CN110856749A - Boron-doped graphene quantum dot-containing nanocomposite and application thereof - Google Patents
Boron-doped graphene quantum dot-containing nanocomposite and application thereof Download PDFInfo
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Abstract
The present invention relates to a nano-carrier for drug delivery and applications thereof, and more particularly, to a boron-doped graphene quantum dot-containing nano-composite and applications thereof. The present invention provides a nanocomposite comprising a plurality of polymer molecules assembled into an aggregate and a plurality of boron-doped graphene quantum dots located inside the aggregate. The polymer molecule is preferably an acid-base reactive dendrimer; the polymer molecules and the boron doped graphene quantum dots can be respectively combined with different drugs; and the nanocomplex may further comprise a target molecule, such as rabies virus glycoprotein. The present invention also provides a method for controlling the disintegration of a nanocomposite, comprising applying a high frequency magnetic field to the nanocomposite to induce the disintegration thereof. The present invention further provides a use of the nanocomposite for preparing a tumor-penetrating drug carrier.
Description
Technical Field
The present invention relates to a nano-carrier for drug delivery and applications thereof, and more particularly, to a boron-doped graphene quantum dot-containing nano-composite and applications thereof.
Background
For the purpose of disease diagnosis or treatment, researchers have widely utilized nanocarriers as drug delivery platforms in vivo. In the field of cancer treatment, nano-sized delivery systems have been demonstrated to increase the accumulation of drugs at the tumor site, thereby increasing the therapeutic effect of the drugs at the affected site and reducing the side effects on normal tissues. In addition, the modification of nanocarriers with target molecules that can be recognized by target cells also facilitates the specific delivery of drugs to specific tumor sites.
However, based on statistics published in the book of natural-Materials by Wilhelm et al in 2016, the accumulation of therapeutic nanocarriers in tumors that developed in the last decade was only about 0.7% of the injected dose, indicating that most nanocarriers were not effective in retaining tumor regions (s.wilhelm, a.j.tavares, q.dai, s.ohta, j.audet, h.f.dvorak, w.c.w.chan, Nature Review Materials 2016, 1, 16014). This result can be attributed to the dense extracellular matrix (tumor-associated extracellular matrix) and the high interstitial fluid pressure (interstitial fluid pressure) in the tumor that prevents the drug from diffusing in the tumor and stably remaining in the tumor. These characteristics of the tumor microenvironment also result in the inability of the drug to be uniformly distributed around and within the tumor, ultimately resulting in limited therapeutic efficacy. To solve this problem, scientists have tried to reduce extracellular matrix formation or decrease interstitial fluid pressure, but these strategies are still under investigation.
Chemotherapy of brain tumors presents more difficulties because the dense vascular endothelium that forms the Blood Brain Barrier (BBB) blocks most drugs from entering the brain, and only hydrophobic molecules with molecular weights below 500Da can pass through the blood brain barrier. Therefore, the treatment of brain tumors mostly requires a combination of surgical removal and chemotherapy, but the survival rate of brain tumor patients receiving such a combination therapy is still low, such as the median survival of glioblastoma multiforme (GBM) patients is only ten-odd months.
Currently, there are many kinds of nano-carriers for tumor therapy, for example, graphene (graphene) is a commonly used drug carrier material. Graphene is a two-dimensional carbon material with a net structure, and can absorb a drug through pi-pi acting force on the surface and emit stable fluorescence under light excitation, so that the graphene is suitable for being used as a drug carrier for diagnosis and treatment. By limiting the size of graphene to the nanometer scale to form Graphene Quantum Dots (GQDs), it has lower cytotoxicity and better biocompatibility. Previous studies found that graphene resonates under near infrared light (usually 808nm) to generate a photothermal conversion effect, so that the method combining drug-loaded graphene and near infrared light irradiation can kill tumor cells by therapeutic drugs and heat. In addition, studies have shown that visible light irradiation promotes graphene to release the carried-in-doxorubicin (doxorubicin). However, since the visible light has a limited penetrating power to the body, the conventional graphene or graphene quantum dots can only be applied to the treatment of tumors close to the surface of the body, but cannot be applied to the treatment of deep tumors or brain tumors.
Accordingly, there is a need to develop a novel nano-preparation to overcome the aforementioned drawbacks of tumor therapy.
Disclosure of Invention
Accordingly, one objective of the present invention is to provide a nanocomposite comprising a plurality of polymer molecules assembled into an aggregate and a plurality of boron-doped graphene quantum dots (B-GQDs) located inside the aggregate.
Another object of the present invention is to provide a method for controlling the disintegration of nanocomplexes, comprising: (a) obtaining the nanocomposite; and (b) applying a High Frequency Magnetic Field (HFMF) of 50 to 600kHz to the nanocomposite to induce disintegration thereof.
It is another object of the present invention to provide a use of the above-mentioned nanocomplex for preparing a drug carrier for penetrating a tumor. In one embodiment of the invention, the tumor is a brain tumor.
In one embodiment of the present invention, the polymer molecule is a stimuli-responsive polymer molecule that changes the volume of the nanocomposite in response to environmental changes. For example, an acid-base reactive dendrimer (dendrimer) having a molecular weight of about 1,000 to 60,000 Da. The dendrimer may be further covalently or non-covalently bonded to a hydrophilic or hydrophobic drug via hydrophilic or hydrophobic functional groups. The drug may be an anti-tumor drug, such as cyclin-dependent kinases (CDKs) inhibitors, such as Palbociclib.
In an embodiment of the present invention, the boron-doped graphene quantum dots are further combined with a hydrophobic drug, such as an anti-tumor drug, including a nucleic acid (DNA) synthesis inhibitor, such as erythromycin, an angiogenesis inhibitor, a tumor metastasis inhibitor, and the like.
In one embodiment of the present invention, the nanocomplex further comprises a target molecule that directs the nanocomplex to target tissue. For example, a targeted nervous system Rabies glycoprotein (RVG) or peptide fragment thereof is conjugated to nanocomplexes to direct penetration of the blood brain barrier to brain tumors.
Compared with the conventional nanocomposite containing graphene quantum dots, which has inconsistent size and loose structure, only coats a single drug and usually needs strong acid in the preparation process, the nanocomposite disclosed by the invention is easy to prepare in a large amount in a short time without strong acid and can be self-assembled into a stable structure, so that the phenomenon that the drug loaded in the nanocomposite is released to non-target tissues prematurely after entering the inside of a body can be avoided, and toxic and side effects on normal tissues can also be avoided. In addition, because the polymer molecules and the boron-doped graphene quantum dots can be respectively combined with drugs, the nanocomposite disclosed by the invention can simultaneously carry drugs with different properties, such as hydrophilic drugs and hydrophobic drugs.
The nanocomplexes of the invention can comprise polymer molecules that are stimuli-responsive to a target tissue. By stimuli-responsive is meant a change in physical or chemical properties in response to a change in the environment in which the target tissue is located, for example a change in aggregation state in response to a change in pH of the environment in which the target tissue is located. When the nanocomplex comprises acid-base reactive polymer molecules that can form aggregates of larger volume in a weakly acidic environment, it is more likely to be retained within the tumor, thus increasing the accumulation of the drug in the tumor.
The drug carrier is controlled to release the drug at the specific part of the body, so that the treatment effect of the target tissue can be improved, and the toxic and side effects on normal tissues can be reduced. Compared with the conventional method for controlling drug release, such as photoinduced release, the boron-doped graphene quantum dots contained in the nanocomposite disclosed by the invention can generate induced current in an external high-frequency magnetic field, and hundreds of nanometers of the composite (primary carrier) can be disintegrated into composite units (such as a single dendrimer or a single secondary carrier of the boron-doped graphene quantum dots) with the length of about 2-5nm by the energy, so that the drug-loaded units can be more easily diffused into the interior from the periphery of a tumor, and a uniform tumor treatment effect is achieved. In addition, since the high frequency magnetic field can penetrate deep into the body, the nanocomplexes of the present invention can be induced to disintegrate even when reaching deep tumors such as brain tumors.
The following embodiments are provided to illustrate the features and applications of the present invention, rather than to limit the scope of the invention, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.
Drawings
Fig. 1 shows the self-assembly process and structure of nanocomplex according to one embodiment of the present invention, in which acid-base reactive dendrimers (pH-Den) bound to palbociclib are assembled into an aggregate, boron-doped graphene quantum dots (B-GQD) bound to epothilone are located inside the aggregate, and rabies virus glycoprotein fragment (RVG) is adsorbed on the surface of the aggregate through electrostatic force;
fig. 2A shows a Transmission Electron Microscope (TEM) image of boron-doped graphene quantum dots, with a scale bar of 10 nm;
FIG. 2B shows a TEM image of phenylboronic acid-modified polyethylenediamine dendrimer, wherein the scale bar indicates 100 nm;
fig. 3A shows a TEM image of the boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite at pH 7.4, with a scale bar representing 200 nm;
fig. 3B shows a TEM image of the boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite at pH 6.6, wherein the scale bar represents 500 nm;
fig. 3C is an enlarged image of fig. 3A, with a scale bar representing 50nm and white arrows indicating boron-doped graphene quantum dots;
fig. 3D is an enlarged image of fig. 3B, with a scale bar representing 50nm and white arrows indicating boron-doped graphene quantum dots;
FIG. 4A shows TEM image of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite subjected to High Frequency Magnetic Field (HFMF) for 15 minutes, wherein the scale bar represents 50 nm;
fig. 4B shows a TEM image of the boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite subjected to a high frequency magnetic field for 30 minutes, wherein the scale bar represents 20 nm;
fig. 5A shows the fluorescence distribution in the collagen solid hydrogel before exposure of the fluorescence-labeled boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite to a high-frequency magnetic field;
fig. 5B shows the fluorescence distribution in the collagen solid hydrogel after the fluorescence-labeled boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite was exposed to the high-frequency magnetic field for 15 minutes;
fig. 5C shows the fluorescence distribution in the collagen solid hydrogel after 30 minutes exposure of the fluorescence-labeled boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite to the high-frequency magnetic field;
fig. 6A shows the fluorescence distribution in ALTS1C1 tumor microspheres before exposure of a fluorescently labeled boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite to a high frequency magnetic field;
fig. 6B shows the fluorescence distribution in ALTS1C1 tumor microspheres after exposure of a fluorescently labeled boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite to a high frequency magnetic field for 5 minutes;
fig. 7 shows the distribution of boron-doped graphene quantum dots/acid-base reactive dendrimer nanocomplexes in mice with or without rabies virus glycoprotein fragment (RVG), with the color bar on the right indicating the color representing the high or low fluorescence intensity; and
fig. 8 shows the distribution of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomplexes in mouse spinal cord with or without rabies virus glycoprotein fragment (RVG), with the color bar on the right indicating the color representing the high or low fluorescence intensity.
Detailed Description
The present invention provides a nanocomposite comprising a plurality of polymer molecules assembled into an aggregate and a plurality of boron-doped graphene quantum dots located inside the aggregate. The polymer molecule and the boron doped graphene quantum dot can be combined with different drugs respectively. The present invention also provides a method of controlling the disintegration of a nanocomplex, comprising: (a) obtaining the nanocomposite, and (b) applying a high frequency magnetic field to the nanocomposite to induce disintegration thereof. The following examples illustrate the preparation of boron-doped graphene quantum dots, and illustrate the self-assembly ability of the nanocomposites of the present invention using dendrimers with acid-base reactivity as an example of polymer molecules. The nanocomplexes demonstrate high stability and low leakage rates in aqueous solution and swell with decreasing pH in the tumor environment. Furthermore, the nanocomplex is induced to disintegrate into complex units in an external high frequency magnetic field, so that the complex units carry the drug to penetrate the dense extracellular matrix and uniformly distribute around and inside the tumor. In addition, when the nano-complex is modified by rabies virus glycoprotein fragments to form a rabies virus-like particle, the modification endows the nano-complex with the capability of penetrating the blood brain barrier and increasing the accumulation of the loaded drug in brain tumors.
Based on the aforementioned characteristics, the nanocomplexes of the present invention are suitable for preparing a tumor-penetrating drug carrier. When a drug-loaded nanocomplex having a particle size of about 200nm is administered to treat a cancer patient, it is presumed that it can specifically accumulate in a tumor through the vascular pores (about one hundred to several hundred nanometers) of the tumor via blood circulation, via an enhanced permeability and retention Effect (EPR). Due to the weak acidic environment (pH 6.5-7.0) of the tumor, the volume of the nanocomposite comprising the acid-base sensitive polymer molecules increases to about 450nm, and thus effectively remains in the tumor, increasing the accumulation of the loaded drug in the tumor. Thereafter, when exposed to a high frequency magnetic field, the nanocomplex is induced to disintegrate into approximately 2-5nm complex units, thereby allowing the carried drug to be transported to the tumor site by the complex units having better penetration, expanding the distribution of the drug in the tumor and improving the therapeutic effect.
Definition of
As used herein, the numerical values are approximations and all numerical data are reported to be within the 20 percent range, preferably within the 10 percent range, and most preferably within the 5 percent range.
As used herein, the term "polymer molecule" refers to a macromolecule formed by polymerization of one or more unit molecules, such as dendrimers, polyethylene glycols, polysaccharides (e.g., hyaluronic acid), and lipid molecules (e.g., phospholipids). The polymer molecules may be chemically modified to be stimuli-responsive, preferably having a molecular weight of between 1,000 and 60,000 Da. The polymer molecules can self-assemble into a stable structure in water or aqueous solution. Materials and methods
Material
4-vinylphenylboronic acid (4-vinylphenylboronic acid), boric acid (boric acid), 4- (Bromomethyl) phenylboronic acid (4- (Bromomethyl) phenylboronic acid), erythromycin hydrochloride (a water-soluble salt of erythromycin), ethanol, acetone, dimethyl sulfoxide (DMSO), and second generation (G2) dendrimers of polyethylene diamine (PAMAM) are commercially available from Sigma-Aldrich corporation (st. Palbociclib hydrochloride (Palbociclib hydrochloride, which is a water-soluble salt of Palbociclib) is commercially available from MedChemExpress. Fluorescent dyes Cy5.5(cyanine 5.5) and RITC (rhodamine Bithiocyanine) are commercially available from Molecular Probes, Inc. (Eugene, Oreg., USA).
Transmission electron microscope
Transmission Electron Microscope (TEM) images of the nanocomposites and their constituent units are obtained from JEOL JEM-2010(200kV, Tokyo, Japan). When preparing the sample, 5 μ L of sample aqueous solution (2mg/mL) prepared in real time was dropped to a copper grid covered with a polyvinyl formal resin (Formvar) layer, settled for 30 seconds, and the excess droplets were sucked up with filter paper. After this procedure was repeated 3 times, the samples were dried overnight at room temperature.
Cell culture
Mouse astrocytic tumor cell line ALTS1C1(ATCC CRL-2541) was purchased from the American Type Culture Collection (ATCC). The cells were incubated at 37 ℃ with 5% CO2Cultured in DMEM medium (Gibco Dulbecco's modified Eagle's medium; Thermo Fisher scientific) supplemented with 10% fetal bovine serum (FBS; Biologica Industrie, Cromwell, Connecticut, USA) and 1% penicillin and streptomycin (Gibco, Thermo Fisher scientific).
Animal experiments
Brain inoculation of C57BL/6 mice 2. mu.L of 5X 107ALTS1C1 tumor cells were pooled per mL and randomly grouped (6 mice per group). 100 μ L of the indicated nanocomplex (1mg/mL) was administered via the tail vein on day 14 post tumor inoculation. The nanocomposite was dissolved in phosphate buffered saline (PBS; 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate in deionized water, pH 7.4).
Statistical analysis
Statistically significant differences in experimental data were analyzed with student's T-test.
Example 1
Preparation of boron doped graphene quantum dots
This example illustrates a method for preparing boron-doped graphene quantum dots (abbreviated as B-GQD), which are the main constituent molecules of the nanocomposite of the present invention. 75mg of 4-vinylphenylboronic acid and 150mg of boronic acid were dissolved in a mixed solution of 2.5mL of ethanol and 15mL of acetone. After 0.5 to 1 hour of ultrasonic oscillation, 2.5mL of 30% hydrogen peroxide solution was slowly added. And carrying out ultrasonic oscillation on the obtained mixed solution for 10 minutes, and reacting at 150-400 ℃ for 2-48 hours to obtain a crude product of the boron-doped graphene quantum dot. The crude product was then dialyzed against deionized water for three days at room temperature, once a day, using a dialysis membrane (OrDial D-ClearDNA Membranes MWCO: 1000; Orange Scientific) having a molecular weight cut-off of 1000 Da. Collecting and drying the boron doped graphene quantum dots attached to the dialysis membrane to obtain B-GQD powder, and storing the B-GQD powder at-20 ℃.
Example 2
Preparation of nanocomposites
2.1 preparation of acid-base reactive dendrimers
The nanocomplexes of the invention preferably comprise polymer molecules with stimulatory reactivity, which expand the volume of the nanocomplexes in the tumor environment, e.g., in the weakly acidic environment of the tumor (about pH 6.5-7.0). This example illustrates the preparation of a polymer molecule with stimulatory reactivity by an acid-base reactive dendrimer (abbreviated as pH-Den) with a molecular weight of 1,000 to 60,000Da, 4.4mg of 4- (bromomethyl) phenylboronic acid and 100mg of a second generation polyethylene diamine dendrimer are dissolved in dimethyl sulfoxide and reacted at 80 ℃ for 24 hours to obtain a crude product of phenylboronic acid modified polyethylene diamine dendrimer (PBA-PAMAM), which is an example of an acid-base reactive dendrimer. The crude product was cooled and then dialyzed against deionized water at room temperature for three days in a Dialysis membrane (OrDial D-Clean Dialysis Membranes MWCO: 1000; Orange scientific) having a molecular weight cut-off of 1000Da, with water being changed once a day. Freeze drying the dialyzed PBA-PAMAM solution to obtain acid-base reactive dendrimer powder, and storing at-20 deg.C.
2.2 preparation of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite
The nanocomposite according to an embodiment of the present invention can be prepared by mixing the boron-doped graphene quantum dots described in embodiment 1 with the acid-base reactive dendrimer described in embodiment 2.1. Before the mixing step, the two nanocomposite units can be combined with a drug to form a drug-loaded form. In one example, 1mg of palbociclib hydrochloride is dissolved in 10 μ L of ethanol, followed by addition of two times water to obtain a 1mg/mL palbociclib solution; and 1mg of the erythromycin hydrochloride was dissolved in 1mL of secondary water to obtain a 1mg/mL solution of the erythromycin. And then, dissolving 1mg of boron-doped graphene quantum dots in deionized water until the concentration is 1mg/mL, mixing with the above-mentioned erythromycin solution, and subjecting the mixture to ultrasonic oscillation for 30 minutes to obtain the boron-doped graphene quantum dot solution combined with the erythromycin through pi-pi acting force. Meanwhile, 1mg of acid-base reactive dendrimer is dissolved in ethanol to reach the concentration of 1mg/mL, and then is mixed with the palbociclib solution, the mixture is subjected to ultrasonic oscillation and vacuum drying for 30 minutes, and is then dissolved back to 1mg/mL by deionized water, so that the acid-base reactive dendrimer solution which is combined with palbociclib through hydrophobic force can be obtained, wherein the palbociclib is located in the hydrophobic core of the acid-base reactive dendrimer. Finally, the boron doped graphene quantum dot solution loaded with the epothilone and the acid-base reactive dendrimer solution loaded with the palbociclib are mixed according to the volume ratio of 1: 2 to 1: 8, and the mixture is vibrated for 10 to 30 minutes by ultrasonic waves, so that the boron doped graphene quantum dot/acid-base reactive dendrimer (B-GQD/pH-Den) nano composite solution can be obtained.
2.3 modification of rabies glycoprotein fragment
To increase targeting to specific tissues, the nanocomplexes of the invention can further comprise a target molecule on the surface, which can be a nucleic acid, a carbohydrate, a lipid, a protein, or a combination thereof. For example, the nervous system targeting and the blood brain barrier penetration of the boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite described in example 2.2 were increased by surface modification of a rabies virus glycoprotein fragment (RVG; having an amino acid sequence of YTIWMPENPRPGTPCDIFTNSRGKRASN GGGGRRRRRRRRR, SEQ ID NO: 1). In one example, 10 μ L of a 200 μ g/mL PBS solution of rabies virus glycoprotein fragments (synthetic peptides from Sigma-Aldrich) was added to a 1mg/mL solution of boron-doped graphene quantum dots/acid-base reactive dendrimer nanocomplex and mixed with shaking for 30 seconds. This step allows the positively charged rabies virus glycoprotein fragments to be adsorbed to the negatively charged acid-base reactive dendrimers on the nanocomposite surface layer by electrostatic force.
FIG. 1 shows the self-assembly process and structure of the nanocomposite. In fig. 1, 1 is a boron-doped graphene quantum dot (B-GQD), 2 is erythromycin, 3 is RVG, 4 is palbociclib, 5 is an acid-base responsive dendrimer (pH-Den), and 1, 2, 3, 4, and 5 together constitute nanoparticles; an acid-base environment with a pH of 7.4 is used as 6, and an acid-base environment with a pH of 6.6 is used as 7. The nano-composite comprises an aggregate formed by self-assembling a boron doped graphene quantum dot core and an acid-base responsive dendritic polymer from inside to outside and an outermost layer of a rabies virus glycoprotein fragment. The boron doped graphene quantum dots and the two nano materials of the acid-base responsive dendrimer form two environments, so that the nano composite assembled by the boron doped graphene quantum dots and the acid-base responsive dendrimer can carry medicines with different chemical structures and properties, such as the epothilone and the palbociclib.
Example 3
Structure and characteristics of nanocomposites
In order to examine the structure and characteristics of the nanocomposite of the present invention, the morphology and behavior of the boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite and the constituent units thereof described in example 2.2 were observed by a Transmission Electron Microscope (TEM). Fig. 2A shows a TEM image of boron-doped graphene quantum dots; fig. 2B shows a TEM image of a phenylboronic acid-modified polyethylenediamine dendrimer (an example of an acid-base reactive dendrimer). Referring to fig. 2A, the boron-doped graphene quantum dots are agglomerated in water, and each quantum dot has a particle size of about 2 to 5 nm. According to FIG. 2B, the acid-base reactive dendrimers self-assemble into spheres with a particle size of about 50 to 80nm in water. Fig. 2A and 2B demonstrate that boron-doped graphene quantum dots and phenylboronic acid-modified polyethylene diamine dendrimers can be successfully synthesized.
Fig. 3A and 3B show TEM images of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposites at pH 7.4 and pH 6.6, respectively; fig. 3C and 3D are enlarged images of fig. 3A and 3B, respectively. According to FIGS. 3A-3B, the nanocomposites self-assemble into structurally stable and uniformly sized spheres with a particle size of about 160 to 250nm at pH 7.4, but increasing to about 450nm at pH 6.6. This property of changing volume in response to environmental pH improves the ability of the nanocomplex to reside in tumors.
FIGS. 4A and 4B show TEM images of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite applied with a high frequency magnetic field (Power Cube 32/900high frequency generator; Taiwan macrography, China) for 15 minutes and 30 minutes, respectively. These images demonstrate that the presence of boron-doped graphene quantum dots allows the nanocomposite to be completely disintegrated into single boron-doped graphene quantum dots having a particle size of about 2 to 5nm and single acid-base reactive dendrimers when subjected to an external high-frequency magnetic field, because the energy of induced current generated by the boron-doped graphene quantum dots destroys the aggregation force between the boron-doped graphene quantum dots and the acid-base reactive dendrimers. This feature can promote the release and diffusion of the drug from the nanocomposite to various sites of the tumor, thereby enhancing the therapeutic effect of the drug on the affected area.
Example 4
Interstitial penetration of nanocomposites upon induced disintegration
In order to evaluate the penetration force of each complex unit released by induced disintegration of the nanocomposite of the present invention in the extracellular matrix, a laser scanning confocal microscope (confocal laser scanning microscope) zeiss sm slm 780 (germany) was used to observe the nanocomposite (prepared as a 1% aqueous solution and with an addition amount of 10 μ L) assembled by the boron-doped graphene quantum dots (labeled with RITC, in which NCO functional groups are bonded to defects on the surface of the quantum dots through covalent bonds) and the acid-base reactive dendrimers (labeled with cy5.5, embedded in the dendrimers through hydrophobic forces) labeled with fluorescent dyes, which was added into 10 wt% collagen solid hydrogel and then subjected to a high frequency magnetic field (frequency of 50 to 600kHz) for 15 minutes and 30 minutes before and after three-dimensional imaging. The excitation wavelength of RITC is 420nm, and the detection wavelength is 600 nm. The excitation wavelength of Cy5.5 is 640nm, and the detection wavelength is 720 nm.
Compared with the fluorescence distribution in the collagen of cubic space with a length of about 800 μm before the high-frequency magnetic field is applied (0 min) as shown in fig. 5A, the fluorescence distribution is expanded by exposing the collagen to the high-frequency magnetic field for 15 min (fig. 5B) or 30 min (fig. 5C), which indicates that the complex units released by the induced disintegration of the nanocomplex can penetrate the dense collagen. This result may be due to either the disintegration forces of the nanocomplexes and the ultra-small size of the composite units, which may increase penetration. Therefore, the nanocomplexes of the present invention can be used to enhance the penetration of the loaded drug in the dense extracellular matrix based on this property.
Example 5
Tumor distribution of nanocomplexes
In order to test the distribution of the nanocomposite in tumors before and after induced disintegration, a laser scanning confocal microscope ZEISS LSM 780 (germany) was used to observe the fluorescent images of the nanocomposite (prepared as a 1% aqueous solution and added in an amount of 50 μ L) assembled by boron-doped graphene quantum dots (labeled with RITC) labeled with a fluorescent dye and acid-base reactive dendrimers (labeled with cy 5.5), which was added to tumor microspheres formed by a mouse astrocytic tumor cell line ALTS1C1 for 4 hours and then subjected to a high-frequency magnetic field (frequency of 50 to 600kHz) for 5 minutes. Tumor microspheres are prepared by mixing 106ALTST1 cells were injected into a PDMS mold at 37 ℃ with 5% CO2The cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin for one day to form tumor microspheres. The PDMS mold has a hole with a diameter of 300 mm and a depth of 300 mm.
As shown in fig. 6A, at different tumor depths, the nanocomplexes are mainly distributed around the tumor before the high frequency magnetic field stimulation. In addition, the distribution of boron-doped graphene quantum dots and acid-base reactive dendrimers almost overlap, indicating high stability and low drug leakage rate of the nanocomposite. However, as shown in fig. 6B, when the nanocomposite is disintegrated by the high frequency magnetic field, the boron-doped graphene quantum dots and the acid-base reactive dendrimers are uniformly distributed throughout the tumor microsphere, especially deep into the inner region of the tumor, which is consistent with the increased penetration of the composite unit due to the application of the high frequency magnetic field disclosed in example 4. The results also show that the nanocomplexes of the present invention can enhance the therapeutic effect of drugs on affected parts.
Example 6
Body distribution of nanocomplexes comprising target molecules
To examine the distribution of the nanocomposites of the present invention in animals with or without binding to target molecules, 100 μ L of PBS solution of boron-doped graphene quantum dot/acid-base reactive dendrimer nanocomposite with or without rabies virus glycoprotein fragment (RVG) was intravenously injected into mice with ALTS1C1 tumor inoculated into the brain, and the distribution and accumulation of the two Cy5.5-labeled nanocomposites in mice were detected 72 hours after injection using in vivo molecular imaging system (IVIS; PerkinElmer). The excitation wavelength of Cy5.5 is 640nm, and the detection wavelength is 720 nm. Control mice were injected with PBS solution only.
As shown in fig. 7, the amount of the modified rabies virus glycoprotein fragment nanocomplex in brain tumors was about twice as large as the amount of the rabies virus glycoprotein fragment-free nanocomplex in brain tumors, indicating that the modification of the rabies virus glycoprotein fragment can promote the nanocomplex to cross the blood brain barrier and reduce its accumulation in the non-nervous system, thereby reducing the side effects of brain tumor therapy. In addition, as shown in fig. 8, modification of rabies virus glycoprotein fragment increased accumulation of nanocomplex in spinal cord, suggesting that nanocomplex can deliver drug to brain tumor via cerebrospinal fluid of central nervous system without being affected by blood brain barrier.
In summary, the nanocomposite of the present invention has high stability and low drug leakage rate in aqueous solution, and can simultaneously carry different drugs. The boron-doped graphene quantum dots contained in the nano-composite can cause the nano-composite to be disintegrated into composite units in an external high-frequency magnetic field, so that the loaded drug can penetrate through compact extracellular matrixes and can diffuse from the periphery of a tumor to the inside of the tumor more easily. In addition, when the nanocomplex comprises the acid-base reactive polymer molecules, the volume and retention rate of the nanocomplex in the weakly acidic tumor are increased, so that the accumulation amount of the loaded drug in the tumor is increased. When further modified with rabies glycoprotein fragments, the nanocomplexes can penetrate the blood brain barrier and accumulate more in brain tumors. Thus, the nanocomplexes of the invention are useful as a multi-drug delivery platform for tumor penetration, particularly for the treatment of deep tumors.
Claims (16)
1. A nanocomposite includes a plurality of polymer molecules assembled into an aggregate and a plurality of boron-doped graphene quantum dots located inside the aggregate.
2. The nanocomposite of claim 1, wherein the polymer molecule is a stimuli-responsive polymer molecule.
3. The nanocomposite of claim 2, wherein the stimuli-responsive polymer molecule is an acid-base-reactive dendrimer.
4. The nanocomposite of claim 1, wherein the polymer molecule is further conjugated to a hydrophilic or hydrophobic drug.
5. The nanocomposite of claim 1 or 4, wherein the boron-doped graphene quantum dots are further conjugated with a hydrophobic drug.
6. The nanocomposite of claim 1, further comprising a target molecule.
7. The nanocomplex of claim 6, wherein the target molecule is a rabies glycoprotein or a peptide fragment thereof.
8. A method of controlling the disintegration of a nanocomplex, comprising:
(a) obtaining a nanocomposite of claim 1; and
(b) applying a magnetic field of 50 to 600kHz to the nanocomposite to induce disintegration thereof.
9. The method of claim 8, wherein the polymer molecule is a stimuli-responsive polymer molecule.
10. The method of claim 9, wherein the stimuli-reactive polymer molecule is an acid-base-reactive dendrimer.
11. The method of claim 8, wherein the polymer molecule is further conjugated to a hydrophilic or hydrophobic drug.
12. The method of claim 8 or 11, wherein the boron-doped graphene quantum dots are further conjugated with a hydrophobic drug.
13. The method of claim 8, wherein the nanocomplex further comprises a target molecule.
14. The method of claim 13, wherein the target molecule is a rabies glycoprotein or a fragment thereof.
15. Use of the nanocomposite of any one of claims 1-7 for preparing a drug carrier for penetrating a tumor.
16. The use of the nanocomplex of claim 15, wherein said tumor is a brain tumor.
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