CN111617246A - Self-assembled nanoparticles of pure photosensitizer and preparation and application thereof - Google Patents
Self-assembled nanoparticles of pure photosensitizer and preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and relates to a pure photosensitizer self-assembly nanoparticle, which realizes 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 anti-tumor activity. The invention provides a pure photosensitizer self-assembly nanoparticle, which is formed by independently self-assembling a photosensitizer or self-assembling the photosensitizer and a PEG (polyethylene glycol) modifier through core-shell matching. 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
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 photosensitizer 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 them, chemotherapy remains the primary treatment of cancer in the clinic, especially for 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 for most chemotherapeutic agents. Therefore, the control and treatment of local diseases by site-specific local treatment regimens is a very good option.
In contrast to systemic chemotherapy, photodynamic therapy (PDT) has been widely studied as a non-invasive 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, the photosensitizer is almost non-cytotoxic without laser treatment. 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 hampered by 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 formed by self-assembling of a small molecule drug or a prodrug without a carrier has become a promising nano platform for effective drug delivery. Moreover, 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 specifically release drugs 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-PEG2KModified As a control, different PEGylated modified nanoparticles were examinedThe difference of the particles in the aspects of antitumor activity and the like, and the influence on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of the PPa self-assembled nanoparticles.
The invention aims to design pure PPa self-assembled nanoparticles (PPa/PPa-PEG)2KNanoparticles) including nanoparticles formed by independently self-assembling a photosensitizer or formed by self-assembling a photosensitizer and a PEG (polyethylene glycol) modifier. 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 aim through the following technical scheme:
the invention provides a pure photosensitizer self-assembly nanoparticle, which is formed by independently self-assembling a photosensitizer or self-assembling the photosensitizer and a PEG (polyethylene glycol) modifier through core-shell matching.
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-PEG2K、PCL500-PEG2K。
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-PEG2KOr PCL-PEG2KSelf-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-assembled nanoparticles provided by the invention comprises the following steps:
dissolving a certain amount of PPa or a mixture of PPa and a PEG modifier into a proper amount of mixed solvent of ethanol and tetrahydrofuran, slowly dripping the solution into water under stirring, and spontaneously forming 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-PEG2KAnd PCL-PEG2K。
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: dissolving a certain amount of PPa into a proper amount of ethanol and tetrahydrofuran (3: 2), 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.
(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)2KOr PCL-PEG2K) 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 shell2KModified PPa self-assembled nanoparticles (PPa/PPa-PEG)2KNanoparticles) and PCL-PEG2KModified PPa self-assembled nanoparticles (PPa/PCL-PEG)2KNanoparticles). (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; (3) the stability of different PEGylation to PPa self-assembled nanoparticles is investigatedIn vitro singlet oxygen production, cellular uptake, intracellular reactive oxygen species production, cytotoxicity, pharmacokinetics, tissue distribution, and pharmacodynamic effects. The prescription with the best effect is comprehensively screened out, 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 that the unique self-assembly phenomenon of commonly used photosensitizers for PDT (pyropheophorbide a, PPa) can self-assemble into nanoparticles alone. Furthermore, amphiphilic polymers (PPa-PEG)2K) By PPa and PPa-PEG2KHydrophobic and pi-pi accumulation interaction between the two components realizes 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-PEG2KIs destroyed, PPa/PPa-PEG2KThe 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-PEG2KThe nanoparticles show good anti-tumor effect in the anti-tumor 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 PEG-modified PPa self-assembled nanoparticles of example 3 of the present invention under different laser irradiation.
Fig. 5 is a diagram of in vitro singlet oxygen generation of PEG-modified PPa self-assembled nanoparticles of example 4 of the 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 diagram of PEG-modified PPa self-assembled nanoparticles of example 7 without direct illumination of drug-containing culture solution
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 quantification 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 change in body weight of mice in an in vivo anti-tumor experiment of PEG-modified PPa self-assembled nanoparticles of example 10 of the present invention.
Fig. 17 is a pathological section view of PEG-modified PPa self-assembled nanoparticles 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 is precisely weighed, dissolved by 200 mu L of mixed solution (3: 2) of ethanol and tetrahydrofuran, and slowly dropped into 2mL of deionized water under stirring to spontaneously form uniform nano-particle PPa nano-particles. 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: dissolving a certain amount of PPa into a proper amount of ethanol and tetrahydrofuran (3: 2), 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.
(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)2KOr PCL-PEG2K) 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 diameters of the nanoparticles are all between 90 and 150nm, the Zeta potential is about-20 mV, and the drug loading is all over 50 percent. Wherein Pa/PCL-PEG2KNanoparticles and PPa/PPa-PEG2KThe particle size and comprehension distribution of the nanoparticles are small, the drug-loading rate is large, and the preparation form is good. PPa/PCL-PEG is preliminarily optimized2KNanoparticles and PPa/PPa-PEG2KAnd (3) nanoparticles.
As shown in Table 2, the particle diameters of the nanoparticles are all between 100 and 160nm, the Zeta potential is about-20 mV, and the drug loading is over 30 percent. Wherein the molar ratio PPa: the PEG modifier is 10: 1, Pa/PCL-PEG2KNanoparticles and PPa/PPa-PEG2KThe particle size and comprehension distribution of the nanoparticles are small, and the nanoparticles have a good 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 microscopy2KNanoparticles and PPa/PPa-PEG2KThe results of the particle size and morphology of the nanoparticles are shown in fig. 1, and the transmission electron microscope 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 undergoes energy minimization under MMFF94 force field to obtain 3D structure, forming stable nano-assembly. 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 groups2KNanoparticles and PPa/PPa-PEG2KThe stability of the nano particle colloid is good, and the particle size does not change obviously within 24 hours. Further preferably selects Pa/PCL-PEG2KNanoparticles and PPa/PPa-PEG2KAnd (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-PEG2KThe 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 exposure to laser light2KThe 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-PEG2KThe nanoparticles exhibit laser-triggered decomposition properties due to PPa-PEG under laser irradiation2KThe photobleaching of the medium PPa component is destroyed. As a result, PPa-PEG2KThe amphiphilic structure of the polymer is destroyed, resulting in PPa/PPa-PEG2KNanoparticle PPa-PEG2KThe stabilization effect decreases. In contrast, laser on PCL-PEG2KWith little effect of PCL-PEG with or without laser treatment2KAll the PEGylation can keep PPa/PCL-PEG2KGood stability of the nanoparticles. PPa/PCL-PEG with laser irradiation for 8 minutes2KThe 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-PEG2KThe 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). The PPa solution, PPa/PCL-PEG, to be mixed with SOSG (1. mu.M)2KNanoparticles or PPa/PPa-PEG2KThe nanoparticles (1. mu.M, PPa equivalent) were diluted in 1ml PBS. Under different laser irradiation time (660nm, 200mWcm-2) or no irradiation,singlet oxygen production was detected in each group of formulations. 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 compared to the PPa solution, and the PPa/PPa-PEG was observed to increase over time at 4 min and 8 min2KThe singlet oxygen generation amount of the nanoparticle is obviously more than that of PPa/PCL-PEG2KThe 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
The uptake of PPa self-assembled nanoparticles in 4T1 cells was determined by flow cytometry, 4T1 cells were treated with 1 × 105Inoculating cells/mL to a 12-hole 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 50 nM. After incubation at 37 ℃ for 0.5h or 2h, cells were washed, collected and dispersed in PBS and the uptake of each preparation by cells was examined 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
Measuring the active oxygen generation condition of PPa self-assembled nanoparticles in 4T1 cells by using an inverted fluorescence microscope, and mixing 4T1 cells with 5 × 104Inoculating 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 20 nM. 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 0.5 h. Followed by laser irradiation 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. 7Shows that the fluorescence intensity of the two nanoparticles is obviously higher than that of the solution, and in addition, PPa/PPa-PEG2KThe fluorescence intensity of the nanoparticles is higher than that of PPa/PCL-PEG2KNanoparticles, illustrative of PPa/PPa-PEG2KThe nanoparticles effectively slow down the ACQ effect and improve the generation efficiency of ROS.
Example 7: cytotoxicity of PEG-modified small-molecule prodrug self-assembled nanoparticles
The cytotoxicity of the PPa self-assembled nanoparticles on mouse breast cancer (4T1) cells is examined 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 parallel wells per concentration. In the control group, 100 mul of culture solution is singly supplemented without adding the liquid medicine to be detected, and the control group is placed in an incubator to be incubated with cells together. Directly illuminating 4 hours after dosing or illuminating again after replacing a new culture solution without drugs, illuminating for 44 hours, 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, then throwing the plate, reversely buckling the 96-well plate on filter paper to fully absorb the residual liquid, adding 200 mu L DMSO into each well, and oscillating for 10 minutes on an oscillator to dissolve the bluish purple crystals. The A1 well (containing only 200. mu.L DMSO) was set as the zeroing well. 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, 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 illumination, and after removal, the cytotoxicity of the two PPa nanoparticles was significantly stronger than that of the solution, probably due to the higher uptake of the nanoparticles. Compared with two PPa nanoparticles, PPa/PPa-PEG2KThe cytotoxicity of the nanoparticle is stronger than that of PPa/PCL-PEG2KNanoparticles, this may be due to PPa/PPa-PEG2KThe 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 the body weight between 200-250g are taken and randomly grouped, and are fasted for 12h before administration and are free to drink water. PPa solution and PPa self-assembly nanoparticles prepared in the implementation are respectively injected into the vein. The dose of PPa was 2 mg/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 spectrometer.
The results are shown in FIG. 10, where PPa is cleared more rapidly from the blood due to the short half-life. Compared with the two PPa self-assembly nanoparticles, the circulation time of the two PPa self-assembly nanoparticles is obviously prolonged. Furthermore, due to PPa/PPa-PEG2KThe core-shell matching stabilization of the nanoparticles enables PPa/PPa-PEG2KIn vivo circulation time ratio of nanoparticles PPa/PCL-PEG2KThe nanoparticles are more elongated.
Example 9: tissue distribution experiment of PEG modified PPa self-assembled nanoparticles
The 4T1KB cell suspension was inoculated into BALB/c mice when the tumor volume reached 350mm3In time, tail vein administration: the administration dose of the PPa solution and the PPa self-assembly nano-particle PPa is 1 mg/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-PEG2KThe tumor accumulation of the nanoparticles is obviously more than that of PPa/PCL-PEG2KAnd (3) nanoparticles. This result is in full agreement with its pharmacokinetic behavior, PPa/PPa-PEG2KThe 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)6cells/100 μ L) were inoculated subcutaneously ventrally in female mice. When the tumor volume grows to 150mm3At the same time, mice were randomly grouped into groups of five mice eachOnly, physiological saline, a PPa solution and PPa self-assembled nanoparticles prepared in examples were separately administered. The administration was 1 time every 1 day and 5 times continuously, and the administration 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. Mice were sacrificed one day after the last dose, 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 exhibited a certain tumor-inhibiting activity compared to the saline group. PPa/PCL-PEG2KThe nano particle shows stronger anti-tumor activity than PPa solution, and the tumor volume is slowly increased. As expected, PPa/PPa-PEG2KThe 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 (10)
1. The self-assembled nanoparticles of the pure photosensitizer are characterized in that the nanoparticles are formed by independently self-assembling the photosensitizer or self-assembling the photosensitizer and a PEG (polyethylene glycol) modifier.
2. The self-assembled nanoparticle of pure photosensitizer according to claim 1, wherein the photosensitizer is a porphyrin photosensitizer, preferably one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, pyropheophorbide a hexyl ether, and chlorin e 6.
3. The self-assembled nanoparticle of pure photosensitizer according to claim 1 or 2, wherein the PEG modifier is an amphiphilic polymer of PEG and photosensitizer or an amphiphilic polymer without photosensitizer.
4. The self-assembled nanoparticle of a pure photosensitizer as claimed in claim 3, wherein the PEG is one or more selected from PPa-PEG, PCL-PEG, DSPE-PEG, PLGA-PEG and PE-PEG, and the molecular weight of PEG is 2000-20000, preferably 2000-5000.
5. The self-assembled nanoparticle of claim 4, wherein the PEG modifier is PPa-PEG2K、PCL500-PEG2KOne of DSPE-PEG, PLGA-PEG and PE-PEG, preferably PPa-PEG2K、PCL500-PEG2K。
6. The method for preparing the self-assembled nanoparticle of pure photosensitizer according to claim 1,
dissolving a certain amount of photosensitizer or a mixture of the 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 in the preparation by a dialysis method to obtain a nano colloidal solution.
7. The method of claim 6, wherein the weight ratio of photosensitizer to PEG modifier is 10:0.5-10:3, the volume ratio of ethanol to tetrahydrofuran is 3: 2-3: 4.
8. use of the self-assembled nanoparticles of a pure photosensitizer as defined in any one of claims 1 to 5 for the preparation of a drug delivery system.
9. The use of the self-assembled nanoparticles of a photosensitizer of any one of claims 1 to 5 in the preparation of an anti-tumor drug.
10. Use of the self-assembled nanoparticles of a pure photosensitizer as defined in any one of claims 1 to 5 for the preparation of a system for injection, oral or topical administration.
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