CN113599537B - Nano aggregate and preparation method and application thereof - Google Patents
Nano aggregate and preparation method and application thereof Download PDFInfo
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- CN113599537B CN113599537B CN202110911464.8A CN202110911464A CN113599537B CN 113599537 B CN113599537 B CN 113599537B CN 202110911464 A CN202110911464 A CN 202110911464A CN 113599537 B CN113599537 B CN 113599537B
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- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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Abstract
The invention discloses a nano aggregate and a preparation method and application thereof, wherein the nano aggregate comprises CPUL1 and triphenylphosphine derivatives; respectively dissolving CPUL1 and a triphenylphosphine derivative in an organic solvent to obtain a CPUL1 solution and a triphenylphosphine derivative solution; adding deionized water into the triphenylphosphine derivative solution, stirring for the first time to obtain a mixed solution, adding the CPUL1 solution into the mixed solution, and stirring uniformly again to obtain the nano-aggregate. The nano particles of the invention have uniform particle size, can prolong the in vivo circulation period, improve the bioavailability of hydrophobic drugs, are easy to accumulate at tumor parts through the EPR effect of the tumor parts, reduce the exposure to normal tissues and better exert the anti-tumor effect.
Description
Technical Field
The invention relates to the field of medicines, in particular to a nano aggregate and a preparation method and application thereof.
Background
The incidence of malignant tumors has risen year by year in recent years, and the malignant tumors have become serious diseases threatening the life and health of human beings. At present, many malignant tumors still have no good medicine for targeted treatment, and chemotherapy is one of the most widely used cancer treatment modes in clinic at present. However, most chemotherapeutic drugs often suffer from poor water solubility and lack of selectivity or specificity, which may lead to unsatisfactory therapeutic results and serious side effects, such as low accumulation in tumor tissue, poor bioavailability, severe drug resistance, side effects on healthy tissue, and the like.
TrxR1(thioredoxin reductase 1), a seleno-enzyme that can be used to support cell growth and protect them from oxygen radicals that cause oxidative stress. TrxR1 is highly expressed in cells of various primary tumors (such as liver cancer) and is associated with poor prognosis of various tumors; the inhibition of the polypeptide can prevent tumor formation, inhibit tumor proliferation and DNA replication, and has important physiological significance for tumor occurrence and development. The structure of CPUL1 is described in patent CN201510894070.0, and the pyrano [3, 2-alpha ] phenazine derivative in the patent is CPUL 1. Studies in Liao J, Wang L, Wu Z, et al.identification of phenazine analog as a novel scaffold for thioredoxin reaction I inhibitors against Hep G2 cancer cell lines [ J ]. Journal of Enzyme Inhibition & Medicinal Chemistry,2019,34(1):1158 show that phenazine derivative CPUL1 is a TrxR1 inhibitor.
Mitochondria are considered to be one of the most important targets for the design of new drugs for cancer, cardiovascular and neurological diseases. When the potential of the in-vitro mitochondrial membrane is 180-200 mV, the inner side is negatively charged; slightly lower in living cells and organisms, and is 130-150 mV. Lipophilic cations are used for drug delivery, of which triphenylphosphine derivatives (TPP) have been the most successful. The TPP contains three benzene rings, so that the molecular surface area can be increased, a delocalized positive charge is formed, and the TPP can penetrate through a mitochondrial double-layer hydrophobic membrane. After TPP modification, many bioactive molecules show mitochondrial targeting.
In order to retain the high-efficiency anticancer activity of chemotherapeutic drugs and simultaneously reduce the toxic and side effects on other parts of the body, the chemotherapeutic drugs are usually used in combination with a nano drug-loaded delivery system. These nanocarrier-assisted drug delivery systems have established a new approach to cancer therapy and have achieved tremendous success in recent years. However, the nanocarrier assisted drug delivery system still has its inevitable disadvantages: for example, the drug loading rate of chemotherapeutic drugs is not ideal and is generally lower than 10% (w/w), so that in order to achieve drug effect, a large amount of nano-carriers need to be injected, the compliance of patients is reduced, and meanwhile most of carriers have no direct treatment effect and cause toxicity and inflammation in the degradation and metabolism process in human bodies; in addition, the drug loading capacity and degradation rate of the nanocarriers of different morphologies are uncertain, and thus, the overall therapeutic effect is unknown.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a nano aggregate with high drug loading rate and uniform particle size, a preparation method and application thereof; another object of the present invention is to provide a method for preparing a nano aggregate; the invention also aims to provide the application of the nano aggregate in the antitumor drugs.
The technical scheme is as follows: the nano aggregate comprises CPUL1 and triphenylphosphine derivatives, CPUL1 and TPP interact to form a stable nano aggregate, and the nano aggregate has the functions of self-assembly, mitochondrial targeting and cell imaging, can enable insoluble active molecules CPUL1 to be well dispersed in an aqueous solution, enables the nano aggregate to be more rapidly aggregated in tumor cell mitochondria by the TPP, can more rapidly release effective drug molecules at a pathological change part, and achieves targeting.
Further, the nano-aggregate is spherical, the particle size of the nano-aggregate is in a nano level, and the particle size is 5-500nm, preferably 90-250 nm.
The preparation method of the nano aggregate comprises the following steps:
(1) respectively dissolving CPUL1 and a triphenylphosphine derivative in an organic solvent to obtain a CPUL1 solution and a triphenylphosphine derivative solution;
(2) and (2) adding deionized water into the triphenylphosphine derivative solution, stirring for the first time to obtain a mixed solution, adding the CPUL1 solution obtained in the step (1) into the mixed solution, and stirring uniformly again to obtain the nano aggregate.
Further, in the step (1), the molar concentration ratio of the CPUL1 solution to the triphenylphosphine derivative solution is 1-100: 1-100.
Further, in the step (1), the organic solvent is any one of ethyl acetate, acetone, methanol, dichloromethane, chloroform, ethyl propionate, propyl acetate, dimethyl sulfoxide or ethanol.
Further, in the step (2), the primary stirring and/or the secondary stirring is magnetic stirring.
Further, in the step (2), the primary stirring temperature is 20-80 ℃, and the stirring speed is 50-2000 rpm.
Further, in the step (2), the stirring temperature is 25-60 ℃, the stirring speed is 100-2500rpm, and the stirring time is 0.5-24 h.
Further, in the step (2), the molar concentration of CPUL1 and/or TPP in the nano-aggregate is 0.16-16 mM/L.
The application of the nano aggregate in tumor treatment medicines. The particle size of the nano aggregate provided by the invention is in a nano level, and the in vivo circulation period can be prolonged. The nanoparticles have smaller particle size, are easy to accumulate at tumor parts through the EPR effect of the tumor parts, reduce the exposure to normal tissues and better exert the anti-tumor effect.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) the drug loading rate reaches 100%, and other carrier materials are not contained in the nano aggregate, so that the problem that a large amount of injection is needed due to low drug loading rate and additional toxicity possibly brought by the carrier materials are avoided;
(2) the nano particles with uniform particle size can prolong the in vivo circulation period, improve the bioavailability of hydrophobic drugs, and are easy to accumulate at tumor parts through the EPR effect of the tumor parts, thereby reducing the exposure to normal tissues and better exerting the anti-tumor effect;
(3) the nano aggregate has targeting property, and compared with single medicine, the nano aggregate improves the anti-tumor effect of active molecules, emits obvious fluorescence in cells and greatly reduces the toxic and side effect of the medicine on normal tissues;
(4) the preparation method of the nano-drug is simple and easy to implement, can save cost, and is safe and pollution-free.
Drawings
FIG. 1 is a transmission electron microscope image of nanoaggregates of example 1 of the invention;
FIG. 2 is a transmission electron microscope image of CPUL1 of example 1 of the present invention;
FIG. 3 is a dynamic light scattering test chart of nanoaggregates of example 1 of the present invention;
FIG. 4 is a dynamic light scattering test chart of CPUL1 of example 1 of the present invention;
FIG. 5 is a hydrated particle size distribution plot of nanoaggregates of example 2 of the invention;
FIG. 6 is a Zeta potential diagram of nanoaggregates of example 2 of the invention;
FIG. 7 is a hydrated particle size distribution plot of nanoaggregates of example 3 of the invention;
FIG. 8 is a Zeta potential diagram of nanoaggregates of example 3 of the invention;
FIG. 9 is a graph of hydrated particle size stability of nano-aggregates of example 3 of the present invention;
FIG. 10 is a hydrated particle size distribution plot of nanoaggregates of example 4 of the invention;
FIG. 11 is a Zeta potential diagram of nanoaggregates of example 4 of the invention;
FIG. 12 is a hydrated particle size distribution plot of nanoaggregates of example 5 of the invention;
FIG. 13 is a Zeta potential diagram of nanoaggregates of example 5 of the invention;
FIG. 14 is a heating curve of a TPP solution titrating CPUL1 solution as determined by isothermal microcalorimetry of a nanoaggregate solution of example 6 of the invention;
FIG. 15A is a graph of the results of the killing of HUH7 cells by CPUL1 and TPP of example 7;
FIG. 15B is a graph showing the killing effect of the nano-aggregates with different ratios on HUH7 cells;
FIG. 16A is a plot of free CPUL1 and CPUL1-TPP nanoaggregates provided in example 8 as a function of time for HUH7 cell fluorescence intensity;
FIG. 16B is a flow cytometer comparison of fluorescence intensity of free CPUL1 and CPUL1-TPP nanoaggregates in HUH7 cells at the same time point in example 8;
FIG. 17 is a flow cytometer used to measure the rate of apoptosis of HUH7 cells induced by CPUL1, CPUL1-TPP nanoaggregates at different concentrations (Q1, dead cells; Q2, late apoptotic or necrotic cells; Q3, early apoptotic cells; Q4, live cells) in example 9;
FIG. 18 is a photograph of the localization of mitochondria of CPUL1, CPUL1-TPP nanoaggregates at different time points in HUH7 cells as measured by confocal laser microscopy in example 10.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
The nano-aggregate provided by the embodiment comprises CPUL1 and TPP, and the hydrated particle size of the nano-aggregate is 104 nm. The preparation method comprises the following steps:
(1) dissolving 4.34mg of CPUL1 in 100. mu.L of dimethyl sulfoxide to obtain solution A with CPUL1 molar concentration of 100 mM/L; dissolving 4.43mg of TPP in 100 mu L of dimethyl sulfoxide to obtain a solution B with the molar concentration of TPP of 100 mM/L;
(2) injecting 100 mu L of the solution B into 4.14mL of deionized water, and magnetically stirring at the rotating speed of 50rpm at 20 ℃ to obtain a solution C; and (3) injecting 100 mu L of the solution A into the solution C, mixing to obtain a solution D, and magnetically stirring at the rotation speed of 50rpm at 20 ℃ for 0.5h to obtain the nano aggregate.
Observing the nano-aggregate provided by the embodiment through a transmission electron microscope, wherein the particle size of the nano-aggregate refers to the particle size of the drug in a powder state after the nano-drug is dried, and the particle size measured under the transmission electron microscope is the particle size of the nano-drug generally; the hydrated particle size of the nano-drug refers to the particle size of a hydrate formed by dispersing the nano-drug in water, and the particle size is generally measured by dynamic light scattering. As shown in fig. 1-4, the Dynamic Light Scattering (DLS) test patterns of the nano-aggregates and the free CPUL1 are consistent with the transmission electron microscope results, and the nano-aggregates provided by this embodiment have small particle size, are at the nano level, and have uniform particle size.
Example 2
The nano-aggregate provided by the embodiment comprises CPUL1 and TPP, and the hydrated particle size of the nano-aggregate is 120 nm. The preparation method comprises the following steps:
(1) dissolving 4.34mg of CPUL1 in 100. mu.L of dimethyl sulfoxide to obtain solution A with CPUL1 molar concentration of 100 mM/L; dissolving 2.22mg of TPP in 100 mu L of dimethyl sulfoxide to obtain a solution B with the molar concentration of TPP being 50 mM/L;
(2) injecting 100 mu L of the solution B into 4.14mL of deionized water, and magnetically stirring at the rotating speed of 150rpm at the temperature of 20 ℃ to obtain a solution C; injecting 100 mu L of the solution A into the solution C obtained in the step (3) for mixing to obtain a solution D, and magnetically stirring at the rotating speed of 150rpm at 25 ℃ for 1h to obtain a nano aggregate;
the synthesized nano-aggregates were detected by DLS, and as shown in fig. 5, CPUL1 and TPP formed nanoparticles having a uniform particle size. As shown in fig. 6, the Zeta potential of the nano-aggregate was 4.7 mV.
Example 3
The nano-aggregate provided by the embodiment comprises CPUL1 and TPP, and the hydrated particle size of the nano-aggregate is 136 nm. The preparation method comprises the following steps:
(1) dissolving 4.34mg of CPUL1 in 100. mu.L of dimethyl sulfoxide to obtain solution A with CPUL1 molar concentration of 100 mM/L; dissolving 3.55mg of TPP in 100 mu L of dimethyl sulfoxide to obtain a solution B with the molar concentration of TPP being 80 mM/L;
(2) injecting 100 mu L of the solution B into 4.14mL of deionized water, and magnetically stirring at the rotating speed of 400rpm at the temperature of 30 ℃ to obtain a solution C; injecting 100 mu L of the solution A into the solution C for mixing to obtain a solution D, and magnetically stirring at the rotating speed of 400rpm at the temperature of 30 ℃ for 2h to obtain a nano aggregate;
the synthesized nano-aggregates were examined by DLS, as shown in fig. 7, CPUL1 and TPP formed nanoparticles having a uniform particle size, and fig. 8 is a Zeta potential diagram of the nano-aggregates. The nano-drug provided in this example was then subjected to a stability test, which tested the hydrated particle size of the nano-aggregates provided in this example during placement. As shown in fig. 9, the nano-aggregates remained stable hydrated particle size over 10 days of standing.
Example 4
The nano-aggregate provided by the embodiment comprises CPUL1 and TPP, and the hydrated particle size of the nano-aggregate is 208 nm. The preparation method comprises the following steps:
(1) dissolving 4.34mg of CPUL1 in 100. mu.L of dimethyl sulfoxide to obtain solution A with CPUL1 molar concentration of 100 mM/L; dissolving 6.65mg of TPP in 100 mu L of dimethyl sulfoxide to obtain a solution B with the molar concentration of TPP being 150 mM/L;
(2) injecting 100 mu L of the solution B into 4.14mL of deionized water, and magnetically stirring at 35 ℃ and 500rpm to obtain a solution C; injecting 100 mu L of the solution A into the solution C, mixing to obtain a solution D, and magnetically stirring at the rotating speed of 500rpm at 35 ℃ for 3h to obtain a nano aggregate;
the synthesized nano-aggregates were examined by DLS, as shown in fig. 10, CPUL1 and TPP formed nanoparticles having a uniform particle size, and fig. 11 is a Zeta potential diagram of the nano-aggregates, showing that the Zeta potential was 41.35 mV.
Example 5
The nano-aggregate provided by the embodiment comprises CPUL1 and TPP, and the hydrated particle size of the nano-aggregate is 208 nm. The preparation method comprises the following steps:
(1) dissolving 4.34mg of CPUL1 in 100. mu.L of dimethyl sulfoxide to obtain solution A with CPUL1 molar concentration of 100 mM/L; dissolving 8.87mg of TPP in 100 mu L of dimethyl sulfoxide to obtain a solution B with the molar concentration of TPP being 200 mM/L;
(2) injecting 100 mu L of the solution B into 4.14mL of deionized water, and magnetically stirring at 40 ℃ and 700rpm to obtain a solution C; injecting 100 mu L of the solution A into the solution C obtained in the step (3) for mixing to obtain a solution D, and carrying out magnetic stirring at the rotating speed of 700rpm at 40 ℃ for 3h to obtain a nano aggregate;
the synthesized nano-aggregates were detected by DLS, as shown in fig. 12, CPUL1 and TPP formed nanoparticles having a uniform particle size, and as shown in fig. 13, the Zeta potential of the nano-aggregates was 46.31 mV.
Example 6
The thermodynamic mechanism was studied by using MicroCal ITC200 (Malven). A0.4 mM aqueous solution of CPUL1 was added to the cuvette. The syringe was filled with 2mM TPP in water. The reference cell contained deionized water. To avoid air bubbles during the titration, all samples were degassed thoroughly for 30 minutes. All experiments were performed at 25 ℃. The TPP solution was injected into the cuvette 20 times, 2.5. mu.L each, with each injection giving rise to a peak on the isotherm. The first data point is ignored due to inaccuracies. The speed of the injector was 250 rpm. Raw data is obtained to generate an energy curve. Ensure complete consistency of the solution in CPUL1 and TPP systems to eliminate the influence of solvent.
As shown in fig. 14, the titration of TPP into CPUL1 gives off a lot of heat. By fitting titration curves, the thermodynamic parameters (Δ S, Δ H, Δ G, Δ H, and Δ L) of TPP and CPUL1 were obtained,n, Ka and Kd). Ka 1.7X 103Δ G ═ 36.5KJ, indicating that the reaction of TPP with CPUL1 is spontaneous and may be explained by the interaction between oppositely charged molecules. A negative Δ S indicates a reduced uniformity of the system energy, which may be due to hydrophobic interaction driven ordered self-assembly. The fitted stoichiometric ratio (n) was 1.092, indicating a binding ratio of TPP to CPUL1 of 1:1, which is direct evidence that CPUL1 and TPP derivatives can self-assemble in water to form nanoparticles.
Example 7
In this example, the effect of the nano-drug provided in example 1 on the cell viability was examined by performing MTT colorimetric assay on HUH7 cells as follows:
(1) cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum and cultured in an incubator at 37 ℃ with 5% carbon dioxide.
(2) Cell viability assay: the cytotoxic effect of CPUL1 and CPUL1-TPP nano-aggregates on HUH7 cells is detected by a tetramethyl azoazolate (MTT) colorimetric method. 6000 cells/well were seeded in 96-well plates and after 12h incubation, 10 μ L of CPUL1-TPP nanoaggregate samples at different concentrations (2.5, 5, 10, 20, 40 μ M) were added. After 48h of incubation, 10. mu.L of MTT (5.0mg/mL) was added. After incubation for 4h, the medium was removed and 150. mu.L of dimethyl sulfoxide was added to dissolve the purple crystals. The absorbance of the solution at 490nm was measured with a multifunctional microplate reader. Cells cultured without any treatment were used as a control. Each set is provided with 3 parallel multiple holes.
As shown in fig. 15A and 15B, compared with the control group, the nano-aggregate prepared in example 1 has a significant effect on the viability of HUH7 cells, and compared with the CPUL1 treatment alone and the TPP treatment alone, the inhibition rate of the CPUL1-TPP nano-aggregate provided by the invention on HUH7 cells is the highest, which indicates that the self-assembled nano-aggregate prepared by the method can achieve a better tumor inhibition effect at a lower drug concentration.
Example 8
In this example, the endocytosis of the CPUL1-TPP nanoaggregates provided in example 1 was observed by flow cytometry observation of HUH7 cells as follows:
the cultured cells were inoculated into 12-well plates, 10 ten thousand cells per well, 1mL of DMEM liquid medium (containing 10% fetal bovine serum) was added thereto, and the mixture was cultured in a 5% carbon dioxide incubator at 37 ℃ for 24 hours. HUH7 cells were treated with CPUL1 and the presented nanoaggregates for 1h, 2h, and 4h, respectively, with drug concentrations of CPUL1 of 2.5. mu.M, 5. mu.M, and 10. mu.M, respectively. After 4h, three washes with PBS buffer, fixation with 4% paraformaldehyde, 3 washes with PBS, and then staining of nuclei with DAPI. And (5) acquiring a cell fluorescence image under a fluorescence microscope. The flow cytometer further measured the amount of intracellular accumulation of free CPUL 1. Under the same other conditions, the uptake of CPUL1-TPP nano-aggregates into cells is also studied, and the fluorescence intensity of the cells is analyzed.
As shown in fig. 16, the fluorescence intensity of HUH7 cells treated with the provided nano-aggregates would be higher than the fluorescence intensity of cells treated with CPUL1 alone. It is demonstrated that the nanoaggregates provided by the present invention are more taken up by HUH7 cells than CPUL1 at the same concentration. The more the taken medicine amount is, the more the medicine with the effect is, the more the tumor cell killing effect is, and the purpose of resisting the tumor is achieved.
Example 9
In this example, nanoaggregates provided by the present invention were tested by killing HUH7 cells with CPUL1 and the set of CPUL1-TPP nanoaggregates provided in example 1 by the following method:
(1) and (3) cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum and cultured in an incubator at 37 ℃ with 5% carbon dioxide.
(2) Observation of cell activity: inoculating the cells cultured in the step (1) into a 12-well plate, adding 1mL of DMEM liquid culture medium (containing 10% fetal calf serum) into each 10 ten thousand cells, placing the cells in a 5% carbon dioxide incubator at 37 ℃ for 12h, and dividing the cells into 3 groups, namely a blank control group, a CPUL1 group and a provided CPUL1-TPP nano aggregate group. Then adding CPUL1 and CPUL1-TPP nano aggregates with the concentration of 2.5, 5 and 10 mu M respectively into a newly configured DMEM medium containing 10% fetal calf serum, placing the DMEM medium in a 5% carbon dioxide incubator at 37 ℃ for 12h, and not performing any treatment on a blank control group. Discarding the culture medium, digesting the cells by trypsin without EDTA, centrifuging at 2000rpm for 5min, washing the cells twice by PBS, and staining by Annexin V-APC/7-AAD apoptosis kit; in normal cells, phosphatidylserine is distributed only inside the lipid bilayer of the cell membrane, whereas in the early stages of apoptosis, phosphatidylserine of the cell membrane is turned outside from inside the lipid membrane. Annexin V is a protein that relies on phospholipid binding and has a high affinity for phosphatidylserine, and thus can bind to the cell membrane of early apoptotic cells through the extracellular exposure of phosphatidylserine. Therefore, Annexin V is taken as one of sensitive indicators for detecting early apoptosis of cells. 7-AAD is a nucleic acid dye which can not pass through a normal plasma membrane, the permeability of the plasma membrane to the 7-AAD is increased along with the processes of apoptosis and cell death, and the dye can emit bright red light under the excitation of proper wavelength by combining the controlled degradation of DNA in the process of apoptosis. Therefore, using Annexin V in combination with 7-AAD allows differentiation between cells at different apoptotic stages). And then detected by flow cytometry. Ten thousand cells were collected and partitioned, and the percentage of cells in each region was counted. The manner of cell death was analyzed.
As shown in fig. 17, the nano-aggregates of the present invention can cause apoptosis and necrosis, and compared with the CPUL1 group alone, the nano-aggregates of the present invention have the highest apoptosis rate on HUH7 cells, which indicates that the nano-aggregates synthesized by the method of the present invention can achieve better tumor suppression effect with lower drug concentration.
Example 10
In this example, the nanoaggregates provided by the present invention were tested by targeting CPUL1 and the CPUL1-TPP nanoaggregate group provided in example 1 to the mitochondria of HUH7 cells as follows:
(1) cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum and cultured in an incubator at 37 ℃ with 5% carbon dioxide.
(2) And (3) observing targeted mitochondria of the nano aggregates: inoculating the cells cultured in the step (1) into a 12-hole plate, adding 1mL of DMEM liquid culture medium containing 10% fetal bovine serum into each hole of 10 ten thousand cells, placing the DMEM liquid culture medium in a 5% carbon dioxide incubator at 37 ℃ for 12h, and dividing the DMEM liquid culture medium into 3 groups, namely a blank control group, a CPUL1 group and a provided CPUL1-TPP nano aggregate group. Then adding 2.5 mu M CPUL1 and CPUL1-TPP nano aggregates into a newly configured DMEM medium containing 10% fetal calf serum respectively, placing the DMEM medium and the CPUL1-TPP nano aggregates into a 5% carbon dioxide incubator at 37 ℃ for culturing for 2h and 4h, and not performing any treatment on a blank control group. Mitochondria of HUH7 cells were stained for detection using the commercial dye MitoTracker Red. After 2h or 4h incubation of the nanoaggregates and 30min dye treatment, localization of mitochondria within the cells was examined with a laser confocal microscope (CLSM). The green fluorescence of CPUL1 itself overlaps with the red fluorescence of the dye to produce yellow fluorescence. The degree of mitochondrial co-localization is expressed as Pearson correlation coefficient.
As shown in fig. 18, the CPUL1-TPP nanoaggregates and free CPUL1 of the present invention can co-localize with dye in mitochondria of HUH7 cells at 2h or 4h, and the fluorescence intensity quantitative profile randomly drawn along white line further confirms that the CPUL1-TPP nanoaggregates target better to mitochondria than the free CPUL 1. The Pearson correlation coefficient of the nanoaggregates incubated for 2h was 0.76, which is higher than 0.65 of the free CPUL1 incubated for 2h, consistent with the results of 4h incubation, and at the same time point, the co-localization fluorescence intensity of the nanoaggregates was significantly higher than that of the free CPUL 1. These results indicate that CPUL1-TPP nanoaggregates have good mitochondrial targeting ability and rapid time-dependent cellular uptake characteristics.
The nano aggregate is a nano particle obtained by co-assembling a hydrophobic drug and a targeting agent. In the co-assembled nano-drug, the ratio of the hydrophobic drug to the targeting agent is adjustable. Therefore, better synergistic effect can be realized by adjusting the proportion of the medicines, and the utilization rate of the medicines is improved. The nano particles can be spherical, the particle size of the nano particles can be adjusted to be 5-500nm, and the nano particles can be highly dispersed.
Claims (10)
1. A nanoaggregate comprising CPUL1 and triphenylphosphine.
2. The nano-aggregate according to claim 1, wherein the nano-aggregate has a spherical morphology and a particle size of 5 to 500 nm.
3. The method for preparing nanoaggregates according to any of claims 1-2, characterized in that it comprises the following steps: (1) respectively dissolving CPUL1 and triphenylphosphine in an organic solvent to obtain a CPUL1 solution and a triphenylphosphine solution; (2) and (2) adding deionized water into the triphenylphosphine solution, stirring for the first time to obtain a mixed solution, adding the CPUL1 solution obtained in the step (1) into the mixed solution, and stirring uniformly again to obtain the nano aggregate.
4. The method for preparing nano-aggregates according to claim 3, wherein in the step (1), the molar concentration ratio of the CPUL1 solution to the triphenylphosphine solution is 1-100: 1-100.
5. The method for preparing nano-aggregates according to claim 3, wherein in the step (1), the organic solvent is any one of ethyl acetate, acetone, methanol, dichloromethane, chloroform, ethyl propionate, propyl acetate, dimethyl sulfoxide or ethanol.
6. The method for producing a nano aggregate according to claim 3, wherein in the step (2), the primary stirring and/or the secondary stirring is magnetic stirring.
7. The method for preparing a nano aggregate according to claim 6, wherein in the step (2), the primary stirring temperature is 20 to 80 ℃ and the stirring speed is 50 to 2000 rpm.
8. The method for preparing nano aggregates according to claim 6, wherein in the step (2), the re-stirring temperature is 25-60 ℃, the stirring speed is 100-2500rpm, and the stirring time is 0.5-24 h.
9. The method for preparing nano-aggregates according to claim 6, wherein in the step (2), the molar concentration of CPUL1 and/or triphenylphosphine in the nano-aggregates is 0.16-16 mM/L.
10. Use of the nanoaggregates according to any of claims 1-2 for the preparation of a medicament for the treatment of tumors.
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