CN113577308A - Nano drug delivery system for cerebral arterial thrombosis targeted therapy and preparation method thereof - Google Patents
Nano drug delivery system for cerebral arterial thrombosis targeted therapy and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a nano drug-loading system for targeted therapy of ischemic stroke and a preparation method thereof, wherein the nano drug-loading system for targeted therapy of ischemic stroke comprises a nanoparticle carrier and a therapeutic drug carried on the nanoparticle carrier; the nanoparticle carrier is synthesized by linking a cell surface chemokine receptor (CXCR4) antagonist with a core nano-skeleton through the action of an auxiliary cross-linking agent and then synthesizing with Glutathione (GSH) at normal temperature and normal pressure; the core nanoscaffold employs a core nanoscaffold having a Maleimide (MAL) stub and an amino (NH2) stub. The invention synthesizes a nano drug-carrying system which has good biocompatibility, is degradable, has higher biological membrane permeability and can carry various compounds, so that the nano drug-carrying system can gather in a cerebral ischemia hypoxic region expressed by a high cell surface chemotactic receptor factor (CXCR4) through a blood brain barrier, and can directionally release therapeutic drugs while scavenging oxygen free radicals.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nano medicine carrying system for targeted therapy of ischemic stroke and a preparation method thereof.
Background
Stroke is the second most common cause of death in the world and is also the leading cause of abnormal limb dysfunction. Epidemiological investigations have shown that the incidence of stroke is increasing year by year, both in developed and developing countries. About 1750 million people die each year from cardiovascular and cerebrovascular diseases and their complications, with a high prevalence of the first mortality in all diseases, with stroke deaths of about 670 million. Due to the high mortality and disability rate of stroke, as well as the long-term nature of the healing process and uncertainty in prognosis, the development of this disease is not only a catastrophic event for the patient and family, but a heavy burden on the medical institutions and society. Due to the existence of blood brain barrier and the limitation of pharmaceutical dosage form, no specific therapeutic drug aiming at the pathological change of ischemic stroke exists clinically at present, and the rapid elimination of the cause of disease, namely thrombolytic therapy, is mainly used.
Therefore, the development of a drug therapy delivery carrier which can efficiently penetrate through a blood brain barrier and has targeting and brain protection properties is very important for improving the survival rate of patients with ischemic stroke.
The nano particles as a novel drug treatment carrier have low toxic and side effects and low administration dosage, can overcome the defects of large dosage and large toxic and side effects of the traditional intravenous/oral administration, and is a research hotspot for treating nervous system diseases. Current research is mainly focused on how efficiently nanoparticles carrying chemical drugs or biological agents reach the brain and cross the blood-brain barrier. The selection of the basic skeleton is mainly divided into inorganic metal nano particles and high molecular organic nano particles. Nano drug delivery vehicle approved by FDA for intracranial glioblastoma treatment is iron nano based
(MagForce), the nanoparticle clinical study shows a certain effect on glioma treatment and has been approved by FDA, but has not been widely used clinically.The reason for this is probably that the metal nanoparticles inevitably have the problem of local heavy metal deposition during long-term use, and have certain central nervous system toxicity and local immunogenicity [ Yarjanli, z., Ghaedi, k., emmaeeili, a., Rahgozar, S ] after long-term use.&Zararabi, A. iron oxide nanoparticles from the neural tissue injury, oxidative stress, and protein aggregation. BMC Neurosci 18,51-51, doi:10.1186/s12868-017 0369-9 (2017). And the physical and chemical properties of the heavy metal material can not be changed or are difficult to change, and the difficulty of surface modification is high, so that the research progress of the metal nano drug-carrying system is influenced. The polymer organic nano-particles have the characteristics of degradability, easy surface modification and the like, but still need to be improved in the aspects of homogeneity and functional stability of biological properties.
In ischemic stroke patients, oxidative stress in ischemic areas is increased, and ischemic brain injury is further aggravated by aseptic inflammation caused by ischemia, resulting in cerebral edema. In order to reduce inflammatory reaction and reduce metabolism of damaged brain tissues, in preclinical animal experiments and clinical trials, the diabetes treatment drug glibenclamide is found to have a certain treatment effect on brain damage after cerebral apoplexy, can obviously inhibit cerebral edema after cerebral ischemic cerebral apoplexy occurs, and effectively reduce mortality, but because glibenclamide itself is used as a hypoglycemic drug, severe hypoglycemia can be caused in the intravenous systemic medication process of cerebral apoplexy treatment dosage, the half-life is short, continuous intravenous administration is needed, and blood sugar is monitored, so the clinical application is still limited by dosage and side effects of traditional systemic administration [ Zhou, J., Atsina, K.B., Himes, B.T., Strohbehn, G.W. & lttzman, W.M.novel therapeutic strategies for gliobblatosoma.cancer J18, 89-99, doi: 10.1097/01820b8ae (443j).
Glutathione (GSH) produced by the body itself under physiological conditions has protective effects on cerebrovascular endothelial functions, including maintaining endothelial cell normal permeability, reducing apoptosis and scavenging reactive oxygen species [ Biswas, S.K., Newby, D.E., Rahman, I. & Megson, I.L. depressed glutathione synthesis and angiogenesis in Apo-E (-/-) micron biochem Biophys Res Commun 338, 1368) -1373, doi:10.1016/j.bbrc.2005.10.098(2005) ]. GSH depletion induces increased levels of cytochrome C, triggering apoptotic signaling pathways, leading to oxidative stress and apoptosis. GSH deficiency is associated with a variety of central nervous system disorders, such as alzheimer's disease, parkinson's disease, and stroke. Several clinical studies have shown that decreased levels of GSH in the brain increase the risk of stroke [ Namba, K., Takeda, Y., Sunami, K. & Hirakawa, M.temporal profiles of the levels of endogenous antioxidants after four-vessel occlusion in rates J neuro angiosurg Anesthesol 13, 131-. GSH, as a medicine for resisting oxidation and relieving inflammatory reaction, has been generally applied to the treatment of hypoxemia, liver damage and other diseases in clinical practice. However, the medicine has no application in the disease of cerebral apoplexy with obvious ischemia, anoxia and oxidative stress reaction because the medicine is difficult to pass through the blood brain barrier. In some reports, nanoparticles carrying GSH as functional group are all made of metal materials (such as gold, silver, copper, etc.), and repeated use of such materials increases the toxicity of heavy metal deposition and the risk of local immunoinflammatory reaction [ Pem, B.et al. sensitivity and safety study of silver and gold nanoparticles with circulating and glutathione J Nanotechnol 10,1802-1817, doi:10.3762/bjnano.10.175(2019) ], which requires higher dosage and toxicological monitoring.
Disclosure of Invention
The invention aims to solve the problem that no specific treatment medicament aiming at the pathological change of ischemic stroke exists clinically at present due to the existence of blood brain barrier and the limitation of medicament dosage form in the prior art, and the problem is mainly that the cause of disease is quickly relieved, namely thrombolytic therapy. Therefore, the invention provides a nano drug delivery system for targeted therapy of ischemic stroke and a preparation method thereof, and the nano drug delivery system has good biocompatibility, is degradable, has higher biological membrane permeability, can carry a plurality of compounds, can be gathered in a cerebral ischemia hypoxic region expressed by a high cell surface chemotactic receptor factor (CXCR4) through a blood brain barrier, and can directionally release therapeutic drugs while scavenging oxygen free radicals.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the nano drug delivery system for the targeted therapy of ischemic stroke comprises a nano particle carrier and a therapeutic drug carried on the nano particle carrier; the nanoparticle carrier is synthesized by linking a cell surface chemokine receptor (CXCR4) antagonist with a core nano-skeleton through the action of an auxiliary cross-linking agent and then synthesizing with Glutathione (GSH) at normal temperature and normal pressure; the core nano-skeleton adopts a structure with Maleimide (MAL) stub and amino (NH)2) Core nanostrucrures of the stub.
In a preferred embodiment of the nano drug delivery system for targeted therapy of ischemic stroke provided by the invention, the therapeutic drug adopts glibenclamide (Glyburide).
In a preferred embodiment of the nano drug delivery system for targeted therapy of ischemic stroke provided by the invention, the auxiliary cross-linking agent is Cystamine Bisacrylamide (CBA).
In a preferred embodiment of the nano drug delivery system for targeted therapy of ischemic stroke provided by the invention, the cell surface chemokine receptor (CXCR4) antagonist is Plerixafor (AMD 3100).
In a preferred embodiment of the drug delivery system for targeted therapy of ischemic stroke, the core nanoscaffold is composed of omega-Pentadecanolide (PDL), N-Methyldiethanolamine (MDEA), diethyl 3, 3 ' - (4, 4 ' -trimethyldipiperidine-1, 1 ' -diyl) dipropionate (TDDP), maleimide polyethylene glycol (MAL-PEG-OH), and aminopolyethylene glycol (NH)2-PEG-OH).
The invention also provides a preparation method for preparing the nano drug delivery system for the targeted therapy of ischemic stroke in the embodiment, which comprises the following steps:
step S1, synthesizing a core nano-skeleton: mixing omega-Pentadecanolide (PDL), N-Methyldiethanolamine (MDEA), diethyl 3, 3 ' - (4, 4 ' -trimethyldipiperidine-1, 1 ' -diyl) dipropionate (TDDP), maleimide polyethylene glycol (MAL-PEG-OH) and aminopolyethylene glycol (NH)2-PEG-OH) was charged into the reactor at a ratio of 30:70:67.5:4.9:4.9 (fed molar ratio) and added with catalystShould give a compound having a Maleimide (MAL) terminus and an amino group (NH)2) Core nanoskeleton of stub (MAL-PEG-PPMTP-PEG-NH)2)；
Step S2, mounting a cell surface chemokine receptor (CXCR4) antagonist to the core nanoscaffold of step S1;
step S3, loading Glutathione (GSH) on the core nano-skeleton in the step S2 to obtain a complete nano-particle carrier;
step S4, synthesizing glibenclamide and the nanoparticle carrier in the step S3 into a nano drug delivery system according to the proportion of 1:20mol, and dialyzing by using a Thermo Snake Skin dialysis membrane of 10K MWCO to remove unbound glibenclamide;
and step S5, obtaining nano drug-carrying systems with different synthesis ratios for standby by the method of step S4.
In a preferred embodiment of the method for preparing a nano drug delivery system for targeted therapy of ischemic stroke, the first reaction stage in step S1 is: stirring the reaction mixture in a nitrogen reaction environment at 90 ℃ and 1atm pressure for 20 hours;
and (3) a second reaction stage: stirring is continued for 70 hours under vacuum of 1.8mmHg atmospheric pressure;
after the two-stage reaction is finished, adding the final product into n-hexane for precipitation; washing the final product with n-hexane twice, dissolving in chloroform, and standing the solution at 30 deg.C under vacuum (pressure less than 1mmHg) overnight to obtain purified product with Maleimide (MAL) residue and amino (NH)2) Core nanoskeleton of stub (MAL-PEG-PPMTP-PEG-NH)2)。
In a preferred embodiment of the preparation method of the nano drug delivery system for targeted therapy of ischemic stroke provided by the present invention, the catalyst in step S1 is Novozym 435 (immobilized candida antarctica lipase B).
In a preferred embodiment of the preparation method of the nano drug delivery system for targeted therapy of ischemic stroke provided by the present invention, the step S2 specifically includes the following steps:
step S21, adding Cystamine Bisacrylamide (CBA) and Plerixafor (AMD3100) into a glass bottle containing a methanol/water mixture (volume ratio 7/3), and carrying out polymerization reaction for 72h in a dark environment at 37 ℃ under the protection of nitrogen;
step S22, dropping the reaction mixture of step S21 into ethanol containing 1.25M hydrochloric acid to keep the pH of the mixture at about 3, centrifuging, discarding the supernatant, collecting the precipitate, washing twice with ethanol and vacuum drying to obtain CBA-converted AMD3100 polymer;
step S23, dissolving the polymer in the step S22 in water, dialyzing for 2 days (MWCO 3.5kDa) for purification, and freeze-drying again;
step S24, the polymer in step S23 is added to the core nano-skeleton (MAL-PEG-PPMTP-PEG-NH) dissolved in step S1 after exposing the disulfide bond residues by shearing with tris (2-carboxyethyl) phosphine hydrochloride (TECP) at normal temperature2) And (3) reacting the solution with sodium acetate (pH 5.2) at room temperature for 2 hours under stirring to obtain a core nano-skeleton carrying AMD3100 functional groups.
In a preferred embodiment of the preparation method of the nano drug delivery system for targeted therapy of ischemic stroke provided by the present invention, the step S3 specifically includes the following steps:
step S31, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into MES buffer solution with pH of 4.6-6.0 to activate the core nano skeleton in the step S2;
and step S32, adjusting the pH of the solution in the step S31 to be more than 7.2 by using concentrated sodium phosphate buffer solution, adding 0.1M sodium phosphate buffer solution with pH value of 7.2 dissolved with Glutathione (GSH) for reaction, and dehydrating and condensing carboxyl of the Glutathione (GSH) and amino of the core nano-skeleton in the step S2 to form a complete nano-particle carrier.
Compared with the prior art, the nano drug delivery system for targeted therapy of ischemic stroke and the preparation method thereof provided by the invention have the beneficial effects that:
the basic raw materials of the nano drug-loading system synthesized by the invention are nontoxic high molecular compounds with biocompatibility verified repeatedly, the cationization degree of the synthesized nano particles is extremely low, the positive charge on the surface is only about 1-3 mv, and the neurotoxicity effect generated by high cationization degree of some nano particles is overcome;
the nano drug delivery system synthesized by the invention has the characteristics of PEG (polyethylene glycol) cytophilic membrane group and extremely small volume (average diameter is about 150nm), so that the nano drug delivery system has high blood brain barrier passing property and better capability of entering cells;
in the nano drug delivery system synthesized by the invention, the maleimide polyethylene glycol (MAL-PEG-OH) and the amino polyethylene glycol (NH)2PEG-OH) provides functional groups on the external surface of the polymer after balling, such as: -MAL residue, -NH2and-OH, -MAL residue is an inert group under normal environment, so that the stability of the whole nanoparticle is ensured; meanwhile, because the-MAL residue has the characteristic of being combined with Cystamine Bisacrylamide (CBA), a functional small molecule compound can be CBA-functionalized and carried on a nanoparticle framework through the combination of the CBA and the MAL residue; in addition-NH on the core nanoskeleton2And the functional group can also be combined with a small molecular compound with-COOH (carboxyl) through a dehydration condensation reaction, so that other functional groups can be introduced on the basis of not changing the main body nano framework structure and the main functional properties of the nano particles, and the biological effect of the nano particles is improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a diagram of a synthesis reaction for a core nanoskeleton provided by the present invention;
FIG. 2 is a CBA reaction scheme of the cell surface chemokine receptor (CXCR4) antagonist (AMD3100) provided by the present invention;
FIG. 3 is a structural formula diagram of a nanoparticle carrier provided by the present invention;
fig. 4 is a flow chart for preparing the nano drug delivery system provided by the invention;
FIG. 5 is a schematic view of the drug delivery system of the present invention efficiently penetrating the blood brain barrier and targeting to the ischemic-anoxic region; wherein: part a shows that the nano drug delivery system is targeted to gather to a cerebral ischemia site and is oxidized and cracked to release glibenclamide (Glyburide); part b shows an enlarged schematic structure diagram of a nano drug-loading system; part c shows that the nano drug delivery system is combined with the cell targeting of the high-expression CXCR4 receptor and is cracked under the action of ROS to release glibenclamide (Glyburide);
FIG. 6 shows the morphology of nanoparticles of the nanoparticle carrier under a scanning electron microscope;
FIG. 7 shows the particle diameter and surface charge of the nanoparticles of the present invention in polarized light scattering measurement;
FIG. 8 is a graph comparing the particle sizes of nanoparticles provided by the present invention;
FIG. 9 is a graph of the effect of nanoparticles provided by the present invention on astrocyte viability.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The nano drug delivery system for targeted therapy of ischemic stroke provided by the embodiment utilizes a nano particle carrier to carry therapeutic drugs to realize a therapeutic effect.
The nanoparticle carrier of this example has a Maleimide (MAL) stub and an amino group (NH)2) The core nano-skeleton at the residue is linked with a cell surface chemokine receptor (CXCR4) antagonist through the action of an auxiliary cross-linking agent and then synthesized with Glutathione (GSH) at normal temperature and normal pressure, and the therapeutic drug of the embodiment can adopt glibenclamide (Gly)buride), glibenclamide has certain treatment effect on brain injury after cerebral apoplexy, can obviously inhibit cerebral edema after cerebral ischemic stroke occurs, and effectively reduce the death rate; the embodiment adopts the nanoparticle carrier to treat the cerebral apoplexy by targeting glibenclamide, thereby avoiding the problem existing in the intravenous whole-body medication process.
The nanoparticle Carrier of this example introduced-MAL stub and-NH2Stub, the outer surface of the polymer after balling provides functional groups such as: -MAL residue, -NH2and-OH, -MAL residue is an inert group under normal environment, so that the stability of the whole nanoparticle is ensured; since the-MAL residue has the property of binding to Cystamine Bisacrylamide (CBA), this example uses Cystamine Bisacrylamide (CBA) (auxiliary cross-linking agent) to carry out CBA-loading of small molecule drug AMD3100 (cell surface chemokine receptor (CXCR4) antagonist) with high ligand-receptor affinity and high specific binding ability to CXCR4 onto the core nanoskeleton, where AMD3100 can bind to the-MAL residue on the core nanoskeleton after CBA, and the reaction can be carried out at normal temperature and pressure without the need of catalyst stabilization [ Li, j.&The method comprises the following steps of Oupicky, D.Dual-function CXCR4 anti-inflammatory polyplexes to a delivery gene therapy and inhibition cancer cell invasion. Angew Chem Int Ed Engl 51,8740-8743, doi:10.1002/anie.201203463(2012), so that AMD3100 can be loaded on a nanoparticle carrier to enable the AMD to be targeted and gathered in a high-expression CXCR4 cerebral ischemia area after crossing a blood brain barrier.
Furthermore, -NH on the core nanoskeleton2Or can be combined with a small molecule compound with-COOH (carboxyl) through dehydration condensation reaction; the small molecule compound adopted in this example is Glutathione (GSH), GSH is a small molecule compound with a molecular weight of 307, and carboxyl groups on GSH molecules can be linked with amino groups (NH) on a core nanometer skeleton2) Amide bond is formed by dehydration and condensation and carried on the nanoparticle skeleton. Since the antioxidant functional group of GSH is Sulfhydryl (SH), GSH is carried on nanoparticles through carboxyl, and does not change the antioxidant property of glutathione [ Pourmbarak Mahnaie, M ].&Mahmoudi,H.Effect of glutathione-stabilized silver nanoparticles on expression of las I and las R of the genes in Pseudomonas aeruginosa strains 25,17, doi 10.1186/s 40001-020-; meanwhile, the molecular weight of glutathione is far smaller than that of the nanoparticles, so that the inherent biological characteristics and molecular polarity [ Khan, N.U., Ali, A., Khan, H., Khan, Z.U ] of the nanoparticles cannot be changed by carrying GSH.&Ahmed, Z.Stablity students and Characterization of Glutathione-Loaded nanoemusion.J. Cosmet Sci 69,257-267 (2018); glutathione is carried on the surface of the nanoparticles and does not react with the drugs wrapped in the nano drug delivery system, and the carrying function of the nano drug delivery system [ Simpson, C.A., Salleng, K.J., Cliffel, D.E ] is not affected.&Feldheim,D.L.In vivo toxicity,biodistribution,and clearance of glutathione-coated gold nanoparticles.Nanomedicine 9,257-263,doi:10.1016/j.nano.2012.06.002(2013)】。
The process flow of the preparation method of the nano drug delivery system of the embodiment is shown in the attached figure 4, and the specific steps are as follows:
step S1, synthesizing a core nano-skeleton: mixing omega-Pentadecanolide (PDL), N-Methyldiethanolamine (MDEA), diethyl 3, 3 ' - (4, 4 ' -trimethyldipiperidine-1, 1 ' -diyl) dipropionate (TDDP), maleimide polyethylene glycol (MAL-PEG-OH) and aminopolyethylene glycol (NH)2PEG-OH) was charged to the reactor in a ratio of 30:70:67.5:4.9:4.9 (feed molar ratio) and catalyst was added: novozym 435 (immobilized Candida antarctica lipase B), and in the first stage, the reaction mixture is stirred for 20 hours in a nitrogen reaction environment at 90 ℃ and 1atm pressure; in the second stage, the mixture is continuously stirred for 70 hours under the vacuum of 1.8mmHg atmospheric pressure, and after the two-stage reaction is finished, the final product is added into n-hexane for precipitation; washing the final product with n-hexane twice, dissolving in chloroform, and standing the solution at 30 deg.C under vacuum (pressure less than 1mmHg) overnight to obtain purified product with Maleimide (MAL) residue and amino (NH)2) Core nanoskeleton of stub (MAL-PEG-PPMTP-PEG-NH)2) The reaction formula is shown as attached figure 1;
step S2, loading a cell surface chemokine receptor (CXCR4) antagonist onto the core nanoscaffold of step S1, the reaction formula is shown in fig. 2, and the specific steps are as follows:
step S21, adding Cystamine Bisacrylamide (CBA) and Plerixafor (AMD3100) into a glass bottle containing a methanol/water mixture (volume ratio 7/3), and carrying out polymerization reaction for 72h in a dark environment at 37 ℃ under the protection of nitrogen;
step S22, dropping the reaction mixture of step S21 into ethanol containing 1.25M hydrochloric acid to keep the pH of the mixture at about 3, centrifuging, discarding the supernatant, collecting the precipitate, washing twice with ethanol and vacuum drying to obtain CBA-converted AMD3100 polymer;
step S23, dissolving the polymer in the step S22 in water, dialyzing for 2 days (MWCO 3.5kDa) for purification, and freeze-drying again;
step S24, the polymer in step S23 is added to the core nano-skeleton (MAL-PEG-PPMTP-PEG-NH) dissolved in step S1 after exposing the disulfide bond residues by shearing with tris (2-carboxyethyl) phosphine hydrochloride (TECP) at normal temperature2) Stirring and reacting for 2h at normal temperature in a sodium acetate solution (pH 5.2) to obtain a core nano-framework carrying AMD3100 functional groups;
step S3, loading Glutathione (GSH) on the core nano-skeleton in step S2 to obtain a complete nano-particle carrier, as shown in fig. 3, the specific steps are as follows:
step S31, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into MES buffer solution with pH of 4.6-6.0 to activate the core nano skeleton in the step S2;
step S32, adjusting the pH of the solution in the step S31 to be more than 7.2 by using concentrated sodium phosphate buffer solution, adding 0.1M sodium phosphate buffer solution with pH being 7.2 dissolved with Glutathione (GSH) for reaction, and dehydrating and condensing carboxyl of the Glutathione (GSH) and amino of the core nano-framework in the step S2 to form a complete nano-particle carrier through linkage;
step S4, synthesizing a nano drug delivery system by the therapeutic drug and the nano particle carrier in the step S3 according to the proportion of 1:20mol ratio, and dialyzing by using a Thermo Snake Skin dialysis membrane of 10K MWCO to remove unbound glibenclamide;
and step S5, obtaining nano drug-carrying systems with different synthesis ratios for standby by the method of step S4.
The invention carries out particle morphology (as shown in figure 6) and particle size (as shown in figures 7 and 8) and surface charge measurement (as shown in figure 7) of polarized light scattering (DLS) measurement under a Scanning Electron Microscope (SEM) on the nano drug delivery system; after adding different concentrations of the nanopharmaceutical particles to the astrocytes and culturing for 72 hours, cell viability was examined and cytotoxicity was evaluated (as shown in fig. 9).
The results show that: the nano drug-loading system is stable in balling, and all indexes are in an ideal range.
In the process of treating cerebral apoplexy, the nano drug delivery system efficiently penetrates through a blood brain barrier and is targeted and concentrated in an ischemic and anoxic region as shown in figure 5, the nano drug delivery system is targeted and concentrated to a cerebral ischemic part and is combined with a high-expression CXCR4 receptor cell in a targeted manner, and the nano drug delivery system is cracked under the action of ROS to release Glyburide (Glyburide).
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A nano drug delivery system for targeted therapy of ischemic stroke comprises a nano particle carrier and a therapeutic drug carried on the nano particle carrier; the method is characterized in that:
the nanoparticle carrier is synthesized by linking a cell surface chemokine receptor (CXCR4) antagonist with a core nano-skeleton through the action of an auxiliary cross-linking agent and then synthesizing with Glutathione (GSH) at normal temperature and normal pressure;
the core nano-skeleton adopts a structure with Maleimide (MAL) stub and amino (NH)2) Core nanostrucrures of the stub.
2. The nano drug delivery system for targeted therapy of ischemic stroke according to claim 1, wherein: the therapeutic drug adopts glibenclamide (Glyburide).
3. The nano drug delivery system for targeted therapy of ischemic stroke according to claim 1, wherein: the auxiliary crosslinking agent adopts Cystamine Bisacrylamide (CBA).
4. The nano drug delivery system for targeted therapy of ischemic stroke according to claim 1, wherein: the cell surface chemokine receptor (CXCR4) antagonist is Plerixafor (AMD 3100).
5. The nano drug delivery system for targeted therapy of ischemic stroke according to claim 1, wherein: the core nanometer skeleton is composed of omega-Pentadecanolide (PDL), N-Methyldiethanolamine (MDEA), diethyl 3, 3 ' - (4, 4 ' -trimethyldipiperidine-1, 1 ' -diyl) dipropionate (TDDP), maleimide polyethylene glycol (MAL-PEG-OH) and amino polyethylene glycol (NH)2-PEG-OH).
6. A preparation method for preparing the nano drug delivery system for targeted therapy of ischemic stroke as claimed in any one of claims 1 to 5, is characterized in that: the method comprises the following steps:
step S1, synthesizing a core nano-skeleton: mixing omega-Pentadecanolide (PDL), N-Methyldiethanolamine (MDEA), diethyl 3, 3 ' - (4, 4 ' -trimethyldipiperidine-1, 1 ' -diyl) dipropionate (TDDP), maleimide polyethylene glycol (MAL-PEG-OH) and aminopolyethylene glycol (NH)2-PEG-OH) is put into a reactor according to the ratio of 30:70:67.5:4.9:4.9 (feed molar ratio) and added with a catalyst to react to obtain a mixture with Maleimide (MAL) stub and amino (NH)2) Core nanoskeleton of stub (MAL-PEG-PPMTP-PEG-NH)2)；
Step S2, mounting a cell surface chemokine receptor (CXCR4) antagonist to the core nanoscaffold of step S1;
step S3, loading Glutathione (GSH) on the core nano-skeleton in the step S2 to obtain a complete nano-particle carrier;
step S4, synthesizing a nano drug delivery system by the therapeutic drug and the nano particle carrier in the step S3 according to the proportion of 1:20mol ratio, and dialyzing by using a Thermo Snake Skin dialysis membrane of 10K MWCO to remove unbound glibenclamide;
and step S5, obtaining nano drug-carrying systems with different synthesis ratios for standby by the method of step S4.
7. The preparation method of the nano drug delivery system for targeted therapy of ischemic stroke according to claim 6, which is characterized in that: the first stage of the reaction in step S1: stirring the reaction mixture in a nitrogen reaction environment at 90 ℃ and 1atm pressure for 20 hours;
and (3) a second reaction stage: stirring is continued for 70 hours under vacuum of 1.8mmHg atmospheric pressure;
after the two-stage reaction is finished, adding the final product into n-hexane for precipitation; washing the final product with n-hexane twice, dissolving in chloroform, and standing the solution at 30 deg.C under vacuum (pressure less than 1mmHg) overnight to obtain purified product with Maleimide (MAL) residue and amino (NH)2) Core nanoskeleton of stub (MAL-PEG-PPMTP-PEG-NH)2)。
8. The preparation method of the nano drug delivery system for targeted therapy of ischemic stroke according to claim 6, which is characterized in that: the catalyst in the step S1 was Novozym 435 (immobilized candida antarctica lipase B).
9. The preparation method of the nano drug delivery system for targeted therapy of ischemic stroke according to claim 6, which is characterized in that: the step S2 specifically includes the following steps:
step S21, adding Cystamine Bisacrylamide (CBA) and Plerixafor (AMD3100) into a glass bottle containing a methanol/water mixture (volume ratio 7/3), and carrying out polymerization reaction for 72h in a dark environment at 37 ℃ under the protection of nitrogen;
step S22, dropping the reaction mixture of step S21 into ethanol containing 1.25M hydrochloric acid to keep the pH of the mixture at about 3, centrifuging, discarding the supernatant, collecting the precipitate, washing twice with ethanol and vacuum drying to obtain CBA-converted AMD3100 polymer;
step S23, dissolving the polymer in the step S22 in water, dialyzing for 2 days (MWCO 3.5kDa) for purification, and freeze-drying again;
step S24, the polymer in step S23 is added to the core nano-skeleton (MAL-PEG-PPMTP-PEG-NH) dissolved in step S1 after exposing the disulfide bond residues by shearing with tris (2-carboxyethyl) phosphine hydrochloride (TECP) at normal temperature2) And (3) reacting the solution with sodium acetate (pH 5.2) at room temperature for 2 hours under stirring to obtain a core nano-skeleton carrying AMD3100 functional groups.
10. The preparation method of the nano drug delivery system for targeted therapy of ischemic stroke according to claim 6, which is characterized in that: the step S3 specifically includes the following steps:
step S31, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into MES buffer solution with pH of 4.6-6.0 to activate the core nano skeleton in the step S2;
and step S32, adjusting the pH of the solution in the step S31 to be more than 7.2 by using concentrated sodium phosphate buffer solution, adding 0.1M sodium phosphate buffer solution with pH value of 7.2 dissolved with Glutathione (GSH) for reaction, and dehydrating and condensing carboxyl of the Glutathione (GSH) and amino of the core nano-skeleton in the step S2 to form a complete nano-particle carrier.
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