WO2023155755A1 - 一种交联聚合物结构及其制备方法与应用 - Google Patents

一种交联聚合物结构及其制备方法与应用 Download PDF

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WO2023155755A1
WO2023155755A1 PCT/CN2023/075705 CN2023075705W WO2023155755A1 WO 2023155755 A1 WO2023155755 A1 WO 2023155755A1 CN 2023075705 W CN2023075705 W CN 2023075705W WO 2023155755 A1 WO2023155755 A1 WO 2023155755A1
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polymer structure
nanoparticles
tpp
group
basic unit
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French (fr)
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张仕勇
廖玉龙
吴潇
廖春燕
卢晓鸾
肖潇
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四川大学
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    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L81/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing sulfur with or without nitrogen, oxygen or carbon only; Compositions of polysulfones; Compositions of derivatives of such polymers
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    • C08J2381/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing sulfur with or without nitrogen, oxygen, or carbon only; Polysulfones; Derivatives of such polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a cross-linked polymer structure and a preparation method and application thereof.
  • nanomedicine Due to the unique performance of quantum size effect and surface interface effect, nanomedicine provides new ideas and new methods for the treatment of many diseases, and greatly promotes the development of personalized medicine and precision medicine.
  • the broad application prospects of nanomedicine have attracted the attention of many scientific researchers and drug R&D companies, and they have devoted themselves to it.
  • nanomedicines are currently facing many problems in the clinical application process, and the representative problem is the delivery efficiency. Every step of the interaction between nanomedicine and organisms is a barrier, and each obstacle may reduce the amount of nanomedicine delivered to the target site.
  • the barriers at the organ level include gastrointestinal digestive system barriers, reticuloendothelial system barriers, and blood circulation system barriers, while barriers at the sub-organ level include vascular extravasation barriers and cell transport barriers.
  • the present invention provides a cross-linked polymer structure and nanomedicine with the polymer structure.
  • the polymer nanomedicine has a simple structure, relying on its special carboxyl (or carboxylate) and disulfide (diselenium) bond structure, to achieve the crossing of multiple biological barriers, effectively promote drug transport to target tissues, and achieve excellent therapeutic effects. conducive to clinical translation.
  • nano-drugs are protonated at a lower pH (1-3) in the stomach to form a dense aggregate structure, avoiding degradation and damage by various enzymes and reducing substances in the stomach, and promoting their arrival in the intestinal tract efficiency;
  • the nanomedicine is partially deprotonated under the condition of intestinal pH (4-6.5), showing negative charge, reducing its electrostatic interaction with negatively charged glycoproteins in the mucus, and avoiding being bound by the mucus , promote its diffusion in the mucus layer and penetrate the mucus layer; at the same time, the disulfide (diselenium) bond rich in the structure of the nano drug interacts with the cysteine-rich receptors on the surface of the intestinal epithelial cells to promote its Adhesion to intestinal epithelial cells, improving its transport in intestinal epithelial cells, enhancing intestinal epithelial cell absorption.
  • the negative charge on the surface can reduce the adsorption with serum protein and reduce the phagocytosis of the reticuloendothelial system; at the same time, its cross-linked structure can maintain the stability of the nano-particles and avoid blood Dilute and dissociate, prolong blood circulation time.
  • the disulfide (diselenium) bond it contains can interact with the protein containing cysteine on the cell surface, promote endocytosis into the cell, and increase the uptake of the nano-drug by the cell.
  • the nano-medicine is convenient to prepare, has stable performance, and is beneficial to industrial production.
  • a cross-linked polymer structure characterized in that it includes several basic units, the basic units are cross-linked by reduction-sensitive bonds, the basic units include basic units A and basic units B, and the structural formula of the basic units A for:
  • the structural formula of the basic unit B is:
  • X is a site available for crosslinking
  • R is a modifying group
  • the reduction-sensitive bond is a disulfide bond or a diselenide bond.
  • the mole percentage of the basic unit A in the basic unit in the polymer structure, n ranges from 0 ⁇ n ⁇ 100%.
  • the basic units form nanoparticles.
  • the particle size of the nanoparticles ranges from 10 to 300 nanometers.
  • R in at least one basic unit B is O ⁇ M + (M + is a metal ion).
  • R in at least one basic unit B is a specific functional group, including but not limited to one or more of targeting groups, membrane-penetrating peptides, and long-circulating molecules. kind.
  • the targeting group can be a cell membrane targeting group (glucose, transferrin, RGD, folic acid, hyaluronic acid), an organelle targeting group (TPP, quaternary One or more of ammonium salts, SS peptide, dexamethasone).
  • the membrane-penetrating peptide can be one or more of TAT, polyarginine, polyhistidine, Pep-7, C105Y and pVEC.
  • the long cycle molecule can be poly(N-(2-hydroxypropyl)methacrylamide, polyethylene glycol, chitosan, povidone, polycarboxylate One or several kinds of acid betaine.
  • the R may be a group with a targeting function, further, a group with a mitochondrial targeting function, such as a triphenylphosphine-containing functional group.
  • the basic unit includes multiple basic units B with different structures.
  • the polymer structure also contains other active substances, and further, the active substances are one or more of growth factors, drugs, and biological targeting substances.
  • the invention also discloses a preparation method of the above-mentioned polymer structure, which is characterized in that it comprises the following steps: suspending the raw material of the basic unit A in an aqueous solution, adding alkali to dissolve it completely, and then adjusting the pH of the solution with an acid to control Ratio of raw materials of basic unit A and its corresponding salts to obtain nanoparticles of different morphologies. Cross-linking the obtained nanoparticles to obtain cross-linked nanoparticles; as an optional method, further modifying the obtained cross-linked nanoparticles, so that part of the basic unit A is converted into a basic unit B containing functional group modification, The cross-linked nanoparticles modified by functional groups are obtained.
  • the present invention also discloses a preparation method of the above-mentioned polymer structure, which is characterized in that it comprises the following steps: firstly modify the raw material of the basic unit A to obtain the raw material of the basic unit B modified by the functional group; The raw material of A is suspended in the aqueous solution, and then the alkali is added to dissolve it completely, and then the pH of the solution is adjusted with acid, the ratio of the raw material of the basic unit A to its corresponding salt is controlled, and the raw material of the basic unit B modified by the functional group is added, and after mixing Cross-linking obtains cross-linked nanoparticles modified by functional groups.
  • This method uses the property that carboxylic acid can undergo protonation and deprotonation under different pH conditions to control the ratio of carboxylic acid and carboxylate, and successfully obtains nanoparticles with various shapes and stable structures, and its concentration can reach 100mg/mL , which is conducive to the realization of scale-up preparation and industrial production.
  • the invention also discloses the use of the above polymer structure, which is characterized in that it is used for preparing medicines.
  • the drug has at least one of the functions of anti-tumor, hypoglycemic, oxidative stress regulation, and metabolic regulation.
  • the polymer nanomedicine of the present invention has a simple structure, relies on its special carboxyl (or carboxylate) and disulfide (diselenium) bond structure to cross multiple biological barriers, and exhibits efficient in vivo transport efficiency and therapeutic effect.
  • the nanoparticles are protonated at a lower pH (1-3) in the stomach to form a dense aggregate structure, which avoids being absorbed by various enzymes or reducing substances in the stomach.
  • the nano-drug when entering the intestinal tract, the nano-drug is partially deprotonated under the condition of intestinal pH (4-6.5), presenting negative charge, reducing its interaction with the negative charge in the mucus Glycoproteins interact electrostatically to avoid being bound by mucus, promote their diffusion in the mucus layer and penetrate the mucus layer; at the same time, the disulfide (diselenium) bonds rich in the nano-drug structure and the cysteine-rich bonds on the surface of intestinal epithelial cells Interact with the receptors of amino acids to promote its adhesion to intestinal epithelial cells, improve its transport in intestinal epithelial cells, and enhance the absorption of intestinal epithelial cells.
  • the negative charge on the surface can reduce the adsorption with serum protein and reduce the phagocytosis of the reticuloendothelial system; at the same time, its cross-linked structure can maintain the stability of the nano-particles and avoid blood Dilute and dissociate, prolong blood circulation time.
  • the disulfide (diselenium) bond it contains can interact with the protein containing cysteine on the cell surface, promote endocytosis into the cell, and increase the uptake of the nano-drug by the cell.
  • the nanomedicine of the present invention may contain different functional groups to facilitate its realization of specific targeting functions, such as cell membrane targeting and organelle targeting.
  • the nano-medicine of the invention has the characteristics of convenient preparation and stable performance, which is beneficial to industrial production.
  • FIG. 1 is a particle size distribution diagram of different nanoparticles in Example 1.
  • Fig. 2 is the control of different pH on the particle size of nanoparticles in Example 1.
  • Fig. 3 is the dilution stability of different nanoparticles of Example 1.
  • Fig. 4 is the serum stability of different nanoparticles of Example 1.
  • FIG. 5 is a particle size distribution diagram of TPP@NP-25 in Example 2.
  • Fig. 6 is the Zeta potential of TPP@NP-25 under different pH conditions in Example 2.
  • Figure 7 shows the adhesion and coagulation efficiency of TPP@NP-25 and different contents of mucin in Example 2.
  • Figure 8 shows the efficiency of TPP@NP-25 across intestinal epithelial cells in Example 2.
  • Fig. 9 shows the intracellular fluorescence intensity of cells incubated with TPP@NP-25 for different time in Example 2.
  • Figure 10 is the fluorescence overlay (a) and colocalization rate (b) of C6#TPP@NP-25 and Mito-Tracker Red in Example 2.
  • Figure 11 is the plasma concentration curve of LA and TPP@NP-25 administered orally in Example 2.
  • Figure 12 is a graph of cell viability after Hela cells were incubated with TPP@NP-25 and NP-25 in Application Example 1.
  • Figure 13 is a graph of the cell viability after incubation of 4T1 cells with TPP@NP-25 and NP-25 in Application Example 1.
  • Figure 14 shows the inhibition rate of TPP@NP-25 and NP-25 on subcutaneous 4T1 tumors in application example 1.
  • Figure 15 is a particle size distribution diagram of GUA@NP-25 in Example 5.
  • Fig. 16 is a particle size distribution diagram of TAT@NP-25 in Example 6.
  • Figure 17 is a dilution stability diagram of TAT@NP-25 in Example 6.
  • Figure 18 is the serum stability graph of TAT@NP-25 in Example 6.
  • Figure 19 is a particle size distribution diagram of poly-HPMA@NP-25 in Example 7.
  • Figure 20 is a dilution stability diagram of poly-HPMA@NP-25 in Example 7.
  • Figure 21 is the serum stability graph of poly-HPMA@NP-25 in Example 7.
  • Fig. 22 is a particle size distribution diagram of GluN@NP-80 in Example 8.
  • Fig. 23 is a diagram of the in vitro blood-brain barrier crossing efficiency of GluN@NP-80 and NP-80 in Example 8.
  • Fig. 24 is the fluorescence image (a) and fluorescence quantitative image (b) of C6#GluN@NP-80 and C6#NP-80 entering tumor cells in Example 8.
  • Fig. 25 is the fluorescence diagram (a) and fluorescence quantitative analysis (b) of surviving cells after GluN@NP-80 and NP-80 acted in Example 8.
  • lipoic acid As an organic acid, lipoic acid has low solubility in water, and the concentration of lipoic acid nanoparticles constructed directly with it is low, easy to agglomerate and settle, and has poor storage stability, making it difficult to realize scale-up production.
  • the invention utilizes a lipoic acid and sodium lipoate mixed system to construct nanoparticles, and the obtained nanoparticles containing lipoic acid and sodium lipoate have good water solubility, high stability and adjustable size, and are easy to realize scale-up preparation and industrial production.
  • a. Dilution Stability Take 2mL of NP-25, NP-80 and NP-220 solutions, dilute them to 10, 100, 1000, 10000 times respectively, and then detect the particle size of the diluted nanoparticles by DLS. The experimental results show that the three nanoparticles still maintain the particle size before dilution when diluted to 10000 times ( FIG. 3 ), indicating that the above-mentioned nanoparticles have good dilution stability.
  • NP-25, NP-80 and NP-220 solutions and incubate with 10% (v/v) fetal bovine serum (FBS) for 1, 2, 6, 12 and 24 h respectively, and detect the concentration of nanoparticles after incubation by DLS. particle size.
  • FBS fetal bovine serum
  • particle size The experimental results showed that the particle size of NP-25, NP-80 and NP-220 did not change significantly even after being incubated with serum for 24 hours ( FIG. 4 ), indicating that the above-mentioned nanoparticles had good serum stability.
  • the proportion of carboxylic acid and carboxylate is controlled by the characteristic that carboxylic acid can undergo protonation and deprotonation under different pH conditions, and successfully obtained nanoparticles with various sizes and stable structures, and their concentration can reach 100mg/ mL, which is conducive to the realization of scale-up preparation and industrial production.
  • TPP-(CH 2 ) 3 -OH 3-Bromopropanol (1.0 g, 7.2 mmol) and triphenylphosphine (TPP) (2.1 g, 8.0 mmol) were dissolved in 25 mL of toluene, then heated to reflux for 24 h. After the reaction, the white precipitate was obtained by filtration, washed three times with ether, and dried in vacuum to obtain the product TPP-(CH 2 ) 3 -OH.
  • (3) The zeta potential of TPP@NP-25 in a pH 7.4 buffer solution (simulating normal body fluid environment such as blood) is about -23mV (Figure 6).
  • RES reticuloendothelial system
  • Mucus penetration assay and intestinal epithelial cell absorption assay were used to verify the ability of nanoparticles to cross the intestinal barrier.
  • Mucus penetration test (1) Incubate mucin and nanomedicine together, and evaluate the interaction between the two by detecting the amount of nanomedicine adsorbed by mucin. Specifically: uniformly disperse equal amounts of TPP@NP-25 in 0.1%, 0.3% and 0.5% mucin solutions, incubate at 37°C for 30 minutes after vortexing, and then centrifuge at 1500rpm for 2 minutes to obtain The pellet was washed twice with PBS. After the precipitate was treated, high performance liquid chromatography (HPLC) was used to measure LA in it to calculate the amount of TPP@NP-25.
  • HPLC high performance liquid chromatography
  • the Ussing chamber test confirmed that the nanomedicine has a good permeability, and the permeability coefficient is 3*10 -6 cm/s.
  • NP-25 was labeled with fluorescent molecule coumarin (C6) to obtain C6-labeled TPP@NP-25 (C6#TPP@NP-25).
  • Colon adenocarcinoma cells (Caco-2) were used as a model to evaluate the absorption of nanomedicine by intestinal epithelial cells.
  • Caco-2 cells in the active phase of logarithmic growth were selected and inoculated in 12-well plates. After 12 h of culture, 100 ⁇ g/mL of C6#TPP@NP-25 was added and incubated for 2, 6, and 12 h, respectively. After the incubation is complete, remove the old medium and wash the cells 3 times with PBS to remove uningested nanoparticles.
  • Intestinal mucus penetration test and epithelial cell uptake test confirmed that the nano-medicine has a better effect of crossing the intestinal barrier.
  • the nanoparticles in this example can effectively cross multiple physiological barriers and achieve effective drug delivery.
  • TPP@NP-25 reported in the present invention is increased by tens of times, which is much higher than that of other reported nano-medicines after introducing TPP (usually less than 5 times).
  • TPP@NP-25 has an excellent inhibitory effect on 4T1 cell line, further exploring the anti-tumor efficacy in vivo.
  • the subcutaneous tumor model was established using 4T1. When the tumor grew to about 50 mm 3 , they were randomly divided into 3 groups (5 rats in each group), which were the normal salt group, the NP-25 group and the TPP@NP-25 group. After administration, the tumor inhibition rate (64.8%) of TPP@NP-25 was 3.2 times that of NP-25 (20.4%) ( Figure 14), showing an excellent in vivo anti-tumor effect.
  • ApoE - / - mice fed with high-fat diet were used to establish an animal model of atherosclerosis to evaluate the effect of TPP@NP-25 on atherosclerosis.
  • 40 ApoE - / - mice were randomly divided into 4 groups (10 mice in each group), model group (normal saline Water treatment), atorvastatin (positive control) group, NP-25 group and TPP@NP-25 group, administered for 2 months. After the treatment, the ApoE - / - mice were euthanized, the aorta was taken out and fixed with paraformaldehyde, then cut longitudinally and stained with 0.3% oil red, and the plaque area was quantitatively analyzed.
  • a rat model of middle cerebral artery occlusion (MCAO) was constructed by suture closure to evaluate the effect of TPP@NP-25 on stroke.
  • Forty SD rats were randomly divided into 4 groups (10 rats in each group), which were model group (normal saline treatment), Edaravone (positive control) group, NP-25 group and TPP@NP-25 group.
  • Edaravone, NP-25 and TPP@NP-25 were administered by intraperitoneal injection 1 hour before model construction. Rats were sacrificed after perfusion for 24 hours, and the brain tissue was taken out and stained with 2,3,5-triphenyltetrazolium chloride (TTC).
  • TTC 2,3,5-triphenyltetrazolium chloride
  • mice 50 male C57BL/6 mice were randomly divided into 5 groups (10 mice in each group) according to body weight, namely sham operation group, model control group, bifenidone (positive control) group, NP-25 group and TPP@
  • the drugs were injected intraperitoneally according to the volume of 10 mL/kg, once a day for 15 consecutive times; the mice in the sham operation group and the model control group were injected intraperitoneally with the same volume of normal saline every day.
  • mice in the sham operation group were sacrificed 21 days after the model was established, and the degeneration and fibrosis of lung tissue cells were detected.
  • the alveolar structure of the mice in the bleomycin model group was disordered, the alveolar wall was significantly thickened, the infiltration of macrophages and lymphocytes in the alveolar septum was obvious, and more fibrous tissue hyperplasia was seen.
  • the inflammatory cell infiltration and parenchymal lesions in the lung tissue of the mice in the biphenidone group, the NP-25 group and the TPP@NP-25 group were significantly reduced, and the alveolar septum collagen fiber hyperplasia was significantly reduced.
  • the structure is basically normal.
  • Masson's trichrome staining showed that only a small amount of blue collagen fibers were deposited in the bronchiole wall and alveolar septum of mice in the sham operation group; a large number of blue-stained collagen fibers could be seen in and around the bronchiole wall, alveolar wall and septum of mice in the model group; The alveolar walls of the nitone group, NP-25 and TPP@NP-25 mice were slightly thickened, with only a small amount of inflammatory cell infiltration and blue-stained collagen fiber deposition.
  • NP-25 is not significantly different from that of the clinical drug Bifenidone, while the therapeutic effect of TPP@NP-25 is significantly better than that of NP-25 and biphenidone.
  • C57BL/KsJ db/db mice were used to establish a model of non-alcoholic fatty liver to evaluate the effect of TPP@NP-25 in the treatment of non-alcoholic fatty liver.
  • 50 C57BL/KsJ db/db mice were randomly divided into 5 groups (10 mice in each group) according to body weight.
  • Control group db/db model group, db/db+berberine (positive control) group, db/db+NP-25 group, db/db+TPP@NP-25 group (intraperitoneal injection of 10 mL/kg different doses of the drug, once a day for four consecutive weeks).
  • db/db mice are used as a diabetes model to evaluate the hypoglycemic efficacy of nanomedicine.
  • mice Thirty 8-week-old db/db mice were selected and randomly divided into 5 groups (6 mice in each group) according to body weight and blood sugar, namely db/db model group, db/db+LA group, db/db+NP- Group 25, db/db+TPP@NP-25 group and db/db+metformin (positive control) group, 6 littermate w/w male mice of the same week age were solvent control group.
  • the above drugs were given by intragastric administration at a volume of 10 mL/kg, and administered daily for 4 consecutive weeks.
  • the solvent control group and the model group were intragastrically administered equal volumes of normal saline. During the experimental period, the blood glucose changes of the mice were detected every 3 days.
  • NP-80 nanoparticles and NP-220 nanoparticles were prepared. EDCI and NHS were respectively added into the prepared nanoparticle solution, and after stirring for 2 hours, 10% molar amount of TPP-(CH 2 ) 3 -OH was added. After reacting for 48 h, the obtained nanoparticles were dialyzed to obtain TPP-containing nanoparticles (TPP@NP-80 and TPP@NP-220).
  • TPP@NP-80 and TPP@NP-220 had similar dilution stability and serum stability to TPP@NP-25.
  • the results show that TPP@NP-80 and TPP@NP-220 in this example have similar multi-organism spanning ability to TPP@NP-25 in Example 2.
  • Example 2 Its stability was verified by referring to the method of Example 1, and the results showed that GUA@NP-25 had excellent dilution stability and serum stability. Referring to the method described in Example 2 for performance and effect verification, the results show that GUA@NP-25 exhibits excellent ability to cross multiple biological barriers. Referring to Application Example 1, the efficacy of the nanoparticles in this example on tumors was verified, and the results showed that GUA@NP-25 and TPP@NP-25 in Example 2 had similar tumor inhibitory effects.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • TAT cell penetrating peptide
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • the poly-HPMA@NP-25 obtained above was labeled with coumarin (C6#poly-HPMA@NP-25) for macrophage uptake experiments.
  • Mouse-derived macrophages (RAW264.7) in the active phase of logarithmic growth were inoculated into 6-well plates, incubated in fresh serum-free medium for 2 hours, and then added C6#poly-HPMA@NP-25 and incubated for 2, 4, 8 hours. After incubation, remove the medium, wash with PBS 3 times, remove the unincorporated material, then collect the cells and perform quantitative analysis of the intracellular fluorescence intensity by flow cytometry. Cells, with the extension of the incubation time, the amount of cell entry only slightly increased, indicating that nanomedicine can effectively escape the uptake of phagocytes, thereby improving the long-term circulation ability.
  • the 4T1 tumor cell model was used to evaluate the in vitro anti-tumor effect of poly-HPMA@NP-25
  • the subcutaneous 4T1 tumor model was used to evaluate the in vivo anti-tumor effect of poly-HPMA@NP-25.
  • the experimental results show that the in vitro/in vivo anti-tumor effect of poly-HPMA@NP-25 in this example is significantly better than that of NP-25, which is consistent with the trend of TPP@NP25 and NP25.
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • Tanswell was placed in a 6-well plate, and mouse brain microvascular endothelial cells (bend3) were added to the upper layer of the chamber (1 ⁇ 10 5 cells).
  • the cell medium was changed every 2 days until the cell transmembrane resistance was greater than 200 ⁇ , which indicated that the in vitro blood-brain barrier was successfully established.
  • An equal amount of C6-loaded GlcN@NP-80 nanoparticles (C6#GlcN@NP-80) and C6-loaded NP-80 nanoparticles (C6#NP-80) were added to the upper layer of the chamber. After incubation for 6 hours, 2 mL of the medium in the lower layer of the chamber was taken to detect the fluorescence intensity, and the efficiency of nanomedicine to penetrate the blood-brain barrier was calculated.
  • I a is the fluorescence intensity of the lower chamber after the nanoparticles are incubated
  • I b is the fluorescence intensity of the nanoparticles when the nanoparticles are added
  • I c is the fluorescence intensity of the culture medium.
  • Tanswell was placed in a 6-well plate, and bend3 cells were added to the upper layer of the chamber (1 ⁇ 10 5 cells). Add 2 mL of complete medium to the upper layer of the small chamber, and 2.75 mL of complete medium to the lower layer of the small chamber. The cell medium was changed every 2 days until the cell transmembrane resistance was greater than 200 ⁇ , which indicated that the in vitro blood-brain barrier was successfully established. Selection of active human glioma cells (U87) Inoculate in 6-well plates and incubate for 24h at 37°C/5%CO 2 . After incubation, the medium was removed, and the constructed in vitro blood-brain barrier was placed in it, and two groups were set up.
  • group 1 equal amounts of C6#GlcN@NP-80 and C6#NP-80 were added to the upper layer of the above-mentioned small chamber, and after incubation for 24 hours, the amount of the above two nanoparticles entering U87 cells was analyzed by confocal laser.
  • group 2 the same amount of C6#GlcN@NP-80 and C6#NP-80 were added to the upper layer of the above-mentioned small chamber, and the surviving U87 cells were stained (viable cell dye) after incubation for 72 hours.
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • the nanoparticles obtained in this example also have excellent dilution stability and serum stability.
  • the performance and effect verification was carried out with reference to the method described in Example 2, and the results showed that the nanoparticles in this example have better ability to cross multiple biological barriers than the nanoparticles in the previous examples.
  • Example 1 Referring to the method of Example 1 for stability evaluation, the results show that the PTX#TPP@NP-25 prepared in this example has good dilution stability and serum stability; refer to Application Example 1 to verify the nanoparticles in this example The results show that at the same dose, PTX#TPP@NP-25 exhibits a stronger tumor inhibitory effect than PTX#NP-25.
  • the nanoparticles prepared in this example were tested for dilution stability and serum stability.
  • the results showed that the nanoparticles prepared in this example exhibited a dilution similar to that of the nanoparticles prepared in Example 1.
  • stability and serum stability Perform performance and effect verification with reference to the method described in Example 2.
  • the results show that the nanoparticles in this example can undergo charge changes under simulated different pH conditions and can successfully cross multiple biological barriers, indicating that cross-linking with diselenide bonds can achieve basically the same effect as cross-linking with disulfide bonds.

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Abstract

交联聚合物结构及其应用，属于生物医药技术领域；所述聚合物结构包含若干基本单元，所述基本单元之间通过还原敏感的二硫(或二硒)键连接；由此得到的交联聚合物纳米粒子具有跨越器官、亚器官、细胞和亚细胞层面等多重生物屏障的能力，增加纳米药物的递送效率从而提升疗效。

Description

一种交联聚合物结构及其制备方法与应用 技术领域
本发明属于生物医药技术领域，具体涉及一种交联聚合物结构及其制备方法与应用。
技术背景
纳米药物因量子尺寸效应和表界面效应等表现出独特的性能，为许多疾病治疗提供了新思路、新方法，大大促进了个性化医学与精准医疗的发展。纳米药物的广阔应用前景受到了广大科研工作者与药物研发企业的关注，纷纷投身其中。但纳米药物目前在临床应用过程中面临诸多问题，其中具有代表性的问题是递送效率。纳米药物与生物体之间的每一步相互作用都是一道障碍，而每一道障碍都可能使递送到目标位点的纳米药物数量降低。以口服纳米药物为例，其面临器官层面的屏障包括胃肠道消化系统屏障、网状内皮系统屏障、血液循环系统屏障等，而亚器官层面的屏障中则有血管外渗屏障以及细胞转运屏障等，进入靶部位后需要面临各种细胞层面的屏障，甚至还有亚细胞结构层面的屏障，如内含体逃逸和细胞器靶向等。针对每道屏障特点给纳米粒子引入相应克服屏障的功能是目前的主要解决方案，但所得纳米药物会变得相当复杂，不但大大增加制备难度，繁复的设计还限制了纳米药物的优势发挥，难以实现临床应用。
发明内容
针对以上问题，本发明提供了一种交联聚合物结构以及具有该聚合物结构的纳米药物。该聚合物纳米药物结构简单，依靠其特殊的羧基(或羧酸根)和二硫(二硒)键结构，实现多重生物屏障的跨越，有效地促进药物转运到达靶组织，实现优异的治疗效果，利于临床转化。
口服给药后，纳米药物在胃部较低pH(1～3)条件下发生质子化，形成致密团聚结构，避免被胃部的多种酶及还原性物质降解、破坏，提升其到达肠道的效率；当进入肠道后，纳米药物在肠道pH(4～6.5)条件下部分去质子化，呈现负电性，减少其与粘液中负电性的糖蛋白发生静电相互作用，避免被黏液束缚，促进其在黏液层的扩散并穿透黏液层；同时，纳米药物结构中富含的二硫(二硒)键与肠上皮细胞表面富含半胱氨酸的受体发生相互作用，促进其与肠上皮细胞粘附，提高其在肠上皮细胞中的转运，增强肠上皮细胞吸收。纳米药物经肠道吸收进入血液循环系统后，表面负电性可使其减少与血清蛋白的吸附，减少网状内皮系统的吞噬；同时，其交联结构可以维持纳米粒子的稳定性，避免因血液稀释而解离，延长 血液循环时间。纳米药物到达病变组织处后，其含有的二硫(二硒)键可与细胞表面含有半胱氨酸的蛋白相互作用，促进内吞入胞，提高细胞对纳米药物的摄取。
同时，该纳米药物制备方便，性能稳定，利于工业生产。
本发明包含以下技术方案：
一种交联聚合物结构，其特征在于，包括若干基本单元，所述基本单元之间通过还原敏感键交联，所述基本单元包括基本单元A和基本单元B，所述基本单元A的结构式为:
所述基本单元B的结构式为:
其中，X为可供交联的位点，R为改性基团。
作为可选方式，在上述聚合物结构中，所述还原敏感键为二硫键或二硒键。
作为可选方式，在上述聚合物结构中，所述聚合物结构中基本单元A在基本单元中所占摩尔百分比例n的范围为0≤n<100％。
作为可选方式，在上述聚合物结构中，所述基本单元形成纳米粒子。
作为可选方式，在上述聚合物结构中，纳米粒子的粒径范围为10～300纳米。
作为可选方式，在上述聚合物结构中，至少一种基本单元B中的R为O-M+(M+为金属离子)。
作为可选方式，在上述聚合物结构中，至少一种基本单元B中的R为具体的功能基团，包括但不限于靶向基团、穿膜肽、长循环分子中的一种或几种。
作为可选方式，在上述聚合物结构中，所述靶向基团可以为细胞膜靶向基团(葡萄糖、转铁蛋白、RGD、叶酸、透明质酸)、细胞器靶向基团(TPP、季铵盐、SS肽、地塞米松)中的一种或几种。
作为可选方式，在上述聚合物结构中，所述穿膜肽可为TAT、聚精氨酸、聚组氨酸、Pep-7、C105Y和pVEC中的一种或几种。
作为可选方式，在上述聚合物结构中，所述长循环分子可为聚(N-(2-羟丙基)甲基丙烯酰胺、聚乙二醇、壳聚糖、聚维酮、聚羧酸甜菜碱中的一种或几种。
作为可选方式，在上述聚合物结构中，所述R可为具有靶向功能的基团，进一步的，可以为具有线粒体靶向功能的基团，如含三苯基膦的功能基团。
作为可选方式，在上述聚合物结构中，所述基本单元包含多种具有不同结构的基本单元B。
作为可选方式，在上述聚合物结构中，所述聚合物结构中还含有其他活性物质，进一步的，所述活性物质为生长因子、药物、生物靶向物质中的一种或多种。
本发明还公开了一种上述聚合物结构的制备方法，其特征在于，包含以下步骤：将基本单元A的原料悬浮在水溶液中，随后加碱使其完全溶解后再用酸调节溶液pH，控制基本单元A的原料与其对应的盐的比例，以获得不同形态的纳米粒子。将上述所得纳米粒子进行交联，得到交联纳米粒子；作为可选方式，进一步的对所得交联纳米粒子进行改构处理，使部分基本单元A转变为含有功能基团修饰的基本单元B，得到功能基团修饰的交联纳米粒子。
本发明还公开了一种上述聚合物结构的制备方法，其特征在于，包含以下步骤：先对基本单元A的原料进行改构处理，获得功能基团修饰的基本单元B的原料；将基本单元A的原料悬浮在水溶液中，随后加入碱使其完全溶解后再用酸调节溶液pH，控制基本单元A的原料与其对应的盐的比例，再加入功能基团修饰的基本单元B原料，混合后交联获得功能基修饰的交联纳米粒子。
本方法利用羧酸可在不同pH条件下发生质子化和去质子化的特性控制羧酸和羧酸盐的比例，成功获得了形态多样、结构稳定的纳米粒子，同时其浓度可达100mg/mL，利于实现放大制备和工业化生产。
本发明还公开了一种上述聚合物结构的用途，其特征在于，将其用于制备药品。
作为可选方式，在上述应用中，所述药品具有抗肿瘤、降血糖、氧化应激调节、代谢调节功能中的至少一种。
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了互相排斥的特征和/或步骤以外，均可以以任何方式组合。
本发明有益效果：
本发明的聚合物纳米药物结构简单，依靠其特殊的羧基(或羧酸根)和二硫(二硒)键结构跨越多重生物屏障，展现出高效的体内转运效率及治疗效果。口服给药后，纳米粒子在胃部较低pH(1～3)条件下发生质子化，形成致密团聚结构，避免被胃部多种酶或还原性物 质降解、破坏，提升其到达肠道的效率；当进入肠道后，纳米药物在肠道pH(4～6.5)条件下部分去质子化，呈现负电性，减少其与粘液中带负电性的糖蛋白发生静电相互作用，避免被黏液束缚，促进其在黏液层的扩散并穿透黏液层；同时，纳米药物结构中富含的二硫(二硒)键与肠上皮细胞表面富含半胱氨酸的受体发生相互作用，促进其与肠上皮细胞粘附，提高其在肠上皮细胞中的转运，增强肠上皮细胞吸收。纳米药物经肠道吸收进入血液循环系统后，表面负电性可使其减少与血清蛋白的吸附，降低网状内皮系统的吞噬；同时，其交联结构可以维持纳米粒子的稳定性，避免因血液稀释而解离，延长血液循环时间。纳米药物到达病变组织处后，其含有的二硫(二硒)键可与细胞表面含有半胱氨酸的蛋白相互作用，促进内吞入胞，提高细胞对纳米药物的摄取。
本发明所述纳米药物可含不同功能基团，促进其实现特定的靶向功能，如细胞膜靶向、细胞器靶向。
本发明所述纳米药物具有制备方便，性能稳定的特点，利于工业化生产。
附图说明：
图1为实施例1中不同纳米粒子的粒径分布图。
图2为实施例1中不同pH对纳米粒子粒径的控制。
图3为实施例1不同纳米粒子的稀释稳定性。
图4为实施例1不同纳米粒子的血清稳定性。
图5为实施例2中TPP@NP-25的粒径分布图。
图6为实施例2中TPP@NP-25在不同pH条件下的Zeta电位。
图7为实施例2中TPP@NP-25与不同含量的黏蛋白粘附聚沉效率。
图8为实施例2中TPP@NP-25跨肠道上皮细胞效率。
图9为实施例2中细胞与TPP@NP-25孵育不同时间后的胞内荧光强度。
图10为实施例2中C6#TPP@NP-25与Mito-Tracker Red的荧光重叠图(a)及共定位率(b)。
图11为实施例2中LA和TPP@NP-25口服的血药浓度曲线。
图12为应用例1中Hela细胞与TPP@NP-25和NP-25孵育后的细胞存活率图。
图13为应用例1中4T1细胞与TPP@NP-25和NP-25孵育后的细胞存活率图。
图14为应用例1中TPP@NP-25和NP-25对皮下4T1肿瘤的抑制率。
图15为实施例5中GUA@NP-25的粒径分布图。
图16为实施例6中TAT@NP-25的粒径分布图。
图17为实施例6中TAT@NP-25的稀释稳定性图。
图18为实施例6中TAT@NP-25的血清稳定性图。
图19为实施例7中poly-HPMA@NP-25的粒径分布图。
图20为实施例7中poly-HPMA@NP-25的稀释稳定性图。
图21为实施例7中poly-HPMA@NP-25的血清稳定性图。
图22为实施例8中GluN@NP-80的粒径分布图。
图23为实施例8中GluN@NP-80和NP-80跨体外血脑屏障效率图。
图24为实施例8中C6#GluN@NP-80和C6#NP-80进入肿瘤细胞后的荧光图(a)及荧光定量图(b)。
图25为实施例8中GluN@NP-80和NP-80作用后，存活细胞荧光图(a)及荧光定量分析(b)。
具体实施方式：
以下通过实施例的具体实施方式再对本发明的上述内容作进一步的详细说明。但不应当将此理解为本发明上述主题的范围仅限于以下的实例。在不脱离本发明的精神和原则之内做的任何修改，以及根据本领域普通技术知识和惯用手段做出的等同替换或者改进，均应包括在本发明的保护范围内。
实施例1：
硫辛酸作为一种有机酸，在水中溶解度低，直接利用其构筑的硫辛酸纳米粒子浓度低，易团聚沉降，储存稳定性差，难以实现放大生产。本发明利用硫辛酸和硫辛酸钠混合体系构筑纳米粒子，所得含硫辛酸和硫辛酸钠的纳米粒子水溶性好、稳定性高、尺寸可调，易于实现放大制备和工业化生产。
将1.03g硫辛酸悬浮在8mL水溶液中，随后加入NaOH使其完全溶解，用HCl调节溶液pH，并定容到10mL，再超声振荡1～3h后，形成纳米粒子。在制备过程中，将pH调节到7.4，形成了粒径～25nm的纳米粒子；将pH调节到6.5，形成了粒径～80nm的纳米粒子；将pH调节到5.8，形成了粒径～220nm的纳米粒子。将上述纳米粒子通过365nm紫外光照交联，分别得到的交联纳米粒子NP-25(图1a)、NP-80(图1b)和NP-220(图1c)。
进一步研究了pH对形成纳米粒子的粒径控制：将412mg(2mmol)硫辛酸悬浮在18mL水溶液中，随后加入NaOH使其完全溶解，再定容到20mL，配制为100mM硫辛酸钠溶液。将上述溶液分为20组样品，每组1mL，分别加入0,0.05,0.1,…,0.9,0.95,1.0当量的盐酸，静置三天后测量各组溶液pH和粒径，绘制pH和粒径的曲线，探究pH对形成纳米粒子的粒 径的影响。结果如图2所示，纳米粒子的尺寸和pH负相关，随着溶液pH值的降低，纳米粒子尺寸增加，表明通过控制pH，可轻易控制形成不同尺寸的纳米粒子。
纳米粒子稳定性测定：
a.稀释稳定性。取2mL的NP-25、NP-80和NP-220溶液，分别将其稀释至10、100、1000、10000倍后通过DLS检测稀释后纳米粒子的粒径。实验结果显示，三种纳米粒子在稀释至10000倍时仍维持稀释前粒径(图3)，表明上述纳米粒子有很好的稀释稳定性。
b.血清稳定性。取2mL的NP-25、NP-80和NP-220溶液，分别与10％(v/v)胎牛血清(FBS)孵育1、2、6、12和24h，通过DLS检测孵育后纳米粒子的粒径。实验结果显示NP-25、NP-80和NP-220即使与血清孵育24h后粒径未发生明显变化(图4)，表明上述纳米粒子有很好的血清稳定性。
本实施例利用羧酸可在不同pH条件下发生质子化和去质子化的特性控制羧酸和羧酸盐的比例，成功获得了尺寸多样、结构稳定的纳米粒子，同时其浓度可达100mg/mL，利于实现放大制备和工业化生产。
实施例2：
TPP-(CH2)3-OH的合成。将3-溴丙醇(1.0g，7.2mmol)和三苯基膦(TPP)(2.1g，8.0mmol)溶解于25mL甲苯中，随后加热回流24h。反应结束后，过滤得到白色沉淀，并用乙醚洗涤三次，真空干燥得到产物TPP-(CH2)3-OH。
在制备好的NP-25溶液中加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDCI)和N-羟基琥珀酰亚胺(NHS)，搅拌2h后，再加入10％摩尔量的TPP-(CH2)3-OH。反应48h后，将得到的纳米粒子进行透析，最后得到含有TPP的纳米粒子(TPP@NP-25)，其粒径约为40nm(图5)。
1.不同生理pH条件下TPP@NP-25的zeta电位变化
(1)TPP@NP-25在pH＝3的溶液(模拟胃部pH环境)中发生聚集，表明其具有在较强酸性条件下聚集的特性，防止纳米药物在胃部被各种酶或还原性物质降解、破坏，促进其 通过胃部并蠕动到肠道。(2)TPP@NP-25在pH＝6.5的磷酸盐缓冲液(模拟肠道pH环境)中未发生聚集，其zeta电位约为-15mV(图6)。纳米粒子在pH＝6.5条件下显电负性，可减少其与粘液中带负电荷的糖蛋白发生静电相互作用，避免被黏液束缚，促进其扩散并穿透黏液层，增强其被肠上皮细胞摄取，进而转运进入血液。(3)TPP@NP-25在pH＝7.4的缓冲液(模拟血液等正常体液环境)的zeta电位约-23mV(图6)。纳米粒子在pH＝7.4条件下显示更强的电负性，可有效降低其与血液蛋白的结合，进而避免被网状内皮系统(RES)清除。
2.跨肠道屏障试验
粘液渗透试验和肠道上皮细胞吸收试验用于验证纳米粒子跨肠道屏障能力。
粘液渗透试验：(1)将粘蛋白和纳米药物共同孵育，通过检测粘蛋白吸附纳米药物的量，评估二者的相互作用。具体为：将等量的TPP@NP-25分别均匀分散在0.1％、0.3％和0.5％的粘蛋白溶液中，涡旋振荡后在37℃条件下孵育30min，之后以1500rpm转速离心2min，得到的沉淀用PBS洗涤两次。沉淀物处理后，采用高效液相色谱仪(HPLC)测定其中LA用以计算TPP@NP-25的量。结果显示仅有较少量纳米药物与粘蛋白相互作用形成聚集体，即使在0.5％粘蛋白浓度下，仅有11.2％的纳米药物与粘蛋白发生相互作用(图7)，表明纳米药物不易与粘蛋白吸附。(2)通过尤斯灌流室(Ussing chamber)评估TPP@NP-25在粘液层的渗透能力。具体为：取10uL粘液均匀地放入尤斯灌流室中覆盖有膜的长方形端口中，向供体填充3mL的含有TPP@NP-25的Krebs-ringer缓冲液，受体用3mL空白Krebs-ringer缓冲液填充。将两侧的溶液连续通入气体(95％O2和5％CO2)，并使用循环水浴将装置保持在37℃。在确定的时间点，从受体取出等分样(0.2mL)，并补充相同体积(0.2mL)Krebs-ringer缓冲液。通过HPLC测定渗透的纳米药物的量，并通过公式计算渗透系数：
Ussing chamber试验证实，纳米药物具有较好的渗透作用，渗透系数为3*10-6cm/s。
上述试验结果显示，TPP@NP-25具有优异的粘液渗透作用。
上皮细胞吸收试验：将NP-25用荧光分子香豆素(C6)标记，得到C6标记的TPP@NP-25(C6#TPP@NP-25)。以结肠腺癌细胞(Caco-2)为模型评估肠道上皮细胞对纳米药物吸收情况。选择处于对数生长活跃期的Caco-2细胞接种于12孔板，培养12h后，加入100μg/mL的C6#TPP@NP-25分别孵育2、6、12h。孵育完成后，去除旧培养基，用PBS洗涤细胞3次，以清除未摄取的纳米粒子。采用酶标仪测定每个孔C6(λex＝430nm，λem＝485nm) 的荧光强度(FL)并计算摄取率。细胞摄取率E＝FL胞内纳米粒子/FL初始纳米粒子。结果显示Caco-2细胞对TPP@NP-25具有较好的摄取作用，在2h已有约40％的摄取率，且摄取率随孵育时间增加而增加(图8)。
肠道粘液渗透试验和上皮细胞摄取试验证实，该纳米药物具有较好的跨越肠道屏障的作用。
4.跨细胞屏障
通过细胞流式仪定量分析纳米药物入胞情况。选择处于活跃期的人肾上皮细胞(293T)细胞接种于6孔板中，在37℃/5％CO2条件下孵育24h。孵育后，去除培养基，加入含C6#TPP@NP-25的新鲜培养基，未加任何材料的细胞为空白对照。分别孵育2、4、8h后，去除培养基，用PBS洗3次，以除去未入胞的材料。采用胰酶消化细胞，将每孔细胞分别收集在1.5mL EP管中。将收集好的细胞再用PBS洗2次，最后将所得的细胞分散在300μLPBS中。通过细胞流式仪进行细胞内荧光强度定量分析，如图9所示，纳米药物与细胞作用2h后能有效入胞，且入胞量随孵育时间的延长而增加，表明其具有较好的跨细胞屏障的能力。
5.跨细胞器屏障
通过激光共聚焦(CLSM)对纳米粒子进入线粒体情况进行分析。选择处于活跃期的人肝癌细胞(HpG-2)细胞接种于玻底皿中，在37℃/5％CO2条件下孵育24h。孵育后，去除培养基，加入含C6#TPP@NP-25的新鲜培养基孵育相应时间。孵育后，再用Mito-Tracker Red将线粒体标记为红色。采用CLSM观察，结果显示，C6#TPP@NP-25与Mito-Tracker Red的荧光高度重叠，相关系数可达0.70(图10)，表明纳米粒子能较好的富集于线粒体，具有跨线粒体屏障能力。
6.药代动力学研究
选择正常雄性SD大鼠(体重250±10g)适应性喂养，一周后分别口服给药相同剂量的硫辛酸(LA)和TPP@NP-25。给药后，在固定时间点从眼眶静脉丛取血0.5mL于含EDTA的采血管中。血液样本经理后，通过HPLC测定相应药物浓度，并绘制药时曲线(图11)。结果显示，LA的血液半衰期仅0.5h，而TPP@NP-25的半衰期为14h。进一步，分别静脉注射LA和TPP@NP-25，得到二者的药时曲线下面积(AUC)，并计算二者的绝对生物利用度(F)。
F＝AUC口服/AUC静脉
最后计算得到LA和TPP@NP-25的绝对生物利用度分别为15％和40％。上述实验结果 表明本实施例制备得到的纳米药物(TPP@NP-25)相对于常规的口服药物(LA)具有更好的跨越生理屏障进入血液系统能力和长循环能力。
综上所述，本实施例中纳米粒子可有效跨越多重生理屏障，实现有效的药物递送。
应用例1：
1.纳米药物对肿瘤细胞抑制作用
选择处于活跃期的Hela细胞接种于96孔板中，在37℃/5％CO2条件下孵育24h。孵育后，去除培养基，分别加入含NP-25和TPP@NP-25的新鲜培养基，未加任何材料的细胞为空白对照。孵育72h后，采用MTT法对细胞存活率进行评估，并计算IC50(半数抑制浓度)。如图12显示，随着NP-25和TPP@NP-25浓度的增加，Hela细胞的存活率降低。特别地，TPP@NP-25(IC50＝50.31μM)的抑制效果相较于NP-25(IC50＝2859.37μM)提高了54.5倍。
选择处于活跃期的小鼠乳腺癌细胞(4T1)接种于96孔板中，在37℃/5％CO2条件下孵育24h。孵育后，去除培养基，分别加入含NP-25和TPP@NP-25的新鲜培养基，未加任何材料的细胞为空白对照。孵育72h后，采用MTT法对细胞存活率进行评估，并计算IC50。如图13显示，随着NP-25和TPP@NP-25浓度的增加，4T1细胞的存活率降低。特别地，TPP@NP-25(IC50＝32.56μM)的抑制效果相较于NP-25(IC50＝1523.42μM))提高了46.9倍。
本发明报道的TPP@NP-25抗肿瘤效果提升数十倍，远高于已报道的其他纳米药物引入TPP后的提升幅度(通常小于5倍)。
2.纳米药物体内肿瘤抑制作用
TPP@NP-25对4T1细胞系具有优异的抑制作用，进一步进行体内抗肿瘤疗效探索。采用4T1进行皮下肿瘤模型建立。当肿瘤长至约50mm3时，随机将其分为3组(每组5只)，分别为生理盐数组、NP-25组和TPP@NP-25组。给药结束后，TPP@NP-25的肿瘤抑制率(64.8％)是NP-25的(20.4％)的3.2倍(图14)，展现了优异的体内抗肿瘤效果。
应用例2：
探究纳米药物在氧化应激相关疾病治疗中的应用，以动脉粥样硬化、中风、肺纤维化和非酒精性脂肪肝为例。
以高脂饲料喂养的ApoE-/-小鼠构建动脉粥样硬化动物模型，以评估TPP@NP-25治疗脉粥样硬化的效果。将40只ApoE-/-小鼠随机分成4组(每组10只)，分别为模型组(生理盐 水处理)、阿托伐他汀(阳性对照)组、NP-25组和TPP@NP-25组，给药治疗2个月。治疗结束后，ApoE-/-小鼠被处安乐死，取主动脉并用多聚甲醛固定，然后纵向剖开后用0.3％油红染色，并对斑块面积进行定量分析。实验结果显示，与模型组相比，NP-25和TPP@NP-25组均显示出油红染色区域(阳性区域)降低的趋势。其中，NP-25的治疗效果与阳性对照阿托伐他汀无显著性差异，TPP@NP-25的疗效明显优于NP-25和阿托伐他汀。
采用缝合封闭法构建大鼠大脑中动脉闭塞(MCAO)模型，以评估TPP@NP-25治疗中风的效果。将40只SD大鼠随机分成4组(每组10只)，分别为模型组(生理盐水处理)、依达拉奉(阳性对照)组、NP-25组和TPP@NP-25组，在模型构建前1小时通过腹腔注射给予依达拉奉、NP-25和TPP@NP-25。灌注24h后处死大鼠，取出脑组织用2,3,5-三苯基氯化四唑(TTC)染色。结果显示，相对于模型组，NP-25和TPP@NP-25组大鼠的脑组织梗死区域明显减小。其中，NP-25组的治疗效果与阳性对照依达拉奉相当，而TPP@NP-25的治疗效果显著优于NP-25和依达拉奉。
以博莱霉素气道滴注方式构建实验性肺纤维化小鼠模型，以评估TPP@NP-25治疗肺纤维化的效果。将50只雄性C57BL/6小鼠按体重随机分为5组(每组10只)，分别为假手术组、模型对照组、比菲尼酮(阳性对照)组、NP-25组和TPP@NP-25组，造模第7天起，按l0mL/kg体积腹腔注射给予分组设置药物，每天1次，连续15次；假手术组及模型对照组小鼠每日腹腔注射同体积生理盐水。实验动物于造模21天后处死小鼠，对肺组织细胞变性和纤维化状况进行检测。结果证实，假手术组小鼠肺组织结构清晰，肺泡呈多边形囊状小体，肺泡腔内无分泌物，肺泡上皮结构完整，肺泡壁未见增厚，肺泡隔中未见炎性细胞，可见丰富毛细血管。博莱霉素模型组小鼠肺泡结构紊乱，肺泡壁明显增厚，肺泡隔中巨噬细胞和淋巴细胞浸润明显，并可见较多的纤维组织增生。与模型组相比，比菲尼酮组、NP-25组和TPP@NP-25组小鼠肺组织内炎性细胞浸润及肺组织实质病变明显减少，肺泡隔胶原纤维增生灶明显降低，肺泡结构基本正常。Masson's trichrome染色显示，假手术组小鼠细支气管壁及肺泡隔仅有少量蓝色胶原纤维沉积；模型组小鼠细支气管管壁及周围，肺泡壁及间隔可见大量的蓝染胶原纤维；比菲尼酮组、NP-25和TPP@NP-25小鼠肺泡壁有轻度增厚，仅有少量炎症细胞浸润和蓝染的胶原纤维沉积。进一步分析定量，针对上述肺纤维化小鼠病理特点，相同剂量下，NP-25的治疗效果与临床药物比菲尼酮无显著性差异，而TPP@NP-25治疗效果显著优于NP-25和比菲尼酮。
以C57BL/KsJ db/db小鼠构建非酒精性脂肪肝的模型，以评估TPP@NP-25治疗非酒精性脂肪肝的效果。将50只C57BL/KsJ db/db小鼠按体重随机分为5组(每组10只)分别为db/m 对照组、db/db模型组、db/db+小檗碱(阳性对照)组、db/db+NP-25组、db/db+TPP@NP-25组(按10mL/kg体积分别腹腔注射给予不同剂量的药物，每天一次，连续四周)。实验动物于第4周末给药1h后对肝组织炎症、细胞脂肪样变、附睾脂肪细胞肥大和脂质沉积情况进行检测。结果显示，db/m对照组小鼠肝小叶结构清晰，肝细胞索由中央静脉向四周整齐排列，附睾脂肪细胞大小正常；db/db模型组小鼠可见肝细胞质疏松、空泡变性，肝实质有大量脂滴聚集，同时脂肪细胞明显肥大。小檗碱组、NP-25组和TPP@NP-25均能明显减轻肝脏脂肪变程度，对炎性细胞浸润以及脂肪细胞肥大有明显抑制作用。db/m对照组小鼠肝小叶结构完整，肝细胞索整齐排列，无明显脂质沉积；db/db模型组小鼠肝小叶结构别破坏，肝实质有大量脂质沉积。进一步定量分析显示，NP-25和TPP@NP-25对肝脏脂质蓄积均有不同程度的抑制作用。其中，NP-25组的抑制效果与阳性药物小檗碱相当，而TPP@NP-25的抑制效果显著优于NP-25和小檗碱。
上述结果证实，本发明所述纳米药物能较好的用于氧化应激相关疾病的治疗，具有良好的临床应用前景。
应用例3：
本应用例利用db/db小鼠作为糖尿病模型来评价纳米药物的降糖疗效。
选取8周龄的db/db小鼠30只，按体重和血糖随机分为5组(每组6只)，分别为db/db模型组、db/db+LA组、db/db+NP-25组、db/db+TPP@NP-25组和db/db+二甲双胍(阳性对照)组，同周龄大小的同窝w/w雄性小鼠6只，为溶剂对照组。按10mL/kg体积分别灌胃给予上述药物，每天给药，连续4周，溶剂对照组和模型组灌胃给予等体积的生理盐水。实验周期内，每3天检测一次小鼠血糖变化。结果显示，相比较模型组，LA无降糖效果，NP-25和TPP@NP-25均显著降低了小鼠血糖含量，其中NP-25降糖效果与阳性对照组二甲双胍相当，而TPP@NP-25显著优于二甲双胍。为进一步考察纳米药物对胰岛素抵抗的改善效果，治疗结束后2h后，将小鼠眼球摘除放血处死，常温离心收集血清样本，采用小鼠胰岛素(Insulin)ELISA检测试剂盒检测血清胰岛素含量，计算胰岛素抵抗指数。结果显示，二甲双胍组胰岛素抵抗指数与NP-25组无显著差异，而明显高于TPP@NP-25组，与降糖结果一致。上述结果显示本发明所述纳米药物能较好的用于糖尿病的降糖治疗，具有良好的临床前景。
实施例3：
在氮气条件下将1.24g(6mmol)硫辛酸(LA)、1.44g(7.5mmol)EDCI和0.92g(7.5mmol)4-二甲氨基吡啶(DMAP)溶解于50mL的二氯甲烷(DCM)中，并搅拌2h；随后 加入1.61g(5mmol)TPP-(CH2)3-OH室温搅拌16h。反应混合物通过旋转蒸发仪除去有机溶剂，残留物再用乙醚洗涤，最后真空干燥去除残留溶剂，得到产物LA-TPP。
将0.93g(4.5mmol)硫辛酸悬浮在8mL水中，随后加入NaOH使其完全溶解后再用HCl调节溶液pH为7.4，随后定容到100mg/mL，再加入0.5mmol的LA-TPP，超声振荡1-3h后，使其组装为均一的纳米粒子。将所得纳米粒子通过365nm紫外光照交联，透析48h后得到交联纳米粒子，其粒径约为40nm左右，参照实施例1、2对其性能进行表征，结果显示本实施例制备的纳米粒子与实施例2中的TPP@NP-25基本保持一致，表明两种不同的方法制备的纳米粒子结构和性能基本一致。
实施例4：
参照实施例1制备得到NP-80纳米粒子和NP-220纳米粒子。在制备好的纳米粒子溶液中分别加入EDCI和NHS，搅拌2h后，再加入10％摩尔量的TPP-(CH2)3-OH。反应48h后，将得到的纳米粒子进行透析，最后得到含有TPP的纳米粒子(TPP@NP-80和TPP@NP-220)。
参照实施例1的方法验证其稳定性，结果显示TPP@NP-80和TPP@NP-220具有与TPP@NP-25类似的稀释稳定性和血清稳定性。参照实施例2所述方法进行进行性能和效果验证，结果显示，本实施例中的TPP@NP-80和TPP@NP-220具有与实施例2中的TPP@NP-25相似的跨越多重生物屏障的能力；参照应用例1，验证本实施例中的纳米粒子对于肿瘤的疗效，结果显示TPP@NP-80和TPP@NP-220具有与实施例2中的TPP@NP-25相当的肿瘤抑制效果，表明不同硫辛酸纳米粒子的粒径对其治疗效果影响不大。
实施例5：
将113μL(1.7mmol)乙二胺溶解在30mL无水DCM中，随后加入2.5g(1.7mmol)1H-吡唑-1-甲脒盐酸盐，并在室温条件下搅拌4h。反应后，通过旋转蒸发仪去除有机溶剂。残留物采用0.5mL甲醇溶解，并继续加入10mL乙醚进行结晶沉淀，过滤得到的固体用乙醚洗涤，最后得到乙二胺-GUA。
在制备好的NP-25溶液中加入EDCI和NHS搅拌2h后，再加入10％摩尔量的乙二胺-GUA。反应48h后，将得到的纳米粒子进行透析，最后得到含有胍基的纳米粒子(GUA@NP-25)，其粒径为35nm左右(图15)。
参照实施例1的方法验证其稳定性，结果显示GUA@NP-25具有优异的稀释稳定性和血清稳定性。参照实施例2所述方法进行性能和效果验证，结果显示GUA@NP-25展现了优异的跨越多重生物屏障的能力。参照应用例1验证本实施例中的纳米粒子对于肿瘤的疗效，结果显示GUA@NP-25与实施例2中的TPP@NP-25具有类似的肿瘤抑制效果。
实施例6：
在制备好的NP-25溶液中加入EDCI和NHS，搅拌2h后，再加入10％摩尔量的细胞穿膜肽(TAT)。反应48h后，将得到的纳米粒子进行透析，制得含有TAT的纳米粒子(TAT@NP-25)，其粒径约为50nm(图16)。
参照实施例1的方法验证其稳定性，结果显示TAT@NP-25具有良好的稀释稳定性(图17)和血清稳定性(图18)。参照实施例2所述方法进行性能和效果验证，结果显示，本实施例中的纳米粒子在模拟的不同pH条件下可发生电荷改变，具有与前述TPP@NP-25纳米粒子类似的跨越多重生物屏障的潜力。本实施例中的纳米粒子具有更好的跨膜入胞效果。参照应用例2，验证本实施例中的纳米粒子对于氧化应激相关疾病的治疗效果，结果显示本实施例中的纳米粒子与实施例2中的TPP@NP-25疗效相当。
实施例7：
在25mL小瓶中加入N-(2-羟丙基)甲基丙烯酰胺(HPMA，1.0g)，以及2,2'-[硫代羰酰(硫)]双[2-甲基丙酸](0.0198g)和DMSO(1.2mL)。搅拌2min后加入三乙基硼烷(0.2mL)。最后，在室温下继续搅拌15min，得到聚HPMA(poly-HPMA)。
在制备好的NP-25溶液中加入EDCI和NHS，搅拌2h后加入10％的poly-HPMA。反应48h后，将得到的纳米粒子透析，制得含有poly-HPMA的纳米粒子(poly-HPMA@NP-25)，其粒径约为50nm(图19)。参照实施例1的方法评估所得纳米粒子稳定性，结果显示poly-HPMA@NP-25具有良好的稀释稳定性(图20)和血清稳定性(图21)。
将上述所得的poly-HPMA@NP-25用香豆素标记(C6#poly-HPMA@NP-25)，用于巨噬细胞摄取实验。将处于对数生长活跃期的鼠源巨噬细胞(RAW264.7)接种于6孔板中，在新鲜无血清培养基中孵育2h，然后加入C6#poly-HPMA@NP-25分别孵育2、4、8h。孵育后，去除培养基，用PBS洗3次，除去未入胞的材料，再将细胞收集并通过细胞流式仪进行细胞内荧光强度定量分析，结果显示纳米药物与细胞作用2h后仅少量进入细胞，随孵育时间的延长，其入胞量仅微弱增加，表明纳米药物可有效地逃避吞噬细胞摄取，进而提升长循环能力。
参照应用例1，采用4T1肿瘤细胞模型评估poly-HPMA@NP-25的体外抗肿瘤效果，以皮下4T1肿瘤模型评估poly-HPMA@NP-25的体内抗肿瘤效果。实验结果显示，本实施例中的poly-HPMA@NP-25的体外/体内抗肿瘤效果显著优于NP-25，与TPP@NP25和NP25的趋势一致。
实施例8:
在制备好的NP-80溶液中加入EDCI和NHS，搅拌2h后，再加入10％摩尔量的氨基葡萄糖(GlcN)。反应48h后，将得到的纳米粒子进行透析，制得含有GlcN的纳米粒子(GlcN@NP-80)，其粒径为100nm左右(图22)。
将Tanswell放置于6孔板中，并将小鼠脑微血管内皮细胞(bend3)加入小室上层(1×105个细胞)。小室上层加入2mL含完全培养基，小室下层加入2.75mL完全培养基。每2天进行细胞换液，直至细胞跨膜电阻大于200Ω，表明成功建立体外血脑屏障。将等量的负载C6的GlcN@NP-80的纳米粒子(C6#GlcN@NP-80)以及负载C6的NP-80的纳米粒子(C6#NP-80)分别加入上述小室上层。孵育6h后，分别取2mL小室下层培养基检测其荧光强度，并计算纳米药物穿透血脑屏障效率。
穿透效率(E)＝(Ia-Ic)/(Ib-Ic)*100％
其中Ia为纳米粒子孵育后下室的荧光强度，Ib是纳米粒子纳米粒子加入时的荧光强度，Ic培养基的荧光强度。
实验结果如图23所示，GlcN@NP-80进入小室下层的量是NP-80的4.2倍，表明其具有优异的跨血脑屏障能力。
将Tanswell放置于6孔板中，将bend3细胞加入小室上层(1×105个细胞)。小室上层加入2mL含完全培养基，小室下层加入2.75mL完全培养基。每2天进行细胞换液，直至细胞跨膜电阻大于200Ω，表明成功建立体外血脑屏障。选择处于活跃期的人脑胶质瘤细胞(U87) 接种于6孔板中，在37℃/5％CO2条件下孵育24h。孵育后，去除培养基，并将已构建好的体外血脑屏障置入其中，并设置2组。第1组是将等量的C6#GlcN@NP-80和C6#NP-80分别加入上述小室上层，孵育24h后，通过激光共聚焦分析上述两种纳米粒子进入U87细胞的量。第2组是将等量的C6#GlcN@NP-80和C6#NP-80分别加入上述小室上层，孵育72h后对存活的U87细胞进行染色(活细胞染料)。实验结果显示，GlcN@NP-80的入胞量是NP-80的7.3倍(图24)，细胞毒性是NP-80的7倍(图25)，表明GlcN@NP-80能更有效地穿透血脑屏障进入肿瘤细胞并高效杀死肿瘤细胞。
实施例9：
在制备好的NP-220溶液中加入EDCI和NHS搅拌2h后，再加入TPP-(CH2)3-OH和TAT反应48h，最后透析制备得到含有两种功能基团的纳米药物。
参照实施例1验证其稳定性，本实施例得到的纳米粒子同样具有优异的稀释稳定性和血清稳定性。参照实施例2所述方法进行进行性能和效果验证，结果显示，本实施例中纳米粒子比前述实施例中纳米粒子具有更优异的跨越多重生物屏障的能力。
实施例10：
将1.0g硫辛酸悬浮在8mL水溶液中，随后加入NaOH使其完全溶解后再用HCl调节溶液pH为7.4，随后定容到100mg/mL。再将疏水抗肿瘤药物紫杉醇溶解于甲醇并分散于上述溶液中，再超声振荡1～3h后光照，可制备得到负载紫杉醇的纳米粒子(PTX#NP-25)。进一步在该溶液中加入EDCI和NHS，反应2h后再加入TPP-(CH2)3-OH继续反应48h。反应结束后，将上述溶液透析48h，最后得到含TPP的负载紫杉醇的纳米粒子(PTX#TPP@NP-25)。
参照实施例1的方法进行稳定性评估，结果显示，本实施例制备的PTX#TPP@NP-25具有良好的稀释稳定性和血清稳定性；参照应用例1，验证本实施例中的纳米粒子的抗肿瘤效果，结果显示，相同剂量下，相对于PTX#NP-25，PTX#TPP@NP-25展现更强的肿瘤抑制效果。
实施例11：
1.Na2Se2碱性水溶液的制备。2g(50mmol)的NaOH固体溶于25mL水中，然后加入3.95g(50mmol)硒粉和100mg十六烷基三甲基溴化铵。另取0.25g(6.6mmol)NaBH4固体和0.2g NaOH固体，在冰浴条件下加入5mL水溶解，在N2保护下，将此溶液在搅拌下滴加到上述硒溶液中。室温反应1h后，再升高温度至90℃反应30min使其完全反应，最后得到具有棕红色的Na2Se2碱性水溶液，此溶液不需处理即可用于下一步二硒化物的合成。
2.硒辛酸的制备。将6.01g(25mmol)6,8-二氯辛酸乙酯加入250mL三口瓶中，再依次加入10mL 95％乙醇，2g(50mmol)氢氧化钠和38mL水，升温至50℃反应2h。反应后再向该溶液中滴加Na2Se2水溶液，滴加完毕后继续保持该温度下反应3h。反应后再降温至40℃，并加入4g活性炭，搅拌30min。之后再趁热过滤，除去活性炭。滤液中加水至350mL，冷却至0℃，在快速搅拌条件下滴加5％的稀盐酸溶液，进行酸化处理，调节pH值至2，析出大量棕褐色固体物质，抽滤、烘干，得到最终产物硒辛酸(SeA)。
参照实施例1所述方法，将其中的硫辛酸替换为硒辛酸(SeA)，制备得到硒辛酸纳米粒子。在此基础上分别参照实施例2、5、6、7和8，加入TPP-(CH2)3-OH、乙二胺-GUA、TAT、poly-HPMA和GlcN功能基团中的一种，制备得到含有功能基团修饰的纳米粒子。同时加入TPP-(CH2)3-OH、乙二胺-GUA、TAT、poly-HPMA和GlcN中的任一两种组合，制备得到含有两种功能基团修饰的纳米粒子。
参照实施例1中所述方法对本实施例中制备的纳米粒子进行稀释稳定性和血清稳定性检测，结果显示，本实施例制备的纳米粒子展现出与实施例1中制备的纳米粒子类似的稀释稳定性和血清稳定性。参照实施例2所述方法进行性能和效果验证。结果显示，本实施例中的纳米粒子在模拟不同pH条件下可发生电荷改变且能够顺利跨越多重生物屏障，表明采用二硒键交联可达到采用二硫键交联基本一致的效果。
以上所述仅为本发明的优选实施例，对本发明而言仅是说明性的，而非限制性的；本领域普通技术人员理解，在本发明权利要求所限定的精神和范围内可对其进行许多改变，修改，甚至等效变更，但都将落入本发明的保护范围。

Claims (11)

  1. 一种交联聚合物结构，其特征在于，包括若干基本单元，所述基本单元之间通过还原敏感键交联，所述基本单元为包括基本单元A和基本单元B，所述基本单元A的结构式为:
    所述基本单元B的结构式为:
    其中，X为可供交联的位点，R为改性基团。
  2. 根据权利要求1所述的聚合物结构，其特征在于，所述还原敏感键为二硫键或二硒键。
  3. 根据权利要求1所述的聚合物结构，其特征在于，所述聚合物结构中基本单元A在基本单元中所占摩尔百分比n的范围为0≤n<100％。
  4. 根据权利要求1所述的聚合物结构，其特征在于，所述基本单元形成纳米粒子。
  5. 根据权利要求4所述的聚合物结构，其特征在于，所述纳米粒子的粒径范围为10～300nm。
  6. 根据权利要求1所述的聚合物结构，其特征在于，所述基本单元包含多种具有不同结构的基本单元B。
  7. 根据权利要求1所述的聚合物结构，其特征在于，至少一种基本单元B中所述R基团为O-M+(M+为金属离子)或功能基团。
  8. 根据权利要求1所述的聚合物结构，其特征在于，所述R为功能基团，包括靶向基团、穿膜肽、长循环基团中的一种或几种。
  9. 根据权利要求1所述的聚合物结构，其特征在于，所述聚合物结构中还含有其他活性物质。
  10. 权利要求1所述聚合物结构的用途，其特征在于，将其用于制备药品；作为可选，所述药品具有抗肿瘤、降血糖、氧化应激调节功能中的至少一种。
  11. 一种如权利要求1所述的聚合物结构的制备方法，其特征在于，包含以下步骤：将基本单元A的原料悬浮在水溶液中，随后加碱使其完全溶解后再用酸调节溶液pH，从而控制基本单元A的原料与其对应的盐的比例，以获得不同尺寸的纳米粒子。
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