CN115919766B - Composite nano micelle and preparation method and application thereof - Google Patents
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Abstract
The application provides a composite nano micelle and a preparation method and application thereof, and relates to the technical field of biological medicine. The preparation method comprises the following steps: dissolving self-assembled amphiphilic peptide in water, adding sound-sensitive substances and anti-tumor small molecules, uniformly mixing, and performing ultrasonic cleaning to obtain amphiphilic peptide nano-micelle; transferring the amphiphilic peptide nano micelle into a dialysis bag, dialyzing in ultrapure water, and finally obtaining the composite nano micelle. The carrier of the composite nano micelle is a functional peptide with both an amphiphilic group and an endothelial cell penetrating peptide fragment, and can greatly enhance the specificity of aggregated tumor cells by combining RGD with integrin alpha v beta 3 over-expressed on the surface of brain glioma. The functional peptide is coated with the sound sensitizer of Bengalhong and the natural micromolecular sulforaphane for resisting the glioma, so that the Bengalhong and the natural micromolecular sulforaphane can be precisely targeted to the glioma part, penetrate through the blood brain barrier, implement the sound power therapy mediated by RB and the synergistic tumor killing of SFN enriched to the glioma part, and realize the sensitization glioma treatment.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a composite nano micelle and a preparation method and application thereof.
Background
Gliomas (Gliomas) are the most common primary tumors of the central nervous system, account for about 40-50% of the tumors of the central nervous system, have about 3-8 cases/10 ten thousand people of incidence per year, and have the characteristics of difficult healing, high recurrence, high mortality rate and the like. The world health organization reports about 50 ten thousand new glioma cases worldwide each year, with about 2 ten thousand death cases. Due to its high aggressiveness and recurrence rate, the average survival of patients is about 1-1.5 years, with a proportion of survival exceeding 5 years of less than 5%. At present, the treatment means of the glioma is mainly surgical excision, radiotherapy and chemotherapy. Because the glioma has the characteristics of self-renewing and rapid proliferation of tumor stem cells, normal brain tissues can be affected, and conventional surgical excision is assisted with radiotherapy and chemotherapy to stimulate the rapid proliferation of tumor cells in a dormant period, so that tumor recurrence is caused. In addition, most of chemotherapeutics cannot effectively enter brain tissues due to Blood-brain barrier (BBB), so that the chemoradiotherapy effect of brain glioma is limited. Therefore, how to improve the therapeutic effect of the glioma and reduce the recurrence rate is still a great problem to be solved in clinical treatment of glioma. The blood brain barrier is composed of brain capillary wall and glial cells, is a natural blood brain barrier between blood plasma and brain cells, and only allows the passage of fat-soluble substances with molecular weight less than 400 Da. The presence of the blood-brain barrier isolates about 98% of small molecules and 100% of drug molecules of potential therapeutic utility from the brain parenchyma, the primary bottleneck in the treatment of central nervous system disorders. In order to deliver therapeutic drug molecules into the brain, many studies have explored various methods for increasing the permeability of the blood brain barrier, including ligand receptor binding, formulation engineering, administration of hypertonic drugs via arterial injection, direct intra-brain injection or infusion, and nasal administration, which, although effective, are somewhat invasive and still have low delivery efficiency. In recent years, researchers have attempted to promote drug delivery by opening the blood brain barrier through various means, such as physical, chemical and biological, for example, laser interstitial thermotherapy as a non-invasive treatment for glioma can open the blood brain barrier for 40 days. However, there is a risk that such treatment means, such as heterogeneity of brain tissue and local brain perfusion, may affect heat conduction, laser may damage peripheral functional areas of tumor, etc. Ultrasound, in contrast to lasers, is more widely used for the treatment of brain tumors due to its penetration and safety into dense brain tissue. Repeated ultrasound scans, such as Leinenga et al, were found to transiently open the blood brain barrier and clear beta-amyloid and restore memory and cognitive function in mice models of alzheimer's disease. This discovery provides a non-invasive, safe method for ultrasound to open the blood brain barrier for drug delivery. Accordingly, researchers have focused on developing a new method of treating glioma with ultrasound assistance. One of the techniques is sonodynamic therapy (SDT), a strategy for co-treating diseases with ultrasound in combination with sonosensitizers.
Sonodynamic therapy is a method of treating tumors by combining ultrasound with sonosensitizers. On one hand, focused ultrasound can destroy cells by using the mechanical action of acoustic energy, and on the other hand, the ultrasonic can use the strong penetrating capacity of the ultrasound to biological tissues to activate the sound-sensitive agent of deep tumor tissues at fixed points to generate substances such as active oxygen and the like so as to mediate apoptosis of tumor cells. Various sound-sensitive substances such as 5-ALA, photolon, photofrin and rose bengal, etc., exhibit remarkable tumor killing effect under the action of ultrasound. However, conventional sonosensitizers have some problems: (1) Most sound sensitive agents are hydrophobic small molecules, and have low water solubility; (2) Can not specifically target tumor cells, and has low bioavailability.
Disclosure of Invention
The application aims to provide a composite nano micelle which has the advantages of small particle size, high biocompatibility and high compatibility.
The application also aims at providing a preparation method of the composite nano micelle, which is simple and convenient.
The application also aims at providing an application of the composite nano micelle in preparing a medicine for treating brain glioma.
The application solves the technical problems by adopting the following technical scheme.
In one aspect, the embodiment of the application provides a preparation method of a composite nano micelle, which comprises the following steps:
dissolving self-assembled amphiphilic peptide in water, adding sound-sensitive substances and anti-tumor small molecules, uniformly mixing, and performing ultrasonic cleaning to obtain amphiphilic peptide nano-micelle;
transferring the amphiphilic peptide nano micelle into a dialysis bag, dialyzing in ultrapure water, and finally obtaining the composite nano micelle.
On the other hand, the embodiment of the application provides a composite nano micelle prepared by the preparation method.
In still another aspect, the embodiment of the application also provides an application of the composite nano micelle in preparing a medicine for treating brain glioma.
The application has two functions of specifically killing tumor cells:
(1) Specificity of chemical mechanism: the self-assembled peptide micelle specifically binds with an alpha v beta 3 receptor on the surface of a tumor cell through RGD and enters the cell, and then the coupled SFN induces the tumor cell to generate excessive active oxygen, so that autophagic cell death is induced.
(2) Physical mechanism specificity: in the acoustic power treatment, the focused ultrasonic wave activates the acoustic sensitizer RB to induce the intracellular generation of active oxygen, so that the apoptosis induced by the active oxygen is limited to tumor cells as much as possible from the spatial position. In conclusion, the designed polypeptide self-assembled micelle can achieve accurate targeting drug delivery to tumor tissues and induce apoptosis of tumor cells through specific organic combination of physiological, chemical and physical mechanisms.
Compared with the prior art, the embodiment of the application has at least the following advantages or beneficial effects:
1. the application designs a nano micelle with high biocompatibility and compatibility, the entrapped natural anti-glioma drug molecule natural sulforaphane SFN and the sound sensitizer manglared RB, which pass through the blood brain barrier efficiently by means of ultrasonic means and utilize the targeting glioma characteristics of RGD (RGD and the integrin alpha over-expressed on the glioma surface) v β 3 Combined), plays the synergistic tumor killing effect of the RB-mediated sonodynamic therapy and the SFN enriched to the glioma part, and realizes the sensitization glioma treatment. In the application, chemical modification and physical technology are combined, so that the sulforaphane exerts its biological effect in tumor cells, and the accurate treatment of glioma is realized.
2. The blood brain barrier is a key bottleneck problem that hinders efficient drug delivery into the brain, and nanomaterial drug delivery is considered as a breakthrough in diagnosis and treatment of neurological diseases. The size of the nano material is a key factor affecting the entrance of the nano material into the brain, and the particle size of the nano micelle is as small as 40nm, so that the efficiency of crossing blood brain barriers can be greatly improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a morphological characterization of a composite nano-micelle in experimental examples of the application;
FIG. 2 is an ultraviolet analysis chart and an HPLC chart of the composite nano-micelle of the experimental example of the application;
FIG. 3 is a graph showing comparison of cell viability assays of the experimental example U87MG of the present application;
FIG. 4 is a graph showing apoptosis detection in experimental examples of the present application;
FIG. 5 is an evaluation chart of the acoustic power treatment of in situ brain glioma-bearing mice based on functional nanomicelle in experimental examples of the application;
FIG. 6 is a structural formula of a self-assembled amphiphilic peptide according to an embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to specific examples.
The preparation method of the composite nano micelle comprises the following steps:
dissolving self-assembled amphiphilic peptide in water, adding sound-sensitive substances and anti-tumor small molecules, uniformly mixing, and performing ultrasonic cleaning to obtain amphiphilic peptide nano-micelle;
transferring the amphiphilic peptide nano micelle into a dialysis bag, dialyzing in ultrapure water, and finally obtaining the composite nano micelle.
The application prepares the sound sensitive agent into the nano compound by utilizing the nano technology, realizes the fixed point release of the medicine by ultrasonic triggering, and realizes the multifunctional integrated treatment modes such as tumor treatment and the like. Self-assembled peptides can overcome the disadvantages of being unstable in free form and being readily cleared by enzymes, which have the following advantages: (1) extending the circulation time of the sonosensitizer in the body; (2) Improving the in-vivo distribution of the medicine and the effective concentration of the sound sensitive agent at the tumor part; (3) The surface of the nano-composite can be modified with ligand molecules targeting tumors, so that active transportation of the sound sensitive agent is realized.
In some embodiments of the present application, the hydrophobic end group of the self-assembled amphiphilic peptide is C 18 The hydrophilic end group is targeted integration alpha v beta 3 ligand RGDS, the connecting bridge chain is R8, the structural formula is shown in figure 6, and the structural formula is as follows: (C18) GRRRRRRRRGDS the self-assembled amphiphilic peptide is a functional peptide with amphiphilicity and endothelial cell penetrating fragments, RGD is used as a hydrophilic group, C18 is used as a hydrophobic group and is connected through penetrating peptide R8 to self-assemble into nano materials, and the specificity of aggregated tumor cells can be greatly enhanced by combining RGD with various malignant tumor cell surface αvβ3 receptors. Therefore, the self-assembled nano material is applied to cross-barrier delivery of ultrasound-triggered fixed-point release drugs or sound-sensitive agents to brain tumor tissues, and has important research and application values for thoroughly eradicating brain glioma.
In some embodiments of the application, the sound-sensitive substance is Bengalhon, and the anti-tumor small molecule is small molecule sulforaphane. The sound-sensitive substance has better active oxygen generating capacity, and is especially activated only in tumor microenvironment. In addition, natural product Sulforaphane (SFN) is derived from cruciferous plants (broccoli, etc.), and has been widely reported to have strong inhibitory or killing effects on different types of tumor cells, such as ovarian cancer, oral cancer, prostate cancer, glioma, etc. The functional group-n=c=s in SFN is believed to have antioxidant and anticancer activity. Therefore, the SFN is transported to the tumor part through the self-assembled nano material in a targeted way, and has very important application value for enhancing the treatment of brain glioma.
In some embodiments of the application, the mass ratio of the self-assembled amphiphilic peptide, the sound sensitive substance and the anti-tumor small molecule is 2: (1-2): (1-2).
In some embodiments of the application, the ultrasonic cleaning is performed at a temperature of 22-26℃for 20-40min and at a power of 25-30KHZ.
In some embodiments of the application, the dialysis bag has a molecular weight cut-off of 800-1200 daltons, the ultrapure water is changed every 4-6 hours, and the dialysis time is 36-60 hours.
The composite nano micelle is prepared by the preparation method.
An application of composite nano micelle in preparing medicine for treating brain glioma.
The features and capabilities of the present application are described in further detail below in connection with the examples.
Example 1
The composite nano micelle is prepared by the following method:
in C 18 As a hydrophobic end group, a hydrophilic end group adopts targeted integration of an alpha v beta 3 ligand RGDS, a connecting bridge chain adopts a membrane penetrating peptide R8, and self-assembled amphiphilic peptide is obtained through synthesis; the structural formula of the amphiphilic peptide adopted in the embodiment is designed by the inventor and synthesized by Shanghai blaze biotechnology Co.
Dissolving self-assembled amphiphilic peptide in water, and adding sound-sensitive substances of Bengalhong (RB) and natural anti-tumor micromolecular Sulforaphane (SFN), wherein the mass ratio of the three is 2:1:1, uniformly mixing, placing in an ultrasonic cleaner in a dark place, and setting instrument parameters as follows: the temperature is 25 ℃, the time is 30min, the power is 28KHZ, and the amphiphilic peptide nano-micelle is obtained after ultrasonic cleaning;
transferring the amphiphilic peptide nano micelle into a dialysis bag with the molecular weight cutoff of 1000 daltons, putting into a beaker filled with ultrapure water for dialysis, replacing the ultrapure water every 4 hours, and dialyzing for 48 hours to obtain the composite nano micelle of the entrapment RB and SFN of the embodiment.
Example 2
The composite nano micelle is prepared by the following method:
in C 18 As a hydrophobic end group, a hydrophilic end group adopts targeted integration of an alpha v beta 3 ligand RGDS, and a connecting bridge chain adopts a membrane penetrating peptide R8 to obtain self-assembled amphiphilic peptide;
dissolving self-assembled amphiphilic peptide in water, and adding sound-sensitive substances of Bengalhong (RB) and natural anti-tumor micromolecular Sulforaphane (SFN), wherein the mass ratio of the three is 2:1:2, uniformly mixing, placing in an ultrasonic cleaner in a dark place, and setting instrument parameters as follows: the temperature is 26 ℃, the time is 25min, the power is 25KHZ, and the amphiphilic peptide nano-micelle is obtained after ultrasonic cleaning;
transferring the amphiphilic peptide nano micelle into a dialysis bag with the molecular weight cutoff of 1100 daltons, putting into a beaker filled with ultrapure water for dialysis, replacing the ultrapure water every 4 hours, and dialyzing for 45 hours to obtain the composite nano micelle of the entrapment RB and SFN of the embodiment.
Example 3
The composite nano micelle is prepared by the following method:
in C 18 As a hydrophobic end group, a hydrophilic end group adopts targeted integration of an alpha v beta 3 ligand RGDS, and a connecting bridge chain adopts a membrane penetrating peptide R8 to obtain self-assembled amphiphilic peptide;
dissolving self-assembled amphiphilic peptide in water, and adding sound-sensitive substances of Bengalhong (RB) and natural anti-tumor micromolecular Sulforaphane (SFN), wherein the mass ratio of the three is 2:2:1, uniformly mixing, placing in an ultrasonic cleaner in a dark place, and setting instrument parameters as follows: the temperature is 22 ℃, the time is 30min, the power is 30KHZ, and the amphiphilic peptide nano-micelle is obtained after ultrasonic cleaning;
transferring the amphiphilic peptide nano micelle into a dialysis bag with the molecular weight cutoff of 900 daltons, putting into a beaker filled with ultrapure water for dialysis, replacing the ultrapure water every 4 hours, and dialyzing for 50 hours to obtain the composite nano micelle of the entrapment RB and SFN of the embodiment.
Example 4
The composite nano micelle is prepared by the following method:
in C 18 As a hydrophobic end group, a hydrophilic end group adopts targeted integration of an alpha v beta 3 ligand RGDS, and a connecting bridge chain adopts a membrane penetrating peptide R8 to obtain self-assembled amphiphilic peptide;
dissolving self-assembled amphiphilic peptide in water, and adding sound-sensitive substances of Bengalhong (RB) and natural anti-tumor micromolecular Sulforaphane (SFN), wherein the mass ratio of the three is 2:2:1, uniformly mixing, placing in an ultrasonic cleaner in a dark place, and setting instrument parameters as follows: the temperature is 25 ℃, the time is 30min, the power is 25KHZ, and the amphiphilic peptide nano-micelle is obtained after ultrasonic cleaning;
transferring the amphiphilic peptide nano micelle into a dialysis bag with the molecular weight cutoff of 1000 daltons, putting into a beaker filled with ultrapure water for dialysis, replacing the ultrapure water every 6 hours, and dialyzing for 60 hours to obtain the composite nano micelle of the coating RB and SFN in the embodiment.
Experimental example
Each composite nano micelle in the experimental example adopts the finished product prepared in the example 1.
1. Characterization of composite nanomicelles
(1) Particle size and potential characterization
Taking 20 mu L of the composite nano micelle, adding 1mL of deionized water for dilution, and measuring the particle size and zeta potential of the nano micelle by using a Markov particle sizer Zetasizer NanoZS. As shown in A, B of fig. 1, all micelles had a positive surface charge, and the particle size of the nano-micelle was 50nm.
(2) Electronic microscope characterization
Taking 50 mu L of nano micelle, carrying out morphology characterization by a transmission electron microscope, wherein the particle size of the obtained nano micelle is about 50nm, and the surface of the nano micelle is smooth and has a spherical structure as shown in figure 1C.
(3) Packet loading rates for RBs and SFNs
The entrapment rate of RB can be calculated by plotting a standard curve with an ultraviolet spectrophotometer (fig. 2A). The highest absorption peak of RB is measured at the wavelength 564nm, firstly, standard substances are prepared by using RB (2-10 mug/mL) water solutions with different concentrations, a standard curve is drawn by absorbance measured at 564nm, the concentration of RB can be calculated by the dilution ratio of the standard curve and a sample, and the Encapsulation Efficiency (EE) is calculated according to the following formula:
EE (%) =mass of micelle Chinese medicine/mass of drug administered x 100%
The calculated pack rate of RB is 17%;
the entrapment rate of SFN was obtained by HPLC analysis (fig. 2B), mobile phase was acetonitrile: water=30:70 (v/v), flow rate 0.01mL/min, uv detection wavelength 215nm, column C18 reverse-phase column, column temperature 40 ℃. Peak area was measured, and the entrapment rate of SFN was calculated to be 15% based on the area ratio.
2. Cytotoxicity experiment of nanomicelles under ultrasound
In order to verify that the composite nano micelle can remarkably enhance the anti-tumor activity under the ultrasonic action, the in-vitro culture of human glioma cells U87MG is adopted, the stimulus of single RB and the nano material (SFN@RB@SPM) which encapsulates the RB and the SFN is given, and the ultrasonic irradiation treatment is carried out on the single RB and the nano material. Wherein the frequency of the ultrasonic wave is 1.0MHz, the power is 1.0W/cm2, and the duration is 30s. Cytotoxicity of different treatment groups against brain gliomas can be quantitatively detected by MTT. According to the figure 3, the self-assembled polypeptide nano-micelle can reduce the survival rate of cells when the concentration of the entrapped RB is 3.5 mug/mL, and the toxic effect of the nano-micelle on U87MG is remarkably enhanced by ultrasound.
3. Apoptosis experiment of nano micelle under ultrasonic action
Based on the anti-tumor effect of ultrasonic combined nano micelle SFN@RB@SPM on U87MG cells, which is observed by cytotoxicity experiments, the effect of the ultrasonic combined nano micelle SFN@RB@SPM on inducing apoptosis is further evaluated. As shown in fig. 4, the control group and the RB alone treatment group have no significant difference (8.43%, 11.3%,14.5%, 13.9%) between before and after the ultrasonic treatment, the apoptosis rate of the nano micelle sfn@rb@spm induced cells is 28.1%, slightly higher than that of the control group and the RB alone treatment group, and the apoptosis rate of the nano micelle ultrasonic treatment group is as high as 81.7%, which indicates that the ultrasonic effect can greatly enhance the apoptosis induction effect of the nano micelle on the U87MG, and is also consistent with the cytotoxicity experimental results.
4. Inhibition of self-assembled polypeptide nano-micelle on brain tumor of mice
(1) Establishment of in-situ brain glioma animal model
And establishing an in-situ brain glioma-bearing mouse model through a brain stereotactic instrument, inoculating the U87-Luc cell suspension to the brain of a mouse, and examining the tumorigenesis condition of the nude mouse by using a small animal living body imager after two weeks. Tumor areas of the brain will exhibit fluorescein-stimulated fluorescence after successful inoculation of brain gliomas.
(2) Acoustic dynamic therapeutic evaluation of in situ brain glioma-bearing mice based on functional nano-micelles
The components which are successfully modeled are randomly divided into a control group and an experimental group, the experimental group is used for intravenous injection of nano-micelle into a tumor-bearing mouse, and the living body imaging result of a small animal shows that the nano-micelle successfully penetrates through a blood brain barrier to enter the brain, and the higher fluorescence intensity is maintained for 2-6 hours. Therefore, the post-drug effect test selects 2 hours after drug administration for ultrasonic treatment, as shown in fig. 5, a represents a head fluorescence intensity value, B represents a head fluorescence intensity change chart of a nude mouse, C represents imaging of brain tumor area of the nude mouse after the drug effect test is finished, and D represents a weight change trend of the nude mouse.
Experiments show that the nano micelle treatment composition inhibits the tumor area of brain glioma and maintains the normal weight of mice under the action of ultrasound.
In summary, the composite nano micelle, the preparation method and the application thereof in the embodiment of the application have the following advantages:
1. the application designs a nano micelle with high biocompatibility and compatibility, the entrapped natural anti-glioma drug molecule natural sulforaphane SFN and the sound sensitizer manglared RB, which pass through the blood brain barrier efficiently by means of ultrasonic means and utilize the targeting glioma characteristics of RGD (RGD and the integrin alpha over-expressed on the glioma surface) v β 3 Binding), exert RB-mediated photodynamic therapy and are enriched in the brainThe synergistic tumor killing effect of SFN at the glioma part realizes the sensitization brain glioma treatment. In the application, chemical modification and physical technology are combined, so that the sulforaphane exerts its biological effect in tumor cells, and the accurate treatment of glioma is realized.
2. The blood brain barrier is a key bottleneck problem that hinders efficient drug delivery into the brain, and nanomaterial drug delivery is considered as a breakthrough in diagnosis and treatment of neurological diseases. The size of the nano material is a key factor affecting the entrance of the nano material into the brain, and the particle size of the nano micelle is as small as 40nm, so that the efficiency of crossing blood brain barriers can be greatly improved.
The embodiments described above are some, but not all embodiments of the application. The detailed description of the embodiments of the application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Claims (1)
1. The application of the composite nano micelle in preparing a medicine for treating brain glioma is characterized in that the preparation method of the composite nano micelle comprises the following steps:
dissolving self-assembled amphiphilic peptide in water, adding sound-sensitive substances and anti-tumor small molecules, uniformly mixing, and performing ultrasonic cleaning to obtain amphiphilic peptide nano-micelle; the anti-tumor small molecule is sulforaphane; the sound-sensitive substance is Bengalhong; the mass ratio of the self-assembled amphiphilic peptide to the sound sensitive substance to the anti-tumor small molecule is 2: (1-2): (1-2); the temperature of the ultrasonic cleaning is 22-26 ℃, the time is 20-40min, and the power is 25-30KHZ; the self-assembled amphiphilic peptide has the structural formula:
；
transferring the amphiphilic peptide nano micelle into a dialysis bag, dialyzing in ultrapure water, and finally obtaining the composite nano micelle; the molecular weight cut-off of the dialysis bag is 800-1200 daltons, the ultrapure water is replaced every 4-6 hours, and the dialysis time is 36-60 hours.
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