CN111617246B

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

Info

Publication number: CN111617246B
Application number: CN202010650330.0A
Authority: CN
Inventors: 罗聪; 张申武; 孙进; 何仲贵
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2020-06-01
Filing date: 2020-07-08
Publication date: 2023-01-13
Anticipated expiration: 2040-07-08
Also published as: CN111617246A

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

Self-assembled nanoparticles of pure photosensitizer and preparation and application thereof

Technical Field

The invention belongs to the field of new auxiliary materials and new dosage forms of medicinal preparations, relates to a pure photosensitizer self-assembly nanoparticle, and particularly relates to construction of the pure photosensitizer (pyropheophorbide a, PPa) self-assembly nanoparticle and application of the nanoparticle in medicament delivery.

Background

Cancer is still considered to be one of the most serious diseases threatening human health. Currently, surgery is the most common and effective method of cancer treatment, especially in the early stages of the treatment of solid tumors without metastasis. Other various therapeutic strategies, such as chemotherapy and phototherapy, have also been adopted clinically for solid tumors. Among these, chemotherapy remains the mainstay of clinical treatment for cancer, especially in patients who are inoperable and have metastatic tumors. However, severe toxicity can result due to the narrow therapeutic window and off-target effects in vivo of most chemotherapeutic agents. Therefore, the control and treatment of local diseases by site-specific local treatment protocols is a very good option.

In contrast to systemic chemotherapy, photodynamic therapy (PDT) has been widely studied as a non-invasive method of cancer treatment. Under local laser irradiation of tumor, photosensitizer can induce apoptosis and necrosis of tumor cells by the generated large amount of Reactive Oxygen Species (ROS). Cytotoxic ROS produced by photosensitizers can damage cell membranes and oxidize intracellular macromolecules, thereby affecting the normal physiological function of tumor cells. Notably, without laser treatment, the photosensitizer is almost non-cytotoxic. Therefore, phototherapy is considered as a promising therapeutic approach for non-invasive cancer treatment against tumors. In addition, clinical applications of phototherapy have expanded to the treatment of deep visceral tumors due to the rapid development of new photosensitizers and optical fibers. However, the therapeutic efficacy of phototherapy is still hindered due to insufficient accumulation of photosensitizers in the tumor. Therefore, rational design of a highly effective Drug Delivery System (DDS) of photosensitizers is crucial for effective phototherapy.

With the rapid development of biomedical nanotechnology, various nano-drug delivery systems (nano-DDS) have been developed to improve the delivery efficacy of anticancer drugs, including chemotherapeutic agents and photosensitizers. Most photosensitizers are delivered by being encapsulated in organic or inorganic nanocarriers in a non-covalent manner. However, non-covalent 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, a nano delivery system self-assembled by carrier-free small molecule drugs or prodrugs has become a promising nano platform for effective drug delivery. In addition, researches find that some hydrophobic drugs can be automatically assembled into nanoparticles. However, pure drug-driven nano-delivery systems often have unsatisfactory colloidal stability due to relatively weak intermolecular interactions between small molecules. Furthermore, it remains challenging how to trigger pure drug nano-delivery systems to release drugs specifically at the tumor site.

To address these challenges, we constructed pure photosensitizer-driven nano self-assembly systems with core-shell matched pegylation modifications for effective photodynamic therapy.

Disclosure of Invention

The invention solves the technical problems that PPa is poor in hydrophobicity and insoluble in water, and is encapsulated in a polymer to cause low drug loading rate, drug leakage, poor related toxicity of auxiliary materials and the like, and designs a pure PPa self-assembled nanoparticle matched with a core shell, thereby realizing the effects of high drug loading rate, good stability, low toxic and side effects and fixed-point disintegration of tumor parts, and further improving the anti-tumor activity. Simultaneously, with PCL-PEG _2K The modification is used as a control, and the difference of different PEG modified nanoparticles in the aspects of antitumor activity and the like is investigated, and the influence on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of the PPa self-assembled nanoparticles is also investigated.

The invention aims to design pure PPa self-assembled nanoparticles (PPa/PPa-PEG) _2K Nanoparticles) comprising a photosensitizer which is self-assembled alone, orThe nano-particle is formed by self-assembling a photosensitizer and a PEG modifying agent. The PPa self-assembly nano-drug delivery system is prepared, the influences of the stability, the in vitro singlet oxygen generation amount, the cell uptake, the intracellular active oxygen generation amount, the cytotoxicity, the pharmacokinetics, the tissue distribution and the pharmacodynamics of different PEG modified pure PPa self-assembly nano-particles are discussed, the preparation with the best effect is comprehensively screened out, a new strategy and more choices are provided for developing a carrier-free pure-drug nano-delivery system, and the urgent need of high-efficiency chemotherapy preparations in clinic is met.

The invention realizes the purpose through the following technical scheme:

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, 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 as follows: 10:0.5-10:3.

furthermore, the invention preferably selects the pyropheophorbide a and PEG modifier self-assembled nanoparticles, and more preferably the pyropheophorbide a and PPa-PEG _2K Or PCL-PEG _2K Self-assembled nanoparticles.

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

The preparation method of the PPa self-assembly nanoparticle provided by the invention comprises the following steps:

dissolving a certain amount of PPa or mixture of PPa and PEG modifier in a proper amount of mixed solvent of ethanol and tetrahydrofuran, and stirringThe solution is slowly dripped into water to spontaneously form uniform nanoparticles. Finally, ethanol and tetrahydrofuran in the preparation are removed by a dialysis method to obtain the nano colloidal solution without any organic solvent. The PEG modifier is 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.

in particular, the amount of the solvent to be used,

(1) The preparation method of the non-PEG PPa self-assembly nanoparticle comprises the following steps: a certain amount of PPa was dissolved in a suitable amount of ethanol and tetrahydrofuran (3), and the solution was slowly added dropwise to water with stirring, and PPa spontaneously formed uniform nanoparticles. Removing ethanol and tetrahydrofuran in the preparation by dialysis to obtain nano colloidal solution without any organic solvent.

(2) The preparation method of the PEG modified PPa self-assembled nanoparticle comprises the following steps: adding a certain amount of PEG modifier (PPa-PEG) _2K Or PCL-PEG _2K ) And dissolving PPa into a proper amount of ethanol and tetrahydrofuran, slowly dripping the solution into water under stirring, and spontaneously forming uniform nanoparticles by the PPa. Removing ethanol and tetrahydrofuran from the preparation by dialysis to obtain nano colloidal solution without any organic solvent.

The invention has the following beneficial effects: (1) Designs PPa-PEG matched with core shell _2K Modified PPa self-assembled nanoparticles (PPa/PPa-PEG) _2K Nanoparticles) and PCL-PEG _2K Modified PPa self-assembled nanoparticles (PPa/PCL-PEG) _2K Nanoparticles). (2) The uniform PPa self-assembled nanoparticles are prepared, the preparation method is simple and easy to implement, the stability is good, and the PPa is efficiently entrapped and loaded; (3) The influence of different PEGylation on the stability, the in vitro singlet oxygen generation amount, the cellular uptake, the intracellular active oxygen generation amount, the cytotoxicity, the pharmacokinetics, the tissue distribution and the pharmacodynamics of the PPa self-assembly nanoparticles is investigated. The prescription with the best effect is screened out comprehensively, a new strategy and more choices are provided for developing a carrier-free self-assembly nano-drug delivery system, and the urgent need of high-efficiency chemotherapeutic preparations in clinic is met.

In the present invention we have found a unique self-assembly phenomenon of commonly used photosensitizers for PDT (pyropheophorbide a, PPa) that can self-assemble alone into nanoparticles. Furthermore, amphiphilic polymers (PPa-PEG) _2K ) By PPa and PPa-PEG _2K Hydrophobic interaction and pi-pi accumulation interaction between the two components realize core-shell matching PEG modification on the PPa nanoparticles. The self-assembly mechanism and the core-shell matching interaction are researched through computer molecular simulation. In addition, under laser irradiation, external PPa-PEG _2K Is destroyed, PPa/PPa-PEG _2K The stability of the nanoparticles is reduced and specific disintegration occurs. The core-shell matched nanoparticle has multiple drug delivery advantages, including ultrahigh drug loading efficiency (74.8%, w/w), high stability, long systemic circulation, high tumor accumulation, good cellular uptake and laser-triggered release at tumor sites. Thus, PPa/PPa-PEG _2K The nanoparticles show good antitumor effect in antitumor treatment of tumor-bearing mice. The method is the first discovery that pure PPa can be self-assembled into nanoparticles, and the effective transfer of PPa self-assembled nanoparticles can be obviously improved by the core-shell matching design.

Drawings

FIG. 1 is a transmission electron microscope image of PPa and PEG-modified PPa self-assembled nanoparticles of example 1.

FIG. 2 is a computer simulation of the PPa molecule of example 2 of the present invention.

FIG. 3 is a fetal calf serum stability chart and particle size variation chart for PEG-modified PPa self-assembled nanoparticles of example 3 of the present invention under different illumination time.

Fig. 4 is a graph of the particle size change of the PEG-modified PPa self-assembled nanoparticle of example 3 of the present invention under different laser irradiation.

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

Fig. 6 is a cell uptake map of PEG-modified PPa self-assembled nanoparticles of example 5 of the invention.

FIG. 7 is a graph showing the effect of PEG-modified PPa self-assembled nanoparticles of example 6 on ROS levels in tumor cells.

FIG. 8 is a cytotoxicity chart of PEG-modified PPa self-assembled nanoparticles of example 7 without removing the drug-containing culture solution directly irradiated with light

FIG. 9 is a cytotoxicity diagram of PEG-modified PPa self-assembled nanoparticles of example 7 of the present invention after removing the light irradiation of the drug-containing culture solution.

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

Fig. 11 is a 4-hour histodistribution plot of PEG-modified PPa self-assembled nanoparticles of example 9 of the invention.

Fig. 12 is a 4-hour tissue distribution quantification plot of PEG-modified PPa self-assembled nanoparticles of example 9 of the invention.

Fig. 13 is a 12-hour histodistribution plot of PEG-modified PPa self-assembled nanoparticles of example 9 of the invention.

Fig. 14 is a 12-hour tissue distribution quantitation plot of PEG-modified PPa self-assembled nanoparticles of example 9 of the invention.

Fig. 15 is a tumor growth curve diagram of the PEG-modified PPa self-assembled nanoparticle of example 10 of the present invention in an in vivo anti-tumor experiment.

Fig. 16 is a graph of the body weight change of the mouse in the in vivo anti-tumor experiment of the PEG-modified PPa self-assembled nanoparticle of example 10.

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

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto.

Example 1: preparation of PPa self-assembled nanoparticles and PEG-modified PPa self-assembled nanoparticles

1mg of PPa was weighed precisely, dissolved with 200. Mu.L of a mixed solution of ethanol and tetrahydrofuran (3. The organic solvent in the nano preparation is removed by dialysis with deionized water at 25 ℃.

(1) The preparation method of the non-PEG PPa self-assembly nanoparticle comprises the following steps: a certain amount of PPa is dissolved in a suitable amount of ethanol and tetrahydrofuran (3), and the solution is slowly added dropwise to water with stirring, and the PPa spontaneously forms uniform nanoparticles. Removing ethanol and tetrahydrofuran from the preparation by dialysis to obtain nano colloidal solution without any organic solvent.

(2) The preparation method of the PEG modified PPa self-assembled nanoparticle comprises the following steps: adding a certain amount of PEG modifier (PPa-PEG) _2K Or PCL-PEG _2K ) PPa is dissolved in a proper amount of ethanol and tetrahydrofuran, the solution is slowly dripped into water under stirring, and the prodrug spontaneously forms uniform nanoparticles. Removing ethanol and tetrahydrofuran in the preparation by a dialysis method to obtain a nano colloidal solution without any organic solvent, wherein the molar ratio of PPa to PEG modifier is 10:1.

as shown in Table 1, the particle size of the nanoparticles is between 90 and 150nm, the Zeta potential is about-20 mV, and the drug loading is over 50 percent. Wherein Pa/PCL-PEG _2K Nanoparticles and PPa/PPa-PEG _2K The nano-particles have smaller particle size and understanding distribution, larger drug-loading rate and better preparation form. PPa/PCL-PEG is preliminarily optimized _2K Nanoparticles and PPa/PPa-PEG _2K And (3) nanoparticles.

As shown in Table 2, the particle size of the nanoparticles is 100-160nm, the Zeta potential is about-20 mV, and the drug loading is over 30%. Wherein the molar ratio PPa: the PEG modifier is 10:1, pa/PCL-PEG _2K Nanoparticles and PPa/PPa-PEG _2K The particle size and the comprehension distribution of the nanoparticles are smaller, and the nanoparticles have better preparation form. Further preferred is PPa: the molar ratio of the PEG modifier is 10:0.5-10:3.

Pa/PCL-PEG prepared in example 1 was measured by transmission electron microscopy _2K Nanoparticles and PPa/PPa-PEG _2K The particle size and morphology of the nanoparticles are shown in figure 1, and a transmission electron microscope image shows that the nanoparticles are uniform and spherical, and the particle size is about 100 nm.

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 of different molar ratios

Example 2: analysis of the mechanism of PPa self-Assembly

Through simple computer simulation, the mechanism of PPa self-assembly is explored, and molecular docking calculation is completed by adopting a Vina scheme of an Yiganyun computing platform. The compound PPa is subjected to energy minimization under the MMFF94 force field to obtain a 3D structure, and a stable nano assembly is formed. The results of semi-flexible docking using AutoDock Vina program are shown in fig. 2, and the unique multiple pyrrole ring structures of the PPa molecule and pi-pi stacking and hydrophobic forces between molecules make a great contribution to the self-assembly of the PPa molecule.

Example 3: colloidal stability test of PPa self-assembled nanoparticles

The PEG-modified self-assembled nanoparticles prepared in example 1 were taken out by 1mL, added to 20mL of phosphate buffer solution (PBS, pH 7.4) containing 10% fbs, incubated at 37 ℃ for 24 hours, and the particle size change thereof was measured by a dynamic light scattering method at predetermined time points (0, 1,2,4,6,8, and 12 hours). The results are shown in FIG. 3, PPa/PCL-PEG compared to the other groups _2K Nanoparticles and PPa/PPa-PEG _2K The stability of the nano particle colloid is good, and the particle size does not change obviously within 24 hours. Further preferably selects Pa/PCL-PEG _2K Nanoparticles and PPa/PPa-PEG _2K And (3) nanoparticles.

The PEG-modified self-assembled nanoparticles prepared in example 1 were taken out by 1mL and added to 20mL PBS, and the change of the particle size of the nanoparticles was observed under different laser irradiation times, the result is shown in FIG. 4, PPa/PPa-PEG _2K The particle size of the nanoparticles increased significantly under laser irradiation in a laser dose-dependent manner. In contrast, PPa/PCL-PEG even after 8 minutes of exposure to laser light _2K The particle size of the nanoparticles is hardly increased significantly. Obviously, the colloidal stability can be significantly improved by performing pegylation modification on the PPa nano-assembly. However, PPa/PPa-PEG _2K The nanoparticles exhibit laser-triggered decomposition properties due to PPa-PEG under laser irradiation _2K The photobleaching of the medium PPa component is destroyed. As a result, PPa-PEG _2K The amphiphilic structure of the polymer is destroyed, resulting in PPa/PPa-PEG _2K Nanoparticle PPa-PEG _2K The stabilization effect decreases. In contrast, laser on PCL-PEG _2K With or without laser treatment, PCL-PEG _2K The PEGylation of (A) can maintain PPa/PCL-PEG _2K Good stability of the nanoparticles. PPa/PCL-PEG irradiated by laser for 8 minutes _2K The particle size of the nanoparticles changed slightly (about 30nm increase), which should be due to some photo-bleaching of the core PPa of the nanoparticles. These results demonstrate that core-shell matched PPa/PPa-PEG _2K The nanoparticles not only can obviously improve the colloidal stability of the PPa nanoparticles, but also can reduce the stability of the nanoparticles under the triggering of laser. This laser-triggered destabilization may help to mitigate aggregation-induced fluorescence quenching (ACQ) effects of the PPa nanoparticles, thereby promoting their photoconversion and generation of reactive oxygen species.

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). Mixing with SOSG (1 μ M) PPa solution, PPa/PCL-PEG _2K Nanoparticles or PPa/PPa-PEG _2K The nanoparticles (1. Mu.M, PPa equivalent) were diluted in 1mL PBS. Singlet oxygen generated in each of the formulations was detected at different laser irradiation times (660nm, 200mWcm-2) or without irradiation. The fluorescence signal intensity was analyzed by varioskan lux multimode microplate reader (excitation 498nm, emission 525 nm).

As shown in FIG. 5, the amount of singlet oxygen produced by PPa self-assembled nanoparticles was reduced as compared with that of

PPa solutionAt

4 and 8 minutes with time extension, PPa/PPa-PEG _2K The singlet oxygen generation amount of the nanoparticle is obviously more than that of PPa/PCL-PEG _2K The stability of the nanoparticles is reduced along with the prolonging of the illumination time, the ACQ effect is obviously relieved, and the generation amount of singlet oxygen is improved.

Example 5: cellular uptake of PPa self-assembled nanoparticles

And (3) measuring the uptake condition of the PPa self-assembled nanoparticles in the 4T1 cells by using a flow cytometer. 4T1 cells were plated at 1X 10 ⁵ Inoculating cells/mL to a 12-well plate, placing the plate in an incubator for incubation for 24 hours to allow the cells to adhere to the wall, and adding a PPa solution and PPa self-assembled nanoparticles after the cells adhere to the wall. The concentration of PPa was 50nM. After incubation at 37 ℃ for 0.5h or 2h, cells were washed, collected and dispersed in PBS and examined for uptake of various agents by flow cytometry.

Experimental results as shown in fig. 6, two PPa-assembled nanoparticle-treated cells exhibited higher intracellular fluorescence intensity than free PPa-treated cells. 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

And (3) measuring the active oxygen generation condition of the PPa self-assembly nanoparticles in 4T1 cells by adopting an inverted fluorescence microscope. 4T1 cells were plated at 5X 10 ⁴ Inoculating cells/mL to a 24-well plate, placing the plate in an incubator for incubation for 24h to allow the cells to adhere to the wall, and adding a PPa solution and PPa self-assembled nanoparticles after the cells adhere to the wall. The concentration of PPa was 20nM. After incubation at 37 ℃ for 4h, the drug-containing culture medium was discarded, and the culture medium containing the active oxygen detection kit (DCFH-DA, 20. Mu.M) was added and incubation continued for 0.5h. Then, the mixture was irradiated with laser light for 5 minutes (660nm, 60mW cm) ^-2 ) Washed three times with PBS and observed by an inverted fluorescence microscope.

The results are shown in FIG. 7, in which the fluorescence intensity of two nanoparticles is significantly higher than that of the solution, and in addition, PPa/PPa-PEG _2K The fluorescence intensity of the nanoparticle is higher than that of PPa/PCL-PEG _2K Nanoparticles, illustrative of PPa/PPa-PEG _2K The nanoparticles effectively slow down the ACQ effect and improve the generation efficiency of ROS.

Example 7: cytotoxicity of PEG (polyethylene glycol) -modified small-molecule prodrug self-assembled nanoparticles

And (3) inspecting cytotoxicity of the PPa self-assembly nanoparticles on mouse breast cancer (4T 1) cells by adopting an MTT method. Digesting the cells in a good state, diluting the cells to 5000cells/mL by using a culture solution, uniformly blowing the cells, adding 100 mu L of cell suspension into each hole of a 96-hole plate, and placing the cells in an incubator for incubation for 24 hours to adhere to the walls. After the cells are attached to the wall, the PPa solution or the nanoparticles prepared in example 1 are added. In the experiment, the preparation and dilution of the drug solution and the nanoparticle preparation are carried out by using 1640 culture solution and sterile filtration by using a 0.22 mu m filter membrane. Test solution was added at 100. Mu.L per well, 3 wells per concentration in parallel. And in the control group, the liquid medicine to be detected is not added, 100 mu L of culture solution is singly added, and the control group is placed in an incubator to be incubated with cells. And (3) directly illuminating 4 hours after adding the medicines or illuminating again after replacing a new culture solution without medicines, taking out the 96-well plate, adding 20 mu L of 5mg/mL MTT solution into each well, putting the plate in an incubator for incubation for 4 hours, throwing the plate, reversely buckling the 96-well plate on filter paper, fully sucking the residual liquid, adding 200 mu L DMSO into each well, and oscillating the DMSO on an oscillator for 10 minutes to dissolve the bluish purple crystals. The A1 wells (containing only 200. Mu.L DMSO) were set as zeroed wells. The absorbance value after zeroing of each well was measured at 570nm using a microplate reader.

Cytotoxicity results are shown in fig. 8 and fig. 9, and there was almost no cytotoxicity between the various formulations when protected from light, however, there was no significant difference between the formulations when the drug-containing culture solution was not removed before light irradiation, and after removal, the cytotoxicity of the two PPa nanoparticles was significantly stronger than that of the solution, which is probably due to the higher uptake of the nanoparticles. Compared with two PPa nanoparticles, PPa/PPa-PEG _2K The cytotoxicity of the nanoparticle is stronger than that of PPa/PCL-PEG _2K Nanoparticles, this may be due to PPa/PPa-PEG _2K The nanoparticles effectively relieve the ACQ effect, and more ROS are generated in cells.

Example 8: pharmacokinetics research of PPa self-assembled nanoparticles

SD rats with body weight of 200-250g were randomly grouped and fasted for 12h before administration, and water was freely available. PPa solution and PPa self-assembly nanoparticles prepared in the implementation are respectively injected into the vein. The dose of PPa was 2mg/kg. Blood was collected from the orbit at the prescribed time points and separated to obtain plasma. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

The results are shown in FIG. 10, where PPa is cleared more rapidly from the blood due to the short half-life. In contrast, the circulation time of two PPa self-assembled nanoparticles is significantly prolonged. Furthermore, due to PPa/PPa-PEG _2K The core-shell matching stabilization of the nanoparticles leads PPa/PPa-PEG _2K In vivo circulation time ratio PPa/PCL-PEG of nanoparticles _2K The nanoparticles are more elongated.

Example 9: tissue distribution experiment of PEG modified PPa self-assembled nanoparticles

The 4T1KB cell suspension is inoculated to BALB/c mice when the tumor volume reaches 350mm ³ In time, tail vein injection administration: the dosage of the PPa solution and the PPa self-assembly nanoparticle PPa is 1mg/kg. After 4 or 12 hours, the mice were sacrificed and the major organs (heart, liver, spleen, lung, kidney) and tumors were isolated and analyzed with a live imager.

The results are shown in fig. 11-14 (4 hours in fig. 11 and 12, and 12 hours in fig. 13 and 14), and the fluorescence intensity of the PPa self-assembled nanoparticle group in tumor tissue was significantly increased compared to the PPa solution. In contrast, PPa/PPa-PEG _2K The tumor accumulation of the nanoparticles is obviously more than that of PPa/PCL-PEG _2K Provided is a nanoparticle. This result is in full agreement with its pharmacokinetic behavior, PPa/PPa-PEG _2K The nanoparticles have the best stability and the longest circulation time in vivo, thereby showing the best tumor accumulation capacity.

Example 10: PPa self-assembled nanoparticle in-vivo anti-tumor experiment

4T1 cell suspension (5X 10) ⁶ cells/100 μ L) were inoculated subcutaneously ventral to female mice. When the tumor volume grows to 150mm ³ At this time, mice were randomly grouped into groups of five mice each, and physiological saline, PPa solution, and PPa self-assembled nanoparticles prepared in examples were given to the mice, respectively. The administration was 1 time every 1 day and 5 times continuously, and the dose was 2mg/kg in terms of PPa. After the administration, the survival state of the mice was observed every day, the body weight was weighed, and the tumor volume was measured. After the last administrationThe mice were sacrificed, organs and tumors were harvested and further evaluated analytically. Major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected and fixed with 4% tissue fixative for H&And E, dyeing.

As shown in fig. 15, PPa showed a certain tumor-inhibiting activity compared to the saline group. PPa/PCL-PEG _2K The nano-particle shows stronger anti-tumor activity than PPa solution, and the tumor volume is slowly increased. As expected, PPa/PPa-PEG _2K The nanoparticle has the most obvious anti-tumor effect, effectively inhibits the tumor growth, and has the trend of even decreasing the tumor volume in the later treatment period. The results show that the stability, cytotoxicity, pharmacokinetics, tissue distribution and the like of the nanoparticles can influence the final anti-tumor effect.

As shown in fig. 16, the small body weights of the groups did not change significantly. As can be seen from fig. 17, there was no significant abnormality in the function of the small major organs in each group. These results indicate that the PPa self-assembled nanoparticles have obvious anti-tumor effect, do not cause significant non-specific toxicity to organisms, and are a safe and effective anti-cancer drug delivery system.

Claims

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.