Ball-in-ball nanoparticles and preparation method thereof
阅读说明：本技术 球包球状纳米颗粒及其制备方法 (Ball-in-ball nanoparticles and preparation method thereof ) 是由 刘兰霞 邓博 冷希岗 马兵 于 2021-03-23 设计创作，主要内容包括：本发明公开了一种球包球状纳米颗粒,是由第一单体分子和第二单体分子自组装得到的球状结构,第一单体分子包括相互连接的第一疏水性分子和亲水性抗肿瘤药物,亲水性抗肿瘤药物用于引起肿瘤细胞的死亡,第二单体分子包括依次连接的第二疏水性分子、环境响应性连接分子、亲水性免疫调节佐剂分子,第一单体分子与第二单体分子穿插分布,第一单体分子形成第一球体,第二单体分子形成第二球体,第一球体与第二球体的球心重合,第二球体的外径大于第一球体的外径,第一疏水性佐剂分子分布于第一球体的内侧,第二疏水性佐剂分子分布于第二球体的内侧。本发明还公开了一种球包球状纳米颗粒的制备方法。(The invention discloses a ball-coated spherical nanoparticle which is a spherical structure obtained by self-assembling a first monomer molecule and a second monomer molecule, wherein the first monomer molecule comprises a first hydrophobic molecule and a hydrophilic anti-tumor drug which are mutually connected, the hydrophilic anti-tumor drug is used for causing the death of tumor cells, the second monomer molecule comprises a second hydrophobic molecule, an environment-responsive connecting molecule and a hydrophilic immune regulation adjuvant molecule which are sequentially connected, the first monomer molecule and the second monomer molecule are distributed in an inserting way, the first monomer molecule forms a first sphere, the second monomer molecule forms a second sphere, the centers of the first sphere and the second sphere are coincided, the outer diameter of the second sphere is larger than that of the first sphere, the first hydrophobic adjuvant molecule is distributed on the inner side of the first sphere, and the second hydrophobic adjuvant molecule is distributed on the inner side of the second sphere. The invention also discloses a preparation method of the spherical nano-particles.)
1. A spherical nanoparticle is characterized in that the spherical nanoparticle is a spherical structure obtained by self-assembling a first monomer molecule and a second monomer molecule, the first monomer molecule comprises a first hydrophobic molecule and a hydrophilic antitumor drug which are connected with each other, the hydrophilic antitumor drug is used for causing the death of tumor cells, the second monomer molecule comprises a second hydrophobic molecule, an environment-responsive linker molecule and a hydrophilic immunoregulation adjuvant molecule which are sequentially connected, the first monomer molecule and the second monomer molecule are distributed in an interpenetration manner, the first monomer molecule forms a first sphere, the second monomer molecule forms a second sphere, the first sphere and the second sphere have a sphere center coincidence, the outer diameter of the second sphere is larger than the outer diameter of the first sphere, and the first hydrophobic adjuvant molecule is distributed on the inner side of the first sphere, the hydrophilic antitumor drug is distributed on the outer side of the first sphere, the second hydrophobic adjuvant molecule is distributed on the inner side of the second sphere, and the hydrophilic immunoregulation adjuvant molecule is distributed on the outer side of the second sphere.
2. The sphere-in-sphere nanoparticle of claim 1, wherein the first sphere has a diameter of 130nm to 145nm and the second sphere has a diameter of 155nm to 170 nm.
3. The sphere-in-sphere nanoparticle of claim 1, wherein the environmentally responsive linker molecule in the second monomer molecule is one or more.
4. The sphere-in-sphere nanoparticle of claim 3, wherein the plurality of environmentally responsive linker molecules in the same second monomer molecule are of the same or different species.
5. The sphere-in-sphere nanoparticle of claim 1, wherein the environmentally responsive linker molecule is selected from the group consisting of a matrix metalloproteinase-responsive polypeptide, an enzymatically active alpha-lactalbumin polypeptide, a glutathione-responsive molecule, and H2O2At least one responsive molecule.
6. The ball-in-ball nanoparticle of claim 5, wherein the environmentally responsive linker molecule is selected from the group consisting of polypeptide molecules having the sequence ESWTKKSPSPEFSGM-GPQGIAGQR.
7. The sphere-in-sphere nanoparticle according to any one of claims 1 to 6, wherein the hydrophilic antitumor drug is at least one selected from a chemotherapeutic drug and a photosensitizing drug.
8. The sphere-in-sphere nanoparticle according to any one of claims 1 to 6, wherein the first hydrophobic molecule is selected from at least one of a lipid molecule, a non-lipid hydrophobic anti-tumor drug, a non-lipid hydrophobic immunomodulatory adjuvant molecule; and/or, the second hydrophobic molecule is selected from at least one of lipid molecules, non-lipid hydrophobic anti-tumor drugs, and non-lipid hydrophobic immunomodulatory adjuvant molecules.
9. The spherical nanoparticle according to any one of claims 1 to 6, wherein the hydrophilic immunomodulatory adjuvant molecule is selected from at least one of cytokine-based adjuvants and small peptide-based adjuvants.
10. The sphere-in-sphere nanoparticle according to any one of claims 1 to 3, wherein the first hydrophobic molecule is selected from 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the hydrophilic antitumor drug is selected from doxorubicin hydrochloride, the second hydrophobic molecule is selected from 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the environment-responsive linker molecule is selected from a polypeptide molecule having the sequence ESWTKKSPSPEFSGMGPQGIAGQR, and the hydrophilic immunomodulatory adjuvant molecule is selected from the sequence: 5 '(SH) -TCCATGAC GTTCCTGACGTT-3' oligodeoxynucleotide.
11. The method for preparing the spherical-in-spherical nanoparticles according to any one of claims 1 to 10, comprising the steps of:
a. mixing the first monomer molecule and the second monomer molecule, and carrying out ultrasonic treatment;
b. stirring the mixture after ultrasonic treatment at 20-30 ℃ for reaction.
12. The method for preparing spherical nanoparticles according to claim 11, wherein the stirring reaction time is 12-24 hours.
Technical Field
The invention relates to the technical field of medicines, in particular to a spherical nanoparticle and a preparation method thereof.
Background
The development of nano-drug preparations or nano-drug carriers has become a hotspot in the international medical field. The nano-drug can improve the stability and the release performance of the drug, improve the bioavailability of the drug, improve the targeting property of the drug, realize the co-delivery of multi-component functional molecules and have unique advantages in the aspect of tumor combination treatment.
However, at present, the nano-carrier is mostly prepared by nano-medical materials, the nano-carrier has small particle size, large specific surface area, rich surface state and high chemical activity, has special properties which are not possessed by a plurality of blocks and common powder, for example, some inorganic nano-materials may cause cell death, form change and chromosome damage, and some organic polymer nano-materials have the problems of difficult degradation and metabolism and the like. Conventional nanocarrier materials therefore have potential safety issues. For drugs with different target of action, programmed staged disintegration and drug release are difficult to achieve when single nanoparticle load delivers the drugs. Moreover, the limited drug loading capacity and the complex preparation process of the nano-carrier limit the clinical application of the nano-carrier drug.
Disclosure of Invention
Based on this, it is necessary to provide a spherical nanoparticle and a preparation method thereof to solve the problems of poor drug safety, low drug loading rate and complex preparation process of the conventional nano-carrier.
A spherical nanoparticle is a spherical structure obtained by self-assembling a first monomer molecule and a second monomer molecule, wherein the first monomer molecule comprises a first hydrophobic molecule and a hydrophilic antitumor drug which are connected with each other, the hydrophilic antitumor drug is used for causing the death of tumor cells, the second monomer molecule comprises a second hydrophobic molecule, an environment-responsive linker molecule and a hydrophilic immunoregulation adjuvant molecule which are connected in sequence, the first monomer molecule and the second monomer molecule are distributed in an interpenetration manner, the first monomer molecule forms a first sphere, the second monomer molecule forms a second sphere, the first sphere and the second sphere are coincided in the sphere center, the outer diameter of the second sphere is larger than the outer diameter of the first sphere, the first hydrophobic adjuvant molecule is distributed on the inner side of the first sphere, and the hydrophilic antitumor drug is distributed on the outer side of the first sphere, the second hydrophobic adjuvant molecule is distributed on the inner side of the second sphere, and the hydrophilic immunoregulation adjuvant molecule is distributed on the outer side of the second sphere.
In some of these embodiments, the first spheres have a diameter of 130nm to 145nm and the second spheres have a diameter of 155nm to 170 nm.
In some of these embodiments, the environmentally responsive linker molecule in the second monomer molecule is one or more.
In some embodiments, the plurality of environmentally responsive linker molecules in the same second monomer molecule are of the same or different species.
In some of these embodiments, the environmentally-responsive linker molecule is selected from the group consisting of a matrix metalloproteinase-responsive polypeptide, an enzymatically-cleaved alpha-lactalbumin polypeptide, a glutathione-responsive molecule, and H2O2At least one responsive molecule.
In some of these embodiments, the environmentally-responsive linker molecule is selected from the group consisting of the polypeptide molecules of sequences ESWTKKSPSPEFSGM-GPQGIAGQR.
In some of these embodiments, the hydrophilic antineoplastic agent is selected from at least one of a chemotherapeutic agent, a photosensitizing agent.
In some of these embodiments, the first hydrophobic molecule is selected from at least one of a lipid molecule, a non-lipid hydrophobic anti-tumor drug, a non-lipid hydrophobic immunomodulatory adjuvant molecule.
In some embodiments, the hydrophobic molecules are selected from at least one of lipid molecules, non-lipid hydrophobic anti-tumor drugs, non-lipid hydrophobic immunomodulatory adjuvant molecules.
In some of these embodiments, the hydrophilic immunomodulatory adjuvant molecule is selected from at least one of cytokine-based adjuvants, small peptide-based adjuvants.
In some of these embodiments, the first hydrophobic molecule is selected from the group consisting of 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the hydrophilic antineoplastic drug is selected from the group consisting of doxorubicin hydrochloride, the second hydrophobic molecule is selected from the group consisting of 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the environmentally responsive linker molecule is selected from the group consisting of the polypeptide molecule of sequence ESWTK KSPSPEFSGMGPQGIAGQR, and the hydrophilic immunomodulatory adjuvant molecule is selected from the group consisting of sequence: 5 '(SH) -TCCATGACGTTCCTGACGTT-3'.
The preparation method of the spherical-in-spherical nano-particles comprises the following steps:
a. mixing the first monomer molecule and the second monomer molecule, and carrying out ultrasonic treatment;
b. stirring the mixture after ultrasonic treatment at 20-30 ℃ for reaction.
In some embodiments, the stirring reaction time is 12-24 h.
The invention relates to an environment-responsive ball-in-ball nanoparticle and a preparation method and application thereof. The nano-particles are 'ball-in-ball' nano-particles prepared by self-assembly by taking amphiphilic monomer molecules with two different lengths as elements. The two amphiphilic monomer molecules comprise a first hydrophobic molecule and a hydrophilic anti-tumor drug which are mutually connected, and a second monomer molecule comprises a second hydrophobic molecule, an environment-responsive connecting molecule and a hydrophilic immunoregulation adjuvant molecule which are sequentially connected. When the spherical nanoparticle reaches the local part of a tumor, the environment responsive connecting molecules are subjected to responsive fracture under the microenvironment condition, the external hydrophilic immunoregulation adjuvant molecules and the first spheres of internal spherical antitumor drugs (chemotherapeutic drugs/photosensitive drugs and the like) are disintegrated and released, the first spheres act on the tumor cells to cause the death of the tumor cells and activate an inflammatory corpuscle passage, and meanwhile, the self antigen generated by the apoptosis of the tumor cells and the free adjuvant molecules act on immune cells in a synergistic manner to activate antigen specific immune reaction. The released immunoregulation adjuvant molecules act on antigen presenting cells of local tumor, enhance tumor immunoreaction and realize the synergistic treatment effect of chemotherapy/photodynamic therapy and the like and immunotherapy. Except that the drug molecules for constructing the nano particles can be used for the combined treatment of immunotherapy and chemotherapy or photodynamic therapy and the like, the nano particles can also be used as nano carriers to load other therapeutic drugs to deliver at different spatial positions of the nano particles, such as hydrophobic molecule positions and the like, so that the multiple combined treatment is realized, the treatment effect of tumors is improved, and the toxic and side effects of the whole body are reduced.
Drawings
FIG. 1 is a schematic diagram of a structure of a ball-in-ball nanoparticle according to an embodiment of the present invention;
FIG. 2A is a graph of the dynamic light scattering particle size of environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 2B is a diagram illustrating the results of the detection of the potential of the environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 3 is a transmission electron microscope image of an environmentally responsive "ball-in-ball" nanoparticle according to one embodiment of the present invention;
FIG. 4 is a graph of the results of confocal imaging of tumor cell uptake of environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 5A is a graph of maturation of environmentally responsive "ball-in-ball" nanoparticles versus CD40 cells, in accordance with an embodiment of the present invention;
FIG. 5B is a graph of maturation of environmentally responsive "ball-in-ball" nanoparticles versus CD86 cells, in accordance with an embodiment of the present invention;
FIG. 6 is a graph of cytokine secretion after incubation of DC cells with environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 7A is a graph of tumor volume versus therapeutic effect of environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 7B is a graph of tumor treatment effect versus survival for environmentally responsive "ball-in-ball" nanoparticles according to one embodiment of the present invention;
FIG. 8 is a comparative plot of the dynamic light scattering particle size of environmentally responsive "ball-in-ball" nanoparticles of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
As shown in fig. 1, an embodiment of the present invention provides a spherical nanoparticle, which is a spherical structure obtained by self-assembling a first monomer molecule and a second monomer molecule, wherein the first monomer molecule includes a first hydrophobic molecule and a hydrophilic antitumor drug that are connected to each other, the hydrophilic antitumor drug is used to cause death of tumor cells, the second monomer molecule includes a second hydrophobic molecule, an environment-responsive linker molecule, and a hydrophilic immunomodulatory adjuvant molecule that are sequentially connected to each other, the first monomer molecule and the second monomer molecule are distributed alternately, the first monomer molecule forms a first sphere, the second monomer molecule forms a second sphere, the first sphere and the second sphere have a center coinciding, the outer diameter of the second sphere is greater than the outer diameter of the first sphere, and the first hydrophobic adjuvant molecule is distributed on the inner side of the first sphere, the hydrophilic antitumor drug is distributed on the outer side of the first sphere, the second hydrophobic adjuvant molecule is distributed on the inner side of the second sphere, and the hydrophilic immunoregulation adjuvant molecule is distributed on the outer side of the second sphere.
The invention relates to an environment-responsive ball-in-ball nanoparticle and a preparation method and application thereof. The nano-particles are 'ball-in-ball' nano-particles prepared by self-assembly by taking amphiphilic monomer molecules with two different lengths as elements. The two amphiphilic monomer molecules comprise a first hydrophobic molecule and a hydrophilic anti-tumor drug which are mutually connected, and a second monomer molecule comprises a second hydrophobic molecule, an environment-responsive connecting molecule and a hydrophilic immunoregulation adjuvant molecule which are sequentially connected. When the spherical nanoparticle reaches the local part of a tumor, the environment responsive connecting molecules are subjected to responsive fracture under the microenvironment condition, the external hydrophilic immunoregulation adjuvant molecules and the first spheres of internal spherical antitumor drugs (chemotherapeutic drugs/photosensitive drugs and the like) are disintegrated and released, the first spheres act on the tumor cells to cause the death of the tumor cells and activate an inflammatory corpuscle passage, and meanwhile, the self antigen generated by the apoptosis of the tumor cells and the free adjuvant molecules act on immune cells in a synergistic manner to activate antigen specific immune reaction. The released immunoregulation adjuvant molecules act on antigen presenting cells of local tumor, enhance tumor immunoreaction and realize the synergistic treatment effect of chemotherapy/photodynamic therapy and the like and immunotherapy. Except that the drug molecules for constructing the nano particles can be used for the combined treatment of immunotherapy and chemotherapy or photodynamic therapy and the like, the nano particles can also be used as nano carriers to load other therapeutic drugs to deliver at different spatial positions of the nano particles, such as hydrophobic molecule positions and the like, so that the multiple combined treatment is realized, the treatment effect of tumors is improved, and the toxic and side effects of the whole body are reduced.
By adopting a staged disintegration mode, after the hydrophilic immunoregulation adjuvant molecules of the second sphere are disintegrated in one step, the first sphere containing the antitumor drug still has the advantage of nanometer, such as increasing the endocytosis of tumor cells to the first sphere, thereby intensively enhancing the lethality of the antitumor drug to tumors. Meanwhile, the immunoregulation adjuvant molecules are also released in a centralized manner and are cooperated with the antitumor drugs, so that the characteristic of intelligent programmed release and the synergistic treatment effect of chemotherapy/photodynamic therapy and the like with immunotherapy are achieved.
The nano-particles are constructed by self-assembly of drug molecules with therapeutic functions, do not relate to other non-functional nano-materials, and have good biological safety. Meanwhile, the nano-particles are disintegrated to have tumor microenvironment responsiveness, and the medicine is only released locally in the tumor, so that the systemic toxic and side effects of the medicine are reduced, and the synergistic treatment effect of different therapies is enhanced.
In some embodiments, the first sphere has a diameter of 130nm to 145 nm. The diameter of the second sphere is 155 nm-170 nm. The diameter of the first spheres may be 130nm, 132nm, 134nm, 136nm, 138nm, 140nm, 142nm, 145 nm. The diameter of the second sphere may be 155nm, 157nm, 160nm, 162nm, 164nm, 167nm, 170 nm. In one embodiment, the diameter of the second spheres is from 159.3nm to 161.9nm, and the diameter of the first spheres is from 134.6nm to 141.8 nm.
In some embodiments, the environmentally responsive linker molecule in the second monomer molecule is one or more. The number of linker molecules determines the size of the nanoparticle, which in turn has a close relationship to its presentation and transfer, when the type of environmentally responsive linker molecule is determined.
In some embodiments, a plurality of the environmentally-responsive linker molecules in the same second monomer molecule are of the same species or of different species. Whether one or more linker molecules are required can be designed depending on the purpose of the experiment (whether one or more environmental responses). When a plurality of the environment responsive connecting molecules in the same monomer molecule are of the same type, the nanoparticle is disintegrated in stages and released with stronger specificity, and the corresponding monomer molecule can respond only when a specific environment is stimulated, so that the phenomenon of mistaken disintegration and release is avoided. When a plurality of environment-responsive connecting molecules in the same monomer molecule are of the same type, the nanoparticle is more flexible in staged disintegration and release, the monomer molecule can respond to multi-environment stimulation, and the requirement on environment change of a specific part is reduced.
The corresponding environment responsive connection is selected according to the design and action principle of the self system. For example, according to the target requirements for drug release. In some embodiments, the environmentally-responsive linker molecule is selected from the group consisting of a matrix metalloproteinase-responsive polypeptide, an enzymatically-cleaved alpha-lactalbumin polypeptide, a glutathione-responsive molecule, H2O2At least one responsive molecule. Wherein the glutathione-responsive molecule can be one or more selected from folic acid, biotin, microorganism B2, microorganism B12, etc.
In some embodiments, the environmentally responsive linker molecule is selected from the group consisting of the polypeptide molecules of sequence ESWTKKSPSPEFSGMGPQGIAGQR.
In some embodiments, the hydrophilic antineoplastic agent is selected from at least one of a chemotherapeutic agent, a photosensitizing agent.
In some embodiments, the first hydrophobic molecule is selected from at least one of a lipid molecule, a non-lipid hydrophobic anti-tumor drug, a non-lipid hydrophobic immunomodulatory adjuvant molecule.
In some embodiments, the second hydrophobic molecule is selected from at least one of a lipid molecule, a non-lipid hydrophobic anti-tumor drug, a non-lipid hydrophobic immunomodulatory adjuvant molecule.
The species of the first hydrophobic molecule and the second hydrophobic molecule may be the same or different.
In some embodiments, the hydrophilic immunomodulatory adjuvant molecule is selected from at least one of cytokine-based adjuvants, small molecule peptide-based adjuvants. The cytokine-based adjuvant may be, for example, GM-CSF. The small molecule peptide adjuvant can be Q11, Hp91 and the like.
The embodiment of the present invention further provides a method for preparing the ball-in-ball nanoparticles in any one of the above embodiments, including the following steps:
a. mixing the first monomer molecule and the second monomer molecule, and carrying out ultrasonic treatment;
b. stirring the mixture after ultrasonic treatment at 20-30 ℃ for reaction.
Research on the self-assembly tendency of the first monomer molecule and the second monomer molecule shows that ultrasonic treatment is carried out before stirring, and then stirring reaction is carried out under the condition of being close to room temperature, so that the self-assembly is facilitated. The inventors believe that this is possible because the sonication breaks up the two monomer molecules that have self-assembled and then under appropriate conditions the self-assembly of the first and second monomer molecules can be better re-performed.
In some embodiments, the sonication of step a may be for 10 seconds to 15 seconds.
The linking reaction between the first hydrophobic molecule and the hydrophilic antitumor drug molecule on the first monomer molecule may be a reaction in which a hydroxyl group forms an ester with a carboxylic acid, an amidation reaction, or the like, without limitation. The linking reaction between the second hydrophobic molecule, the environment-responsive linking molecule, and the hydrophilic immunomodulatory adjuvant molecule on the second monomer molecule is also not limited, and may be a reaction in which a hydroxyl group forms an ester with a carboxylic acid, an amidation reaction, or the like. As long as the basic attachment is achieved and the reaction mechanism of the sphere-in-sphere nanoparticles is not affected. The type of reaction may be selected as appropriate depending on the type of group of each molecule attached.
The ball-in-ball nanoparticles are obtained by self-assembling the first monomer molecule and the second monomer molecule, so that the reaction time and temperature of the first monomer molecule and the second monomer molecule have important influence on whether the ball-in-ball nanoparticles can be self-assembled into an expected ball-in-ball structure.
In some embodiments, step b is performed with stirring at 20 ℃ to 30 ℃ for a reaction time of 12 hours to 24 hours. Specifically, the stirring temperature may be 20 deg.C, 22 deg.C, 24 deg.C, 26 deg.C, 28 deg.C, 30 deg.C. The reaction time may be 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours. Preferably, the stirring temperature is 24 ℃ to 26 ℃. The reaction time is 16 to 20 hours.
In a specific embodiment, in the spherical nanoparticle, the first hydrophobic molecule is selected from 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the hydrophilic anti-tumor drug is selected from doxorubicin hydrochloride, the second hydrophobic molecule is selected from 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, the environmentally-responsive linker molecule is selected from a polypeptide molecule having the sequence ESWTKKSPSPEFSGMGPQGIAGQR, and the hydrophilic immunomodulatory adjuvant molecule is selected from the sequence: 5 '(SH) -TCCATGACGTTCCTGACGTT-3'.
In some embodiments, the method of preparing the first monomer molecule thereof comprises the steps of:
x1, carrying out sulfydryl modification on 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine;
x2, carrying out pyridyl dithiol modification on doxorubicin hydrochloride;
and X3, mixing the products obtained in the step X1 and X2, and stirring and reacting at 20-30 ℃.
The stirring reaction time of the step X3 can be 24-48 h. Specifically, the stirring temperature may be 20 deg.C, 22 deg.C, 24 deg.C, 26 deg.C, 28 deg.C, 30 deg.C. The reaction time may be 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours.
In some embodiments, the method of preparing the second monomer molecule thereof comprises the steps of:
y1, activating carboxyl of an environment responsive molecule MMP-9 polypeptide (the sequence is NH2-ESWTKKSPSPEFSGMGPQGIAGQR-COOH into ester;
y2, stirring and reacting the product obtained in the step Y1 with hydrophobic molecule 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine at room temperature for 24-48 h;
y3, carrying out pyridyl dithiol modification on amino on the product molecule obtained in the step Y2;
y4, the product obtained by Y3 and the hydrophilic immune regulation adjuvant molecule are selected from the following sequences: 5 '(SH) -TCCATGACGTTCCTGACGTT-3' and reacting at 20-30 ℃ for 10-14 h.
The activating agent in step Y1 may be any one of 1, 2-dichloroethane and N-hydroxysuccinimide, N-dicyclohexylcarbodiimide, isobutyl chloroformate, etc.
Preferably, the molar ratio of the first monomer molecule to the second monomer molecule in the embodiment is (16-53): 1.
The following are specific examples.
Example (b):
s1: adding 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester and dithiothreitol into hydrophobic molecule 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine for modifying sulfydryl, wherein the mass ratio of the 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester to the dithiothreitol is (5.2-10.4) mg (3-6): (3.9-7.8) mg, and stirring and reacting at room temperature for 12-24 hours;
s2: adding 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester into hydrophilic drug molecule doxorubicin hydrochloride to perform pyridyl dithiol modification, wherein the mass ratio of the doxorubicin hydrochloride to the 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester is (4.0-8.0) mg, and the mass ratio of the doxorubicin hydrochloride to the 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester is (3.0-6.0) mg, and stirring and reacting for 8-16 h at room temperature;
s3: mixing the substances obtained in S1 and S2, stirring at room temperature for reaction for 24-48 h, dialyzing, and freeze-drying;
s4: mixing an environment-responsive linker MMP-9 polypeptide (the sequence is NH2-ESWTKKSPSPEFSGMGPQGIAGQR-COOH) with an activator 1, 2-dichloroethane and N-hydroxysuccinimide to perform substitution reaction to activate carboxyl into ester, preparing for the next step of amide reaction, adding the three components in a mass ratio of (0.9-1.8) mg, (0.6-1.2) mg, (0.7-1.4) mg, and stirring for reaction at 40 ℃ for 4 hours. N, N-dicyclohexyl carbodiimide, isobutyl chloroformate and the like can also be used as an activating agent for activating a carboxyl terminal;
s5: adding hydrophobic molecule 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine (0.5-1) mg into the substance obtained in the S4, stirring and reacting for 24-48 h at room temperature, and connecting the hydrophobic molecule to an activated carboxyl terminal through an amide reaction;
s6: adding 0.5-1 mg of 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester to the substance obtained in S5, performing pyridyl dithiol modification on amino on the molecule of the S5 product, and stirring at room temperature for reaction for 8-16 h;
s7: mixing the substance obtained in S6 with hydrophilic adjuvant functional oligodeoxynucleotide CPG-ODN (with the sequence of 5 '(SH) -TCC ATG ACG TTC CTG ACG TT-3'), adding the mixture to OD (5-10), and reacting at room temperature overnight;
s8: and (3) mixing the substances obtained from S3 and S7 according to the mol of (800-1600) nmol and (27.6-55.2) nmol of monomer molecules of S3 and S7, carrying out ultrasonic treatment for 10-15S, and then stirring at 500rpm at room temperature for 12-24 h to obtain the MMP-9 environment-responsive nanoparticle. As shown in fig. 2A, 2B, and 3. The MMP-9 environmental responsiveness 'ball-in-ball' nanoparticle has the diameter of 159.3-161.9 nm and the potential of-26.2 mV-24.4 mV when measured by the dynamic light scattering particle size. MMP-9 environmental responsiveness "ball package ball" nanoparticle is in the homogeneous distribution under the transmission electron microscope.
Experimental example:
1. cellular uptake experiments of matrix metalloenzyme 9(MMP-9) responsive "sphere-in-sphere" nanoparticles:
in order to observe the uptake of MMP-9 environment responsive 'sphere-coated sphere' nanoparticles by tumor cells, E.G7-OVA tumor cells are paved in a confocal dish, after the tumor cells and the nanoparticles are incubated for 2h (the dosage is calculated according to 2 mu M of doxorubicin hydrochloride), PBS is used for washing, a fixing solution is used for fixing, a laser confocal microscope (Leica, TCS SP5) is used for observing the distribution of the MMP-9 environment responsive 'sphere-coated sphere' nanoparticles in the tumor cells, and the whole process is carried out in a dark place. The same concentration of monomer molecule A was used as a control, and monomer molecule A was the product obtained in S3. As shown in figure 4, MMP-9 environmentally-responsive "sphere-in-sphere" nanoparticles facilitate the uptake of chemotherapeutic drugs by tumor cells compared to chemotherapeutic drug molecules alone.
2. Effect of MMP-9 environmentally responsive "sphere-in-sphere" nanoparticles on maturation of BMDCs:
flow cytometry for detecting the maturation of BMDCs cells by nanoparticles, tumor cells and MMP-9 environment-responsive 'sphere-coated sphere' nanoparticles are incubated for 12h (dosage is calculated according to doxorubicin hydrochloride 2 mu M), then BMDCs and tumor cells containing the nanoparticles are incubated for 24h, cells are collected, flow antibodies such as CD11C, CD40 and CD86 are marked, and detection is carried out by a flow cytometer. PBS, free doxorubicin hydrochloride of the same concentration, and a mixture of free doxorubicin hydrochloride of the same concentration and CPG-ODN were used as controls. (wherein Free doxorubicin hydrochloride is represented by DOX, Free doxorubicin hydrochloride + CPG-ODN is represented by Free DC, and nanoparticles are represented by NPs). As shown in fig. 5A, 5B, MMP-9 environmentally-responsive "sphere-in-sphere" nanoparticles had a greater maturation-promoting effect on DC cells compared to free chemotherapeutic drugs and free chemotherapeutic plus adjuvant.
3. Determination of MMP-9 environmental responsiveness "sphere-coated sphere" nanoparticles on the secretion of BMDCs cytokines by ELISA:
BMDCs were collected and plated in 96-well plates. Tumor cells and MMP-9 environment-responsive 'sphere-in-sphere' nanoparticles are incubated for 12h (dosage is calculated according to doxorubicin hydrochloride 2 mu M), then the tumor cells containing the nanoparticles and BMDC in a 96-well plate are incubated for 24h, and then the supernatant is obtained through centrifugation. The BMDCs supernatant was assayed for the amount of the cytokine TNF-. alpha.according to the ELISA kit protocol. And (3) measuring the absorbance OD value at 450nm by using a microplate reader, drawing a standard curve according to the absorbance and the concentration of the standard substance, and calculating the concentration of the sample. PBS, free doxorubicin hydrochloride of the same concentration, and a mixture of free doxorubicin hydrochloride of the same concentration and CPG-ODN were used as controls. (wherein Free doxorubicin hydrochloride is represented by DOX, Free doxorubicin hydrochloride + CPG-ODN is represented by Free DC, and nanoparticles are represented by NPs). As shown in fig. 6, MMP-9 environmental-responsive "sphere-coated sphere" nanoparticles can significantly promote the secretion of BMDCs cytokines, and have the effect of promoting the activation of antigen-presenting cells.
4. MMP-9 environmental responsiveness "ball package ball" nanoparticle in tumor-bearing mice in vivo anti-tumor curative effect:
female C57BL/6 mice bred in an SPF-level breeding room and 6 weeks old are selected to establish a lymphoma mouse ectopic tumor transplantation model. After the experimental animals were adapted to the breeding environment, the hair on the back and groin of the mice was removed, and EG7-OVA cells (about 5X 10) in logarithmic growth phase and in good culture state were subcutaneously inoculated into the right groin5One/only). When the tumor diameter of the inoculated mice is about 5mm, the tumor-bearing mice with similar growth conditions are randomly divided into four groups of PBS, free doxorubicin hydrochloride + CPG and heterozygosis nanoparticles, and the tumor cells are inoculated on the 7 th day and the 1 st dayEqual volumes of fresh sterile PBS solution, free doxorubicin hydrochloride + CPG, and nanoparticle solution were administered on days 4 and 21, respectively (the amount of doxorubicin hydrochloride was 0.1 mg/vial). Carefully observing the survival condition, the diet condition and the mental state of the mice, measuring and recording the length and the width of the tumor-bearing mice and the tumor volume (mm) of the mice by using a digital vernier caliper every two days3) 1/2 × length × width 2. (wherein Free doxorubicin hydrochloride is represented by DOX, Free doxorubicin hydrochloride + CPG-ODN is represented by Free DC, and nanoparticles are represented by NPs). As shown in fig. 7A and 7B, MMP-9 environmentally responsive "sphere-in-sphere" nanoparticles produced strong inhibition of tumor growth and significantly prolonged mouse survival.
Comparative example 1
Comparative example 1 was prepared substantially the same as the nanoparticles of example 1 except that the monomer molecules according to S3 and S7 were mixed at a molar ratio of 800nmol:82.8nmol as shown in fig. 8, and the monomer molecules or nanoparticles could not self-assemble into a ball-in-ball structure as shown in example 1.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
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