CN113648401B - Hybrid nano-assembly for proteasome inhibition sensitization photodynamic therapy and preparation and application thereof - Google Patents

Hybrid nano-assembly for proteasome inhibition sensitization photodynamic therapy and preparation and application thereof Download PDF

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CN113648401B
CN113648401B CN202110955686.XA CN202110955686A CN113648401B CN 113648401 B CN113648401 B CN 113648401B CN 202110955686 A CN202110955686 A CN 202110955686A CN 113648401 B CN113648401 B CN 113648401B
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CN113648401A (en
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罗聪
杨馥骏
孙丙军
李术萌
何仲贵
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Shenyang Pharmaceutical University
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Abstract

The invention discloses a carrier-free hybrid nano assembly, a preparation method and application thereof, belonging to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations. The carrier-free hybrid nano assembly is formed by co-assembling a porphyrin photosensitizer and Bortezomib (BTZ), or is formed by co-assembling a porphyrin photosensitizer, BTZ and a PEG modifier. The carrier-free hybrid nano assembly has strong anti-tumor activity and high safety. The co-assembled nano preparation provides a new strategy and more choices for developing the combined application of drug delivery and photodynamic chemotherapy, and meets the urgent need of efficient photochemical synergistic treatment in clinic.

Description

Hybrid nano-assembly for proteasome inhibition sensitization photodynamic therapy and preparation and application thereof
Technical Field
The invention belongs to the technical field of novel auxiliary materials and novel dosage forms of pharmaceutical preparations, and particularly relates to construction of co-assembled nanoparticles comprising porphyrin photosensitizers and a proteasome inhibitor Bortezomib (BTZ) and application of the co-assembled nanoparticles in drug delivery.
Background
Photodynamic therapy (PDT) has been widely studied as a non-invasive cancer treatment with spatiotemporal selectivity and high safety. Under tumor-locating laser irradiation, photosensitizers (PSs) produce large amounts of Reactive Oxygen Species (ROS). ROS cause oxidative damage to proteins, lipids, DNA and other intracellular components, and in particular ROS-induced oxidative damage to proteins accelerates the functional inactivation and degradation of cellular biological macromolecules, ultimately leading to tumor cell death. However, there are a number of defense mechanisms in tumor cells, such as accelerated degradation of misfolded proteins; upregulation of antioxidants (e.g., glutathione synthase and glutathione peroxidase). Among them, misfolded proteins are degraded by proteasomes, i.e., ubiquitin-proteasome pathway (UPP), is considered one of the major obstacles to PDT.
The important role of UPP in misfolded protein degradation has greatly motivated the development of proteasome inhibitors, of which Bortezomib (BTZ) is the first approved proteasome inhibitor for myeloma, which effectively inhibits the proteasome by reversible binding to the β5 subunit. In addition to myeloma, BTZ also shows potent antitumor activity against various cancer cell lines (including breast, colorectal, lung, prostate and lymphoma). Although BTZ has strong antitumor activity, its clinical application is still limited by serious adverse reactions. There is increasing evidence that BTZ-mediated proteasome inhibition has excellent synergy with PSs in several human cancer cell lines. In view of the obvious synergy between proteasome inhibition and ROS-induced protein damage, we propose that accurate binding of BTZ and PS not only increases tumor cell sensitivity to PDT, but also is expected to significantly reduce BTZ toxicity by reducing dose. However, effective co-delivery of two or more therapeutic agents is still limited by inefficiency, inconvenience in dose scaling, and adverse in vivo pharmacokinetic and biodistribution, and there is a strong need to develop more effective co-delivery modes.
In recent decades, biological nanotechnology is widely applied to the development of anticancer nano-drugs, and reasonably designed nano-carriers not only effectively improve adverse physicochemical properties of the drugs, but also realize specific accumulation and on-demand release of the drugs in tumors, and simultaneously, the combined application of multiple drugs is promoted. However, conventional nanocarriers (e.g., liposomes and micelles) co-deliver two or more drugs still present many challenges, such as low drug co-delivery efficiency, poor encapsulation stability, premature drug leakage, and the like. Furthermore, flexible adjustment of the dose ratio of multiple drugs to achieve optimal synergy is more challenging, mainly due to the large differences in physicochemical properties of the drug and affinity of the carrier material. Fortunately, unsupported nanoassemblies formed from the drug itself have become promising nanoplatforms. In this unique nanosystem, the drug molecule itself acts as a carrier, both improving the loading efficiency of the drug and reducing the side effects caused by the excipients.
Chemotherapy in combination with PDT is an effective strategy to increase the anti-tumor effect. More importantly, the dosage of the chemotherapeutic medicine can be reduced, so that the toxic and side effects related to chemotherapy can be obviously reduced. Research and development of a photochemical synergistic anti-tumor nano assembly is an important subject to be researched at present.
Disclosure of Invention
Based on the technical problems in the background art, the invention further designs the nanoparticles formed by co-assembling the photosensitizer and the BTZ for solving the problems of low drug loading, drug leakage, auxiliary material related toxicity and the like caused by poor hydrophobicity, indissolvable water and inclusion in the polymer of the BTZ, thereby realizing the technical effects of high drug loading, good stability, toxic and side effects and the like, further solving the problems of poor treatment effect of single pure medicine and quenching (ACQ) effect caused by aggregation induction of the photosensitizer, and improving the combined treatment effect of chemotherapy and photodynamic therapy.
The invention realizes the aim through the following technical scheme:
the carrier-free hybrid nano assembly is formed by co-assembling a porphyrin photosensitizer and Bortezomib (BTZ), or is formed by co-assembling a porphyrin photosensitizer, BTZ and a PEG modifier.
Further, the photosensitizer comprises pyropheophorbide a (PPa), chlorin e6, hypericin, zinc phthalocyanine and BDP.
Further, the PEG modifier is DSPE-PEG, TPGS, PCL-PEG, PLGA-PEG and PE-PEG, the molecular weight of the PEG is 500-10000, and the mass ratio of the medicine (porphyrin photosensitizer and BTZ) to the PEG modifier is 10:90-90:10.
Further, the molar ratio of the BTZ to the porphyrin photosensitizer in the carrier-free hybrid nano-assembly is 5:1-1:5.
The invention provides a preparation method of the carrier-free hybrid nano assembly, which comprises the following steps:
BTZ and porphyrin-based photosensitizers were dissolved in tetrahydrofuran: slowly dripping ethanol into deionized water in a volume ratio of 1:1-1:5 under stirring to spontaneously form uniform nanoparticles, and removing the organic solvent to obtain a carrier-free hybrid nano assembly formed by co-assembling a porphyrin photosensitizer and BTZ;
alternatively, BTZ, porphyrin-based photosensitizer, and PEG modifier are dissolved in tetrahydrofuran: and slowly dripping the mixture into deionized water under stirring in the volume ratio of ethanol of 1:1-1:5 to spontaneously form uniform nanoparticles, and removing the organic solvent to obtain the carrier-free hybrid nano-assembly formed by co-assembling the porphyrin photosensitizer, the BTZ and the PEG modifier.
Further, in the preparation method, the method for removing the organic solvent may be a solvent evaporation method, an ultrafiltration method, and a membrane permeation method.
Further, the photosensitizer is preferably pyropheophorbide a.
Further, the PEG modifier is preferably DSPE-PEG 2K The BTZ and PPa form co-assembled nanoparticles (BTZ@PPa nanoparticles), or PEG modifiers (DSPE-PEG 2K ) Modified nanoparticles (BTZ@PPa PEG) 2K Nanoparticles).
Further, the preferred solvent is tetrahydrofuran to ethanol in a volume ratio of 1:1.
Further, the molar ratio of the BTZ to the porphyrin photosensitizer is 5:1-1:5.
Further, the molar ratio of BTZ to porphyrin photosensitizer is optimized to be 1:4.
The invention provides the application of the carrier-free hybrid nano-assembly in a drug delivery system.
The invention provides application of the carrier-free hybrid nano-assembly in preparing an anti-tumor drug.
Further, the administration form of the drug delivery system or the antitumor drug includes injection administration, oral administration or topical administration.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention prepares the co-assembled nanometer hybrid of BTZ and porphyrin photosensitizer, which is used for treating cancers. The porphyrin photosensitizer and BTZ are used cooperatively, so that the photochemical combined treatment effect is improved.
2. The co-assembled nano hybrid of the BTZ and the porphyrin photosensitizer has the technical effects of high drug loading capacity, good stability, low toxic and side effects and the like, meets the urgent requirements of high-efficiency low-toxicity preparations in clinic, and provides an effective nano platform for developing carrier-free hybrid nano assemblies and photochemical combination treatment.
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In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 shows BTZ@PPa and BTZ@PPa PEG of example 1 of the present invention 2K Nanoparticle graph.
FIG. 2 shows BTZ@PPa and BTZ@PPa PEG of example 1 of the present invention 2K Transmission electron microscopy of nanoparticles.
FIG. 3 is a schematic diagram showing the molecular docking of BTZ and PPa in example 2 of the present invention.
FIG. 4 shows BTZ@PPa and BTZ@PPa PEG of example 3 of the present invention 2K Stability profile of nanoparticles in PBS of 10% fetal bovine serum.
FIG. 5 is a graph showing in vitro singlet oxygen production under different conditions according to example 4 of the present invention.
FIG. 6 shows the PPa solution, BTZ@PPa nanoparticle and BTZ@PPa PEG of example 5 of the present invention 2K Cellular uptake profile of nanoparticle nanoparticles.
FIG. 7 shows the BTZ solution, PPa solution, BTZ/PPa mixed solution, BTZ/PPa of example 6 of the present inventionMixing solution agent and illumination L, BTZ@PPa nanoparticle and illumination L, BTZ@PPa PEG2K nanoparticle and BTZ@PPa PEG 2K Nanoparticle + 4T1 cytotoxicity profile of light L.
FIG. 8 is an immunofluorescence of example 7 of the present invention. a, physiological saline; b, PPa solution + light; c, BTZ solution; d, BTZ@PPa nanoparticles; e, BTZ@PPa PEG 2K Nanoparticles; f, BTZ@PPa nanoparticles+illumination; g, BTZ/PPa mixed solution + illumination; h, BTZ@PPa PEG 2K Nanoparticles + light irradiation.
FIG. 9 is a quantitative immunofluorescence chart of example 7 of the present invention. a, physiological saline; b, PPa solution + light; c, BTZ solution; d, BTZ@PPa nanoparticles; e, BTZ@PPa PEG 2K Nanoparticles; f, BTZ@PPa nanoparticles+illumination; g, BTZ/PPa mixed solution + illumination; h, BTZ@PPa PEG 2K Nanoparticles + light irradiation.
FIG. 10 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 8 of the present invention 2K A blood concentration versus time profile for nanoparticle nanoparticles.
FIG. 11 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue profile of nanoparticle nanoparticles for 2 h.
FIG. 12 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue distribution quantification of nanoparticle nanoparticles for 2 h.
FIG. 13 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue profile of nanoparticle nanoparticles for 4 h.
FIG. 14 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue distribution quantification of nanoparticle 4 h.
FIG. 15 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue profile of nanoparticle nanoparticles for 8 h.
FIG. 16 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue distribution quantification of nanoparticle nanoparticles for 8 h.
FIG. 17 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa according to example 9 of the present invention PEG 2K Tissue profile of nanoparticle nanoparticles for 12 h.
FIG. 18 shows the PPa solution, BTZ@PPa nanoparticles and BTZ@PPa PEG of example 9 of the present invention 2K Tissue distribution quantification of nanoparticle 12 h.
FIG. 19 is a graph showing the tumor growth in an in vivo anti-tumor assay of example 10 of the present invention. a, BTZ solution; b, physiological saline control group; c, BTZ@PPa nanoparticles; d, PPa solution + illumination; e, BTZ@PPa nanoparticles+illumination; f, BTZ@PPa PEG 2K Nanoparticles; g, BTZ@PPa PEG 2K Nanoparticles + illumination; h, BTZ/PPa mixed solution+light.
FIG. 20 is a graph showing the weight change of mice in an in vivo antitumor test according to example 10 of the present invention. a, BTZ solution; b, physiological saline control group; c, BTZ@PPa nanoparticles; d, PPa solution + illumination; e, BTZ@PPa nanoparticles+illumination; f, BTZ@PPa PEG 2K Nanoparticles; g, BTZ@PPa PEG 2K Nanoparticles + illumination; h, BTZ/PPa mixed solution+light.
Fig. 21 is a diagram showing liver and kidney functions according to example 10 of the present invention. a, BTZ solution; b, physiological saline control group; c, BTZ@PPa nanoparticles; d, PPa solution + illumination; e, BTZ@PPa nanoparticles+illumination; f, BTZ@PPa PEG 2K Nanoparticles; g, BTZ@PPa PEG 2K Nanoparticles + illumination; h, BTZ/PPa mixed solution+light.
FIG. 22 shows a BTZ solution, a physiological saline control group, BTZ@PPa nanoparticles, a PPa solution+light irradiation, a BTZ@PPa nanoparticle+light irradiation, and a BTZ@PPa PEG according to example 10 of the present invention 2K Nanoparticles, BTZ@PPa PEG 2K Nanoparticles + light, BTZ/PPa mixed solution + light pathological section map.
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
Example 1: preparation of BTZ@PPa nanoparticles
BTZ and pyropheophorbide a (PPa) with different molar ratios are dissolved into 200 mu L of tetrahydrofuran and ethanol with the volume ratio of 1:1, the solution is slowly dripped into 2mL of deionized water under stirring, PPa and BTZ spontaneously form uniform nano particles, and then organic solvent in the nano preparation is removed by dialysis in the deionized water under the condition of 25 ℃ to obtain a nano colloid solution without any organic solvent.
The particle size, particle size distribution and synergy index of BTZ and PPa of the prepared nano-preparation were examined, and the results are shown in table 1.
TABLE 1 particle size, particle size distribution of BTZ@PPa nanoparticles and synergy index of BTZ and PPa
Figure BDA0003220390740000051
As shown in Table 1, the particle diameters of the nanoparticles are all between 85 and 135nm, and the synergy index is 0.38 to 1.89. Wherein BTZ: when ppa=1:4, the btz@ppa nanoparticles are distributed uniformly, and the synergy index of the BTZ solution agent and the PPa solution agent is higher. It is initially preferred that the ratio of BTZ to PPa is 1:4.
(1) The preparation method of the non-PEGylated BTZ@PPa nanoparticle comprises the following steps: precisely weighing 1mg of BTZ and 4 times of molar quantity of pyropheophorbide a (PPa), dissolving the pyropheophorbide a (PPa) by using 200 mu L of tetrahydrofuran and ethanol in a volume ratio of 1:1, slowly dropwise adding the solution into 2mL of deionized water under stirring to spontaneously form uniform BTZ@PPa nanoparticles, and then dialyzing the solution with deionized water at 25 ℃ to remove an organic solvent in the nano preparation to obtain a nano colloid solution (figure 1) without any organic solvent.
(2) The preparation method of the PEG modified BTZ@PPa nanoparticle comprises the following steps: precisely weighing 0.2mg of PEG modifier (DSPE-PEG) 2K ) 1mg BTZ and 4 times mole amount of PPa, using 200 mu L tetrahydrofuran and ethanol solution volume ratio (1:1) to dissolve, slowly dripping the solution into 2mL deionized water under stirring to spontaneously form uniform BTZ@PPa PEG 2K And (3) nanoparticles. The organic solvent in the nanofabricated was then removed at 25 ℃ to give a nano-colloidal solution without any organic solvent (fig. 1).
By dynamic meansLight scattering method for detecting prepared BTZ@PPa nanoparticle and BTZ@PPa PEG 2K The particle size, particle size distribution, zeta potential and drug loading of the nanoparticles are shown in Table 2.
TABLE 2 particle size, particle size distribution, zeta potential and drug loading of BTZ@PPa nanoparticles
Figure BDA0003220390740000061
As shown in Table 2, the particle size of the BTZ@PPa nanoparticle is about 88nm, the Zeta potential is about-15 mV, and the PPa drug loading rate is 84.8%; BTZ@PPa PEG 2K The particle diameter of the nanoparticle is about 103nm, the Zeta potential is about-22 mW, and the PPa drug loading rate is 67.8%.
Measurement of BTZ@PPa nanoparticles and PEG-modified BTZ@PPa PEG prepared in example 1 by transmission electron microscopy 2K The particle size and morphology of the nanoparticles are shown in FIG. 2, and the transmission electron microscope chart shows that the nanoparticles are uniform spheres with the particle size of about 100-110 nm.
Example 2: BTZ@PPa assembly mechanism analysis
And exploring the mechanism of BTZ@PPa assembly through computer simulation, and completing molecular docking calculation by adopting a Vina scheme of a Yan Fuyun computing platform. The compound BTZ@PPa performs energy minimization under the MMFF94 force field to obtain a 3D structure, so as to form a stable nano-assembly. Semi-flexible docking was performed using the AutoDock Vina program, resulting in various forces between PPa and BTZ, such as pi-pi stacking, hydrophobic forces, pi cationic forces, and hydrogen bonding, that contributed significantly to the assembly of PPa and BTZ, as shown in fig. 3.
Example 3: colloidal stability test of nanoparticles
BTZ@PPa nanoparticle prepared in example 1 and BTZ@PPa PEG 2K The nanoparticles were removed by 1mL, added to 20mL of phosphate buffer (PBS, pH 7.4) containing 10% fbs, incubated at 37 ℃ for 12 hours, and their particle size change was determined by dynamic light scattering at predetermined time points (0,1,2,4,6,8 and 12 hours). As shown in FIG. 4, compared with the non-PEG modified BTZ@PPa nanoparticle, the BTZ@PPa PEG 2K The nanoparticle colloid has better stability, and the particle size does not change obviously within 12 hours. PEG-modified BTZ@PPa nanoparticles are preferred.
Example 4: in vitro singlet oxygen detection of nanoparticles
Singlet oxygen generated under laser irradiation was detected with a singlet oxygen fluorescent probe (DCFH-DA). The same volume of PPa solution (200 nM) mixed with DCFH-DA (20 nM), BTZ@PPa nanoparticle mixed with DCFH-DA (20 nM) and BTZ@PPa PEG 2K Nanoparticles (200 nM, PPa equivalent) were incubated at 37℃for 30 min, and irradiated with laser light (660 nm,20mW cm) -2 ) The singlet oxygen generated in each set of formulations was detected at different times or without irradiation and the fluorescence signal intensity was analyzed by Nikon Corp inverted microscope.
As a result, as shown in fig. 5, the amount of singlet oxygen generated by the nanoparticles was significantly larger than that by the PPa solution under laser irradiation.
Example 5: cellular uptake of nanoparticles
BTZ@PPa nanoparticle and BTZ@PPa PEG prepared in example 1 were measured using a confocal microscope 2K Uptake of nanoparticles in 4T1 cells. 4T1 cells were plated at 5X10 4 Inoculating cells/mL on a 24-well plate, incubating in an incubator for 24h to adhere cells, and adding PPa solution and BTZ@PPa PEG after the cells adhere 2K The concentration of PPa in the nanoparticle is 2.5 mug/mL, after incubation for 1 hour, 2 hours and 4 hours at 37 ℃, cells are washed, collected and dispersed in PBS, PPa is extracted by ultrasonic disruption, centrifugation and protein precipitation, and finally the uptake of various preparations by the cells is analyzed by a C2, nikon confocal microscope.
The experimental results are shown in fig. 6, and the nanoparticle treated cells had higher intracellular fluorescence intensity than the free PPa treated cells taken up in the 4-hour experimental group. Thus, the prepared BTZ@PPa PEG 2K Nanoparticles have higher cellular uptake efficiency than free PPa.
Example 6: cytotoxicity of nanoparticles
Adopting MTT method to inspect BTZ@PPa PEG 2K The nanoparticle is used for treating mouse breast cancer (4T 1) cells and mouse colon cancer(CT 26) cytotoxicity of cells. The cells in good condition are digested, diluted to the cell density of 2000 cells/ml by using culture solution, and 100 mu L of cell suspension is added into each well of a 96-well plate after being uniformly blown, and the cells are placed in an incubator for incubation for 24 hours to adhere to the cells. After the cells are attached, PPa solution or BTZ@PPa nanoparticle and BTZ@PPa PEG are added 2K And (3) nanoparticles. The experiment uses 1640 culture solution to formulate and dilute drug solutions and nanoparticle formulations and sterile filtration with 0.22 μm filters. 100 μl of each well of test solution was added, 3 wells in parallel per concentration. The control group is not added with the liquid medicine to be detected, and is singly supplemented with 100 mu L of culture solution, and the culture solution is placed in an incubator to be incubated with cells. Relates to a laser irradiation experimental group, and after 4 hours from drug addition, the laser irradiation (660 nm,20mW cm -2 ) After 44 hours, the 96-well plate was taken out, 20. Mu.L of MTT solution of 5mg/mL was added to each well, the plates were thrown out after incubation in an incubator for 4 hours, and after the 96-well plate was back-buckled on filter paper to sufficiently suck the residual liquid, 200. Mu.L of DMSO was added to each well and the mixture was shaken on a shaker for 10 minutes to dissolve the bluish violet crystals. A1 wells (containing only 200. Mu.L DMSO) were set as zeroed wells. Absorbance values after zeroing of each well were determined at 570nm using a microplate reader.
The cytotoxicity results are shown in FIG. 7, when the light is shielded, the PPa solution has almost no cytotoxicity, the BTZ solution and the mixed solution of PPa and BTZ show a certain cytotoxicity, and the BTZ@PPa PEG 2K Nanoparticle cytotoxicity was weaker than the mixed solution. However, after laser irradiation, BTZ@PPa PEG 2K Nanoparticle cytotoxicity was significantly enhanced, showing cytotoxicity stronger than solutions of PPa and BTZ, demonstrating nanoparticle enhanced activation and photodynamic synergistic cytotoxicity.
Example 7: immunofluorescence assay
Cells were plated in 24-well plates (10X 10) 4 Cells/well) for 12 hours, 4T1 cells were incubated with BTZ solution, PPa solution, BTZ/PPa mixed solution, BTZ@PPa nanoparticles and BTZ@PPa PEG 2K The nanoparticles were incubated under the same conditions. The BTZ and PPa concentrations were 25nM and 100nM, respectively. After 4 hours of incubation, a 660nm laser (50 mW cm -2 ) The laser-treated group was irradiated for 5 minutes. Then, the cells were further cultured for 20 hours. Untreated cells were used as controls. Then, the cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde, and then incubated with the anti-ubiquitin antibody for 12 hours in a refrigerator at 4 ℃. The anti-ubiquitin antibody was diluted with PBS solution containing 1% BSA and 0.1% Triton X-100. Thereafter, the cells were incubated with secondary antibodies for 2 hours at room temperature. Finally, cells were incubated with Hoechst 33342 for nuclear staining. Fluorescent signals of ubiquitinated proteins were observed using CLSM (CLSM, C2, nikon, japan).
As shown in fig. 8 and 9, the light-irradiated experimental group had higher fluorescence intensity than the non-light-irradiated experimental group, and the nanoparticle-treated cells of the light-irradiated group had higher intracellular fluorescence intensity than the free PPa-treated cells. Therefore, the light group has stronger proteasome inhibition effect than the non-light group, and the prepared BTZ@PPa/DSPE-PEG 2K The nanoparticle has the strongest proteasome inhibition effect under the illumination condition.
Example 8: pharmacokinetic study of nanoparticles
SD rats weighing 200-250g were randomly grouped and fasted for 12h before dosing, and were given free water. PPa solution for intravenous injection and BTZ@PPa nanoparticle and BTZ@PPa/DSPE-PEG prepared in example 1 2K The nanometer particle and PPa are administered in the dosage of 2mg/kg, and blood is taken from the eye orbit at the specified time point, and the blood plasma is obtained by separation. PPa was then extracted by sonication, centrifugation and protein precipitation, and finally the cells were analyzed for uptake of the various formulations using a varioskan lux multimode microplate reader (excitation 415nm, emission 675 nm).
The experimental results are shown in fig. 10, and PPa in the PPa solution experimental group was cleared from blood at a higher rate due to the short half-life. Compared with PPa solution, BTZ@PPa PEG 2K The circulation time of the nano particles is obviously prolonged, the AUC of PPa is obviously improved, and a good foundation is improved for the accumulation of the medicine in vivo tumor.
Example 9: tissue distribution experiment of nanoparticles
Inoculating 4T1 cell suspension into BALB/c mice when tumor volume reaches 400mm 3 At the time of tail vein injection administration: PPa solution, BTZ@PPa nanoparticle and BTZ@PPa PEG 2K Nanoparticles, and PPa administration agent for bothAfter 2mg/kg,2 hours, 4 hours, 8 hours, 12 hours and 24 hours, mice were sacrificed, and major organs (heart, liver, spleen, lung, kidney) and tumors were isolated and analyzed with a biopsy imager.
The results are shown in FIGS. 11-18, and compared with the PPa solution, BTZ@PPa PEG 2K The fluorescence intensity of the nanoparticle group in tumor tissues is obviously increased. And reached maximum accumulation at 4 hours. This result is in complete agreement with its pharmacokinetic behavior, BTZ@PPa PEG 2K Nanoparticles are most stable and circulate for the longest period of time in vivo, thus exhibiting the best tumor accumulating capacity.
Example 10: in vivo anti-tumor experiments of nanoparticles
4T1 cell suspension (5X 10) 6 Cells/100 μl) were inoculated subcutaneously on the ventral side of female mice. Until the tumor volume grows to 150mm 3 At this time, the mice were randomly grouped, and six mice per group were given physiological saline, PPa solution+laser, BTZ solution, mixed solution of BTZ and ppa+laser, btz@ppa nanoparticles prepared in example 1, and btz@ppa nanoparticles+laser, respectively, btz@ppa PEG 2K Nanoparticles and BTZ@PPa PEG 2K Nanoparticles + laser. The administration was 1 time every 1 day, 5 times in succession, and the administration dose was 10. Mu. Mol/kg calculated as PPa. The light group was irradiated with laser light 4 hours after administration, and the survival state of the mice was observed daily, which was called body weight, and the tumor volume was measured. After the last dose, mice were sacrificed 3 days apart, organs and tumors were obtained 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 fig. 19, and PPa solution showed a certain tumor inhibitory activity compared to the physiological saline group. BTZ@PPa PEG 2K Nanoparticles showed stronger antitumor activity than mixed solutions of BTZ and PPa, and tumor volume increased slowly. As expected, BTZ@PPa PEG 2K The anti-tumor effect of the nanometer particle and laser-administered treatment group is most obvious, the tumor growth is effectively inhibited, and the tumor volume even has a tendency to decrease in the later treatment period. The result shows that the nanoparticle has stability, cytotoxicity,The final anti-tumor effect is affected by both pharmacokinetics and tissue distribution.
As shown in fig. 20, the body weight of each experimental group of mice did not significantly change. As can be seen from FIGS. 21 and 22, the mice of each group had no obvious abnormality in the function of the main organs, and these results indicate that BTZ@PPa PEG 2K The nanoparticle has obvious anti-tumor effect, does not cause obvious toxicity to organisms, and is a safe and effective anticancer drug delivery system.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (5)

1. The carrier-free hybrid nano assembly is characterized by being formed by co-assembling pyropheophorbide a and bortezomib BTZ or by co-assembling pyropheophorbide a, BTZ and DSPE-PEG, wherein the molecular weight of the PEG is 500-10000, and the ratio of the total mass of the pyropheophorbide a and the BTZ to the mass of the DSPE-PEG is 10:90-90:10;
the molar ratio of BTZ to pyropheophorbide a in the carrier-free hybrid nano-assembly is 5:1-1:5;
the preparation method of the carrier-free hybrid nano assembly comprises the following steps:
BTZ and pyropheophorbide a were dissolved in tetrahydrofuran: slowly dripping ethanol into deionized water in a volume ratio of 1:1-1:5 under stirring to spontaneously form uniform nanoparticles, and removing the organic solvent to obtain a carrier-free hybrid nano assembly formed by co-assembling pyropheophorbide a and BTZ;
alternatively, BTZ, pyropheophorbide a, and DSPE-PEG were dissolved in tetrahydrofuran: and slowly dripping the mixture into deionized water under stirring in the volume ratio of ethanol of 1:1-1:5 to spontaneously form uniform nanoparticles, and removing the organic solvent to obtain the carrier-free hybrid nano assembly formed by co-assembling pyropheophorbide a, BTZ and DSPE-PEG.
2. The carrier-free hybrid nano-assembly of claim 1, wherein the method of removing the organic solvent is solvent evaporation, ultrafiltration or membrane permeation.
3. Use of the carrier-free hybrid nano-assembly of any one of claims 1-2 in the preparation of a drug delivery system.
4. Use of the carrier-free hybrid nano-assembly according to any one of claims 1-2 for the preparation of an anti-tumor drug.
5. The use according to claim 3 or 4, wherein the drug delivery system or the form of administration of the anti-neoplastic drug comprises injection, oral administration or topical administration.
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