stability, long systemic circulation, high tumor accumulation, and good cellular uptake and laser targeting at tumor sites. triggered release. Therefore, PPa/PPa-PEG _2K nanoparticles showed good antitumor effect in the antitumor therapy of tumor-bearing mice. This is the first time that pure PPa can self-assemble into nanoparticles, and the core-shell matching design can significantly improve the effective delivery of PPa self-assembled nanoparticles.

Description of drawings

FIG. 1 is a transmission electron microscope image of PPa and PEG-modified PPa self-assembled nanoparticles of Example 1 of the present invention.

Figure 2 is a computer simulation diagram of the PPa molecule in Example 2 of the present invention.

Fig. 3 is a diagram showing the fetal bovine serum stability and the particle size variation of the PEG-modified PPa self-assembled nanoparticles according to Example 3 of the present invention and different light exposure times.

Fig. 4 is a graph showing the particle size change of the PEG-modified PPa self-assembled nanoparticles of Example 3 of the present invention under different laser irradiations.

Fig. 5 is a diagram of in vitro singlet oxygen generation of PEG-modified PPa self-assembled nanoparticles according to Example 4 of the present invention.

Fig. 6 is a graph of cellular uptake of PEG-modified PPa self-assembled nanoparticles according to Example 5 of the present invention.

Fig. 7 is a graph showing the effect of PEG-modified PPa self-assembled nanoparticles of Example 6 of the present invention on the ROS level in tumor cells.

Figure 8 is the cytotoxicity diagram of the PEG-modified PPa self-assembled nanoparticles of Example 7 of the present invention after direct light irradiation without removing the drug-containing culture medium

Fig. 9 is a graph of cytotoxicity of the PEG-modified PPa self-assembled nanoparticles of Example 7 of the present invention after removing the light from the drug-containing culture medium.

Fig. 10 is a blood concentration-time curve of PEG-modified PPa self-assembled nanoparticles according to Example 8 of the present invention.

Fig. 11 is a 4-hour tissue distribution diagram of the PEG-modified PPa self-assembled nanoparticles of Example 9 of the present invention.

Fig. 12 is a 4-hour tissue distribution quantitative diagram of PEG-modified PPa self-assembled nanoparticles according to Example 9 of the present invention.

Fig. 13 is a 12-hour tissue distribution diagram of the PEG-modified PPa self-assembled nanoparticles of Example 9 of the present invention.

Fig. 14 is a 12-hour tissue distribution quantitative diagram of PEG-modified PPa self-assembled nanoparticles according to Example 9 of the present invention.

Fig. 15 is a graph showing the tumor growth curve of the in vivo anti-tumor experiment of the PEG-modified PPa self-assembled nanoparticles of Example 10 of the present invention.

Fig. 16 is a graph showing the change in body weight of mice in an in vivo anti-tumor experiment of PEG-modified PPa self-assembled nanoparticles according to Example 10 of the present invention.

Fig. 17 is a pathological section view of the PEG-modified PPa self-assembled nanoparticles of Example 10 of the present invention.

detailed description

The present invention is further illustrated below by means of examples, but the invention is not therefore limited to the scope of the examples.

Embodiment 1: Preparation of PPa self-assembled nanoparticles and PEG-modified PPa self-assembled nanoparticles

Accurately weigh 1 mg of PPa, dissolve it with 200 μL of a mixed solution of ethanol and tetrahydrofuran (3:2), and slowly add the solution dropwise to 2 mL of deionized water under stirring, to spontaneously form uniform nanoparticles of PPa nanoparticles. The organic solvent in the nano-preparation was removed by dialysis with deionized water at 25°C.

(1) The preparation method of non-PEGylated PPa self-assembled nanoparticles: Dissolve a certain amount of PPa in an appropriate amount of ethanol and tetrahydrofuran (3:2), and slowly add the solution dropwise to water under stirring, and the PPa spontaneously Form uniform nanoparticles. Ethanol and tetrahydrofuran in the preparation are removed by dialysis to obtain a nano colloid solution without any organic solvent.

(2) The preparation method of PEG-modified PPa self-assembled nanoparticles: a certain amount of PEG modifier (PPa-PEG _2K or PCL-PEG _2K ) PPa is dissolved in an appropriate amount of ethanol and tetrahydrofuran, and the solution is slowly stirred under stirring. Slowly added dropwise to water, the prodrug spontaneously formed uniform nanoparticles. Ethanol and tetrahydrofuran in the preparation were removed by dialysis to obtain a nanocolloid solution without any organic solvent, wherein the molar ratio of PPa to PEG modifier was 10:1.

As shown in Table 1, the particle size of the nanoparticles is between 90-150nm, the Zeta potential is about -20mV, and the drug loading is above 50%. Among them, Pa/PCL-PEG _2K nanoparticles and PPa/PPa-PEG _2K nanoparticles have smaller particle size and understanding distribution, and larger drug loading capacity, which has better preparation morphology. Preliminarily, PPa/PCL-PEG _2K nanoparticles and PPa/PPa-PEG _2K nanoparticles were selected.

As shown in Table 2, the particle size of the nanoparticles is between 100-160nm, the Zeta potential is about -20mV, and the drug loading is above 30%. When the molar ratio of PPa:PEG modifier is 10:1, the particle size and understanding distribution of Pa/PCL-PEG _2K nanoparticles and PPa/PPa-PEG _2K nanoparticles are smaller, and they have better formulation morphology. It is further preferred that the molar ratio of PPa:PEG modifier is 10:0.5-10:3.

Measure the particle size and the shape of the Pa/PCL-PEG _2K nanoparticles prepared in Example 1 and the PPa/PPa-PEG _2K nanoparticles by a transmission electron microscope, the results are as shown in Figure 1, and the transmission electron microscope figure shows that the nanoparticles are uniform spherical, The particle size is around 100nm.

Table 1. Particle size, particle size distribution, surface charge and drug loading of PPa self-assembled nanoparticles

Table 2. Particle size, particle size distribution, surface charge and drug loading of PPa self-assembled nanoparticles with different molar ratios

Embodiment 2: The self-assembly mechanism analysis of PPa

Through simple computer simulations, the mechanism of PPa self-assembly was explored, and the molecular docking calculation was completed using the Vina program of the Yinfu cloud computing platform. The compound PPa was energy minimized under the MMFF94 force field to obtain a 3D structure and form a stable nanoassembly. The AutoDock Vina program was used for semi-flexible docking. The results are shown in Figure 2. The unique multiple pyrrole ring structures of PPa molecules, as well as the π-π stacking and hydrophobic interactions between molecules have made great contributions to the self-assembly of PPa molecules.

Embodiment 3: Colloidal stability test of PPa self-assembled nanoparticle

1 mL of the PEG-modified self-assembled nanoparticles prepared in Example 1 was taken out, added to 20 mL of phosphate buffered saline (PBS, pH 7.4) containing 10% FBS, incubated at 37° C. for 24 hours, and At predetermined time points (0, 1, 2, 4, 6, 8 and 12 hours), the particle size change was measured by dynamic light scattering. The results are shown in Figure 3. Compared with other groups, the colloidal stability of PPa/PCL-PEG _2K nanoparticles and PPa/PPa-PEG _2K nanoparticles was better, and the particle size did not change significantly within 24 hours. Pa/PCL-PEG _2K nanoparticles and PPa/PPa-PEG _2K nanoparticles are further preferred.

1 mL of the PEG-modified self-assembled nanoparticles prepared in Example 1 was taken out and added to 20 mL of PBS. Under different laser irradiation times, the changes in the particle size of the nanoparticles were observed. The results are shown in Figure 4. PPa/PPa-PEG The particle size of _2K nanoparticles increases significantly under laser irradiation in a laser dose-dependent manner. In contrast, the particle size of PPa/PCL-PEG _2K nanoparticles hardly increased significantly even after exposure to laser light for 8 minutes. Obviously, PEGylation on PPa nanoassemblies can significantly improve the colloidal stability. However, the PPa/PPa-PEG _2K nanoparticles exhibited laser-triggered decomposition properties due to the destruction of the photobleaching of the PPa component in PPa-PEG _2K under laser irradiation. As a result, the amphiphilic structure of the PPa-PEG _2K polymer was destroyed, resulting in a decrease in the PPa-PEG _2K stabilization effect of the PPa/PPa-PEG _2K nanoparticles. In contrast, laser had little effect on PCL-PEG _2K , and PEGylation of PCL-PEG _2K with or without laser treatment maintained good stability of PPa/PCL-PEG _2K nanoparticles. The particle size of the PPa/PCL-PEG _2K nanoparticles irradiated with laser for 8 minutes changed slightly (about 30nm), which should be due to the photobleaching of the core PPa of the nanoparticles. These results indicate that the core-shell matched PPa/PPa-PEG _2K nanoparticles can not only significantly improve the colloidal stability of PPa nanoparticles, but also destabilize the nanoparticles under laser triggering. This laser-triggered destabilization may help alleviate the aggregation-induced fluorescence quenching (ACQ) effect of PPa nanoparticles, thereby promoting their photoconversion and reactive oxygen species generation.

Example 4: In vitro singlet oxygen detection of PPa self-assembled nanoparticles

Singlet oxygen generated under laser irradiation was detected with a singlet oxygen fluorescent probe (SOSG). A solution of PPa mixed with SOSG (1 μM), PPa/PCL-PEG _2K nanoparticles or PPa/PPa-PEG _2K nanoparticles (1 μM, PPa equivalent) was diluted in 1 mL of LPBS. The singlet oxygen produced in each group of preparations was detected under different laser irradiation times (660nm, 200mWcm-2) or no irradiation. Fluorescence signal intensity was analyzed by varioskan lux multimode microplate reader (excitation 498nm, emission 525nm).

The results are shown in Figure 5. Compared with the PPa solution, the singlet oxygen generation of PPa self-assembled nanoparticles decreased. With the prolongation of time, at 4 minutes and 8 minutes, the PPa/PPa-PEG _2K nanoparticles The amount of singlet oxygen generated was significantly more than that of PPa/PCL-PEG _2K nanoparticles. With the prolongation of the light time, the stability of the nanoparticles decreased, which significantly alleviated the ACQ effect and increased the amount of singlet oxygen generated.

Example 5: Cellular uptake of PPa self-assembled nanoparticles

The uptake of PPa self-assembled nanoparticles in 4T1 cells was measured by flow cytometry. 4T1 cells were inoculated on a 12-well plate at a density of 1×10 ⁵ cells/mL, and incubated in an incubator for 24 hours to allow them to adhere to the wall. After the cells adhered to the wall, PPa solution and PPa self-assembled nanoparticles were added. The concentration of PPa was 50 nM. After incubating at 37°C for 0.5h or 2h, the cells were washed, collected and dispersed in PBS, and the uptake of various preparations by the cells was investigated by flow cytometry.

The experimental results are shown in Figure 6, the cells treated with two kinds of PPa-assembled nanoparticles showed higher intracellular fluorescence intensity than the cells treated with free PPa. Therefore, the prepared PPa self-assembled nanoparticles have higher cellular uptake efficiency than free PPa.

Example 6: Intracellular active oxygen detection of PPa self-assembled nanoparticles

The production of reactive oxygen species by PPa self-assembled nanoparticles in 4T1 cells was measured by an inverted fluorescence microscope. 4T1 cells were inoculated on a 24-well plate at a density of 5×10 ⁴ cells/mL, and incubated in an incubator for 24 hours to allow them to adhere to the wall. After the cells adhered to the wall, PPa solution and PPa self-assembled nanoparticles were added. The concentration of PPa was 20 nM. After incubating at 37° C. for 4 h, the drug-containing culture medium was discarded, and the culture medium containing an active oxygen detection kit (DCFH-DA, 20 μM) was added, and the incubation was continued for 0.5 h. Afterwards, irradiate with laser for 5 minutes (660nm, 60mW cm ^-2 ), wash with PBS three times, and observe with an inverted fluorescence microscope.

The results are shown in Figure 7. The fluorescence intensity of the two nanoparticles was significantly higher than that of the solution. In addition, the fluorescence intensity of the PPa/PPa-PEG _2K nanoparticles was higher than that of the PPa/PCL-PEG _2K nanoparticles, indicating that the PPa/ PPa-PEG _2K nanoparticles effectively slowed down the ACQ effect and improved the production efficiency of ROS.

Example 7: Cytotoxicity of PEG-modified small molecule prodrug self-assembled nanoparticles

The cytotoxicity of PPa self-assembled nanoparticles on mouse breast cancer (4T1) cells was investigated by MTT assay. The cells in good condition were digested, diluted with culture medium to a cell density of 5000cells/mL, blown evenly, and 100 μL of cell suspension was added to each well of a 96-well plate, and incubated in an incubator for 24 hours to make it adhere to the wall. After the cells adhere to the wall, add the PPa solution or the nanoparticles prepared in Example 1. In this experiment, both the preparation and dilution of the drug solution and the nanoparticle preparation were made with 1640 culture solution, and were sterile filtered with a 0.22 μm filter membrane. 100 μL of the test solution was added to each well, and 3 parallel wells were prepared for each concentration. For the control group, without adding the drug solution to be tested, 100 μL of culture solution was added only, and placed in an incubator to incubate with the cells. After 4 hours of adding the drug, directly illuminate or replace with a new drug-free culture medium and then illuminate again. After 44 hours, take out the 96-well plate, add 20 μL of 5 mg/mL MTT solution to each well, incubate in the incubator for 4 hours, and then shake the plate. After the 96-well plate was turned upside down on the filter paper to fully absorb the residual liquid, 200 μL of DMSO was added to each well and shaken on a shaker for 10 minutes to dissolve the blue-purple crystals. Set A1 well (containing only 200 μL DMSO) as the zero well. Use a microplate reader to measure the absorbance value of each well after zeroing at 570 nm.

The results of cytotoxicity are shown in Figure 8 and Figure 9. There was almost no cytotoxicity among the various preparations in the dark. However, when the drug-containing culture medium was not removed before light exposure, there was no significant difference between the preparations of each group. After removal, the cytotoxicity of the two PPa nanoparticles was significantly stronger than that of the solution, which may be due to the higher uptake of the nanoparticles. Compared with the two PPa nanoparticles, the cytotoxicity of PPa/PPa-PEG _2K nanoparticles was stronger than that of PPa/PCL-PEG _2K nanoparticles, which may be because the PPa/PPa-PEG _2K nanoparticles effectively alleviated the ACQ effect, and the cells There are more ROS produced.

Embodiment 8: Pharmacokinetic study of PPa self-assembled nanoparticles

SD rats with a body weight of 200-250 g were randomly divided into groups, fasted for 12 hours before administration, and allowed to drink water freely. The PPa solution and the PPa self-assembled nanoparticles prepared in the practice were intravenously injected respectively. The dose of PPa was 2 mg/kg. Orbital blood was collected at specified time points, and plasma was obtained by separation. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

The experimental results are shown in Figure 10. Due to the short half-life, PPa is cleared from the blood faster. In contrast, the cycle time of the two PPa self-assembled nanoparticles was significantly prolonged. In addition, due to the stabilization of the core-shell matching of PPa/PPa-PEG _2K nanoparticles, the in vivo circulation time of PPa/PPa-PEG _2K nanoparticles is longer than that of PPa/PCL-PEG _2K nanoparticles.

Embodiment 9: Tissue distribution experiment of PEG-modified PPa self-assembled nanoparticles

The 4T1KB cell suspension was inoculated into BALB/c mice, and when the tumor volume reached 350 mm ³ , the tail vein injection was administered: the dosage of PPa solution and PPa self-assembled nanoparticles PPa was 1 mg/kg. After 4 or 12 hours, the mice were sacrificed, and major organs (heart, liver, spleen, lung, kidney) and tumors were isolated and analyzed using an intravital imager.

The results are shown in Figures 11-14 (4 hours in Figures 11 and 12, and 12 hours in Figures 13 and 14), compared with the PPa solution, the fluorescence intensity of the PPa self-assembled nanoparticles group in the tumor tissue was significantly increased. In contrast, the tumor accumulation of PPa/PPa-PEG _2K nanoparticles was significantly greater than that of PPa/PCL-PEG _2K nanoparticles. This result is completely consistent with its pharmacokinetic behavior. PPa/PPa-PEG _2K nanoparticles have the best stability and the longest circulation time in vivo, so they show the best tumor accumulation ability.

Embodiment 10: In vivo anti-tumor experiment of PPa self-assembled nanoparticles

The 4T1 cell suspension (5x 10 ⁶ cells/100 μL) was inoculated subcutaneously in the ventral side of female mice. When the tumor volume grew to 150 mm ³ , the mice were randomly divided into five groups, and were given physiological saline, PPa solution and the PPa self-assembled nanoparticles prepared in the examples. Dosing once every other day, 5 consecutive administrations, calculated in PPa, the dosage is 2mg/kg. After administration, the survival status of the mice was observed every day, the body weight was weighed, and the tumor volume was measured. Mice were sacrificed one day after the last dose, and organs and tumors were harvested for further analysis and evaluation. Major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected and fixed with 4% tissue fixative for H&E staining.

The experimental results are shown in Figure 15, compared with the normal saline group, PPa exhibited certain tumor suppressive activity. PPa/PCL-PEG _2K nanoparticles showed stronger antitumor activity than PPa solution, and the tumor volume increased slowly. As expected, the anti-tumor effect of PPa/PPa-PEG _2K nanoparticles was the most obvious, effectively inhibiting tumor growth, and the tumor volume even showed a downward trend in the later stage of treatment. The results showed that the stability, cytotoxicity, pharmacokinetics, tissue distribution and NPs of nanoparticles all affect the final antitumor effect.

As shown in Figure 16, there was no significant change in body weight in each group. It can be seen from Figure 17 that there was no obvious abnormality in the functions of small major organs in each group. These results indicate that the PPa self-assembled nanoparticles have obvious anti-tumor effects and do not cause significant non-specific toxicity to the body, and are safe and effective anti-cancer drug delivery systems.

Claims (7)

1. The pure photosensitizer self-assembly nanoparticle is characterized in that the nanoparticle is formed by self-assembly of a photosensitizer and a PEG (polyethylene glycol) modifier, the weight ratio of the photosensitizer to the PEG modifier is 10.5-10, the PEG modifier is PPa-PEG and PCL-PEG, the molecular weight of the PEG is 2000-20000, and the photosensitizer is pyropheophorbide a.

2. The self-assembled nanoparticle of claim 1, wherein the PEG modifier is PPa-PEG _2K 、PCL ₅₀₀ -PEG _2K 。

3. The method for preparing the pure photosensitizer self-assembled nanoparticles according to claim 1 or 2,

dissolving a certain amount of mixture of a photosensitizer and a PEG modifier into a proper amount of mixed solvent of ethanol and tetrahydrofuran, slowly dripping the solution into water under stirring to spontaneously form uniform nanoparticles, and finally removing an organic reagent by a dialysis method to obtain a nano colloidal solution.

4. The method of claim 3, wherein the weight ratio of photosensitizer to PEG modifier is 10.5-10: 2-3:4.

5. use of the self-assembled nanoparticles of a pure photosensitizer as defined in claim 1 or 2 for the preparation of a drug delivery system.

6. The use of the self-assembled nanoparticles of a pure photosensitizer as defined in claim 1 or 2 in the preparation of an anti-tumor drug.

7. Use of the self-assembled nanoparticles of a pure photosensitizer as defined in claim 1 or 2 for the preparation of a system for injection, oral or topical administration.

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