CN114939178A - Amino acid/polypeptide coordination polymer and preparation method and application thereof - Google Patents
Amino acid/polypeptide coordination polymer and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an amino acid/polypeptide coordination polymer and a preparation method and application thereof, wherein the coordination polymer is formed by self-assembly of coordination center ions and ligands; the coordination center ion is one of metal ions with the function of adjuvant therapy of arthritis, and the ligand is histidine protected by 9-fluorenylmethoxycarbonyl and macrophage targeting peptide modified by the histidine derivative. The invention provides a nano-scale amino acid/polypeptide coordination polymer with different therapeutic drugs loaded on an inner layer and an outer layer, the nano-scale amino acid/polypeptide coordination polymer has high biological safety, can actively target diseased joints through targeting peptide, accelerate the release of different loaded drugs on the inner layer and the outer layer of nano-particles under the low pH response of an inflammation microenvironment, and carry out treatment in a double-tube manner from the angle of eliminating inflammatory macrophages, thereby realizing economic, low-toxicity and high-efficiency arthritis treatment effects.
Description
Technical Field
The invention belongs to the fields of biomedicine and nanomedicine, and particularly relates to an amino acid/polypeptide coordination polymer and a preparation method and application thereof.
Background
Rheumatoid Arthritis (RA) is currently one of the most serious systemic autoimmune diseases, and is typically characterized by synovitis, an aggressive synovial pannus, and joint cartilage and bone erosion. RA, also known as "immortal cancer", afflicts about 500 million patients in our country and is a significant public health problem. Clinically widely accepted RA treatment methods are mainly chemical drugs and biological drugs, and severe patients can undergo joint replacement surgery and the like. Although China has advanced a lot in diagnosis and treatment level in the aspect of RA in recent years and emphasizes early diagnosis and early intervention, patients still cannot realize continuous alleviation of RA symptoms and need to take medicines for a long time and in large dose. Chemotherapy, the most common clinical treatment strategy, mainly induces apoptosis of inflammatory cells or abnormally proliferating synovial cells by the cytotoxic effect of chemical drugs. However, general chemotherapeutic drugs have poor specificity distribution in human body, insignificant accumulation in inflammatory joints and low treatment efficiency, and drugs in normal tissues can generate strong side effects on human body due to strong cytotoxicity.
Methotrexate (MTX), the most typical anti-rheumatic chemotherapeutic drug that ameliorates the disease, is commonly used as the first choice for the treatment of RA. MTX has an immunosuppressive function, mainly acts on dihydrofolate reductase, inhibits DNA synthesis, further inhibits cell proliferation and induces cell apoptosis. However, MTX has no organ selectivity, and under the condition of systemic administration, MTX causes obvious gastrointestinal reaction, liver function damage, bone marrow suppression and the like, so that the development of a safer and more efficient MTX administration strategy is urgently needed.
The nano-drug is a powerful method for overcoming the existing defects of free drugs, and can improve the bioavailability and the treatment specificity of the treatment drug. For example, due to their nanoscale size, nanomedicines can extravasate from RA-permeable blood vessels and subsequently be sequestered by inflammatory cells, thereby passively targeting the diseased joint of RA. The controllable release of the drug at the inflammatory joint part can be realized by regulating the property of the nano material or utilizing an inflammation microenvironment response strategy and the like. However, the clinical transformation of nano-drugs is more complex, expensive and time consuming than traditional drugs. Such complex factors as uncertainty in vivo metabolism, heterogeneity in mass production, and cost-effectiveness imbalance, make nano-drugs inferior compared to current therapies, despite their promising therapeutic effects. Therefore, attention should be paid to developing nano-drugs with high stability, easy large-scale production, nontoxic components and easy control of physicochemical properties.
In the natural context, many proteins maintain protein structure through coordination of metal ions. For example, there is a strong coordination between metal ions and histidine, cysteine, aspartic acid, glutamic acid and tryptophan that make up the protein. Therefore, the use of metal ions to modulate the self-assembly of amino acids/polypeptides is considered to be an efficient method for the preparation of a variety of supramolecular nanomaterials. Compared with most of nanometer materials, the amino acid/polypeptide as the component of the carrier has the advantages of clear metabolic pathway, good biological safety and the like. And some amino acids/polypeptides have physiological activity and can directly or indirectly participate in regulating and controlling the biological fate of cells. For example, Tuftsin is a natural peptide consisting of L-threonine-L-lysine-L-proline-L-arginine. The peptide is formed by enzyme cutting Fc part of immunoglobulin molecule, and can promote phagocytosis process of macrophage. Tuftsin has 72000 binding sites on the macrophage surface and interacts with Fc receptors on macrophages. In addition, another receptor, neuropilin-1, on the surface of macrophages, has also been shown to interact with Tuftsin. Therefore, the Tuftsin sequence doped in the nano-drug can realize the active targeting of the nano-drug to the macrophage.
Disclosure of Invention
Based on the research background, the invention utilizes the strong coordination effect between metal ions and histidine protected by 9-fluorenylmethyloxycarbonyl to lead the histidine protected by 9-fluorenylmethyloxycarbonyl and the metal ions to be rapidly coordinated and self-assembled in aqueous solution with mild conditions to form the polymer nano-particles. The invention utilizes the characteristic to modify histidine protected by 9-fluorenylmethyloxycarbonyl to the tail end (N end or C end) of the targeting peptide, so that the formed polymer nanoparticles have the capability of targeting RA pathogenic cells in inflammatory joints. The invention further mixes 9-fluorenylmethyloxycarbonyl protected histidine and targeting peptide with the end of the histidine derivative to react with metal ions, so as to construct nanoparticles capable of targeting inflammatory macrophages, and different anti-RA drugs are loaded on the inner layer and the outer layer of the nanoparticles. The nanoparticle shows universal loading performance to drugs with different properties, and can accumulate at inflammatory tissue sites depending on active and passive targeting performance, so that low-pH-response anti-inflammatory targeted chemotherapy in inflammatory microenvironment is realized.
The specific technical scheme of the invention is as follows:
an amino acid/polypeptide coordination polymer, wherein the coordination center ion of the coordination compound is Zn 2+ 、Fe 3+/2+ 、Ca 2 + Or Cu 2+ Is 9-fluorenylmethyloxycarbonyl-protected histidine (Fmoc-H) and/or an inflammatory macrophage targeting peptide terminally modified with the histidine derivative, said peptide comprising a Tuftsin sequence (threonine-lysine-proline-arginine or arginine-proline-lysine-threonine).
The 9-fluorenylmethyloxycarbonyl protected histidine can be either at the N-terminus or the C-terminus of the peptide exemplified below: (Fmoc-H … … RPKT … …, Fmoc-H … … TKPR … …, … … PRKT … … H-Fmoc or … … TKPR … … H-Fmoc). In one particular example of the invention, 9-fluorenylmethyloxycarbonyl protected histidine is at the N-terminus of the peptide.
Preferably, the number of amino acids of the peptide is 4 to 30.
In addition to the Tuftsin sequence, tripeptide RGD or cyclic peptide iRGD containing RGD sequence, Vasoactive Intestinal Peptide (VIP), cell-penetrating peptide TAT and macrophage targeting peptide M2pep have been reported to be applied to RA targeting therapy.
Further preferably, the peptide with the terminal of the 9-fluorenylmethyloxycarbonyl protected histidine can be one or more of Fmoc-H-DGR, Fmoc-H-RGD, Fmoc-H-Tuftsin, Tuftsin-H-Fmoc, Fmoc-H-M2pep, M2pep-H-Fmoc, VIP-H-Fmoc, Fmoc-H-VIP, Fmoc-H-TAT or TAT-H-Fmoc. In a specific example of the present invention, the peptide terminated with 9-fluorenylmethyloxycarbonyl-protected histidine is 9-fluorenylmethyloxycarbonyl-protected histidine-threonine-lysine-proline-arginine.
Preferably, the molar ratio of the 9-fluorenylmethyloxycarbonyl-protected histidine to the targeting peptide terminally modified by the histidine derivative in the ligand is 24:0-10, preferably 49: 1.
Another object of the present invention is to provide an amino acid/polypeptide coordination polymer, which is prepared by self-assembly polymerization of the coordination compound of the present invention. Further, the coordination polymer is a nanoparticle.
Another objective of the invention is to provide a preparation method of the amino acid/polypeptide coordination polymer, wherein 9-fluorenylmethyloxycarbonyl protected histidine and/or targeting peptide with the terminal modified by the histidine derivative and Zn are used 2+ 、Fe 3+/2+ 、Ca 2+ Or Cu 2+ After mixing under low pH conditions (preferably pH of 1-4, more preferably pH of 2), the pH of the aqueous solution is adjusted to neutral or slightly alkaline (preferably pH in the range of 7-8), and the mixture is stirred until the amino acid/polypeptide coordination polymer nanoparticles self-assemble.
Preferably, the molar ratio of the 9-fluorenylmethoxycarbonyl protected histidine to the peptide having a terminal 9-fluorenylmethoxycarbonyl protected histidine is from 24:0 to 12, more preferably 49: 1; ligands and Zn 2+ In a molar ratio of 1:4 to 20, more preferably 5.2: 1.
One specific example is as follows:
(1) mixing the organic ligand Fmoc-H, Fmoc-H-Tuftsin in a molar ratio of 49:1, and mixing with the organic ligand-Zn 2+ Adding a zinc nitrate solution into the system at a molar ratio of 5.2: 1;
(2) adding medicine (such as MTX) into the system according to the required function, adjusting the pH value of the solution to be neutral or alkalescent (preferably the pH range is 7-8) under stirring, inducing ligand to self-assemble to form nano particles, and obtaining the amino acid/polypeptide coordination polymer nano particles with the medicine loaded in the inner layer after centrifugal purification.
(3) Adding another drug (such as platinum ions) into the polymer nanoparticles obtained in the step (2) according to the required functions, adding a reducing agent under vigorous stirring, inducing the nanoparticles to grow on the outer layer of the amino acid/polypeptide coordination polymer nanoparticles, and purifying to obtain the amino acid/polypeptide coordination polymer nanoparticles with the drugs loaded on the inner and outer layers.
Another object of the present invention is to provide the use of the amino acid/polypeptide coordination polymer of the present invention in a drug delivery vehicle. The polymer nano-particles can be used as carriers to carry medicines, probes and nano-materials for disease diagnosis and treatment.
Specifically, the drug is one or more of a small molecular compound, polypeptide/protein, polysaccharide and nucleic acid.
In the specific implementation mode of the invention, the polymer disclosed by the invention is incubated with a proper amount of MTX or platinum ions to obtain coordination polymer nanoparticles with different types of drugs loaded on the inner layer and the outer layer.
The coordination polymer can form nano particles under neutral or alkalescent conditions, the particle size is 55.0-65.0nm, and the release of the inner-layer and outer-layer medicines is accelerated after the coordination polymer is cracked under acidic conditions. The stability and biosafety of the coordination polymer nanoparticle meet the requirements of a drug delivery carrier, and the coordination polymer nanoparticle is particularly suitable for treating RA. The research of the invention proves that the amino acid/polypeptide coordination polymer nanoparticles loaded with MTX and platinum nanoparticles can be enriched in inflammatory joint areas in an active and passive targeting manner, and the anti-RA targeted chemotherapy with low pH response in inflammatory microenvironment is realized.
Another objective of the present invention is to provide a pharmaceutical preparation comprising the amino acid/polypeptide coordination polymer nanoparticles loaded with MTX and platinum nanoparticles of the present invention.
The design principle is as follows:
traditional anti-RA drugs cannot target inflammation areas in a targeted mode, the drugs are short in biological half-life and poor in bioavailability, RA cannot be eradicated, patients still need to keep the drugs for life, and therefore the drugs which are not selective for normal organs are taken for a long time and serious toxic and side effects are caused. In view of the above, it is necessary to develop nanocarriers with targeting properties to arthritic lesions as much as possible, and to ensure that the nanocarriers are non-immunogenic to avoid the aggravation of inflammation, and to ensure that the nanocarriers can be metabolized from the body in time and safely to prevent additional toxic damage caused by the accumulation of the nanocarriers.
The technical key point of the invention is that the coordination center ion of the nano-scale metal coordination polymer selects metal ions beneficial to RA treatment, and the ligand also selects 9-fluorenylmethyloxycarbonyl-protected histidine with anti-inflammatory activity and targeted peptide modified by histidine derivative with targeting performance. Meanwhile, the inner layer and the outer layer of the nano carrier are modified with two different types of drugs with anti-RA activity, and multiple anti-inflammatory means are combined together to realize the treatment of RA.
The peptide having 9-fluorenylmethyloxycarbonyl-modified histidine and 9-fluorenylmethyloxycarbonyl-modified histidine at the end are not necessarily required at the same time in the present invention, and both of these can theoretically form a polymer as a ligand. The targeting function can be achieved by doping only a small part of the polypeptide, simply because the price of the peptide is more expensive than that of the amino acid derivative alone, and the targeting peptide does not generally need to be doped in a large amount to achieve the targeting effect.
Compared with the common nano-drug, the nano-drug is used as a drug carrier, not only has inflammatory environment responsiveness, but also can realize the controlled release of the loaded drug. Moreover, the carrier also has anti-inflammatory physiological activity, so compared with the drug delivery carrier without pharmacological activity, the nano-drug is particularly suitable for treating inflammatory diseases such as RA. In addition, the nano-drug has low price of raw materials, green and environment-friendly preparation conditions and simple preparation steps, and is suitable for industrial-grade amplification production.
The invention has the advantages that:
(1) the amino acid/polypeptide coordination polymer realizes active targeting of RA affected joints by promoting macrophage uptake through Tuftsin sequences.
(2) The drug-loaded amino acid/polypeptide coordination polymer can release drugs under low pH response in inflammatory microenvironment, and realizes efficient treatment of RA.
(3) Researches show that the amino acid/polypeptide coordination polymer has broad-spectrum loading capacity for different drugs and can effectively load small-molecule drugs and nano materials. And the inner layer and the outer layer of the nano-particles can be loaded with drugs, and the nano-particles have good stability. Therefore, the invention has good application prospect in the aspect of anti-RA combination therapy.
Drawings
FIG. 1 shows the results of potentiometers for the particle size of amino acid/polypeptide coordination polymer nanoparticles prepared in examples 1 and 2 of the present invention. FIG. 1A is the hydrodynamic diameter and FIG. 1B is the Zeta potential diagram.
FIG. 2 is a FT-IR chart of amino acid/polypeptide coordination polymer nanoparticles prepared in examples 1 and 2 of the present invention.
FIG. 3 is a TEM image of amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention.
FIG. 4 is a UV-vis spectrum of amino acid/polypeptide coordination polymer nanoparticles prepared in examples 1 and 2 of the present invention.
FIG. 5 is an XRD pattern of amino acid/polypeptide coordination polymer nanoparticles prepared in example 3 of the present invention.
FIG. 6 is EDS spectrum of amino acid/polypeptide coordination polymer nanoparticle prepared in example 2 of the present invention.
FIG. 7 is an XPS spectrum of amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention.
FIG. 8 is a graph showing the hydrodynamic diameter stability results of amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention.
FIG. 9 is a graph showing the drug release results of the amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention.
FIG. 10 is a diagram showing the results of a blood compatibility experiment of amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention.
Fig. 11 is a laser confocal imaging diagram of the amino acid/polypeptide coordination polymer nanoparticles prepared in example 2 of the present invention in macrophage RAW 264.7.
FIG. 12 shows the pharmacodynamic results of amino acid/polypeptide coordination polymer nanoparticles prepared in examples 1 and 2 of the present invention. Fig. 12A shows the primary paw thickness and fig. 12B shows the rat body weight.
FIG. 13 is a schematic diagram of the construction of an amino acid/polypeptide coordination polymer according to the present invention.
Detailed Description
The invention constructs an amino acid/polypeptide coordination polymer which has higher water solubility, stability and biocompatibility and shows the universal loading capacity for different types of medicines. The amino acid/polypeptide coordination polymer nanoparticles loaded with MTX and platinum nanoparticles are suitable for in vivo anti-RA therapy. The invention proves the anti-RA treatment potential of the amino acid/polypeptide coordination polymer through modes of representation, in-vitro stability and drug release research, cell uptake research, in-vivo pharmacodynamic research and the like.
EXAMPLE 1 amino acid/polypeptide coordination Polymer nanoparticles TZFH (Fmoc-H/Fmoc-H-Tuftsin) -Zn 2+ Construction of
First, the required Fmoc-H was precisely weighed and dissolved in 0.08M hydrochloric acid to prepare a 0.026M Fmoc-H solution, and a 0.026M Fmoc-H-Tuftsin aqueous solution, a 1M Tris (hydroxymethyl) aminomethane (Tris) aqueous solution and a 0.01M zinc nitrate hexahydrate aqueous solution were prepared. 850. mu.L of water, 98. mu.L of Fmoc-H solution, 2. mu.L of Fmoc-H-Tuftsin solution and 50. mu.L of zinc nitrate hexahydrate solution were mixed and stirred for 5 minutes under magnetic stirring, and then 1M Tris solution was added dropwise to adjust the pH of the system to neutral. Then the solution is centrifuged and purified under the condition of 10000rpm/10min to obtain the nano-particle (Fmoc-H) 98％ /Fmoc-H-Tuftsin 2％ )-Zn 2+ (abbreviated as TZFH), resuspended in water and stored in a refrigerator at 4 ℃.
In addition, Fmoc-H-Zn was constructed by changing the charge ratio of Fmoc-H to Fmoc-H-Tuftsin 2+ (abbreviated as ZFH) for referenceThe specific implementation is as follows. After 850. mu.L of water, 100. mu.L of Fmoc-H solution and 50. mu.L of zinc nitrate hexahydrate solution were mixed and stirred for 5 minutes under stirring, 1M Tris solution was added dropwise to the reaction system to adjust the pH of the solution to neutral. The post-treatment was as above.
Hydrodynamic diameter & Zeta characterization:
the appropriate amount of freshly prepared ZFH and TZFH in example 1 was taken and measured for their hydrodynamic diameter and Zeta potential on a particle size potentiometer.
The hydrodynamic diameter test results (fig. 1A) show that the hydrodynamic diameter of ZFH is 116.8-119.1nm and the hydrodynamic diameter of TZFH is 275.1-276.5 nm. The results of the Zeta test (FIG. 1B) show that TZFH has a higher surface potential (-17.3-18.2 mV) than ZFH (-16.2-16.9 mV), due to the positive charge of the Tufstin sequence.
FT-IR measurement:
FT-IR measurement was carried out by KBr pellet method using 2mg each of Fmoc-H as a starting material and ZFH and TZFH freshly prepared in example 1.
FT-IR (FIG. 2) demonstrated that ZFH and TZFH were prepared from Fmoc-H and/or Fmoc-H-Tuftsin, and that the nanoparticles showed peaks in the pattern corresponding to the starting material, such as imidazole rings (3190 cm) -1 ) Aromatic ring (1606 cm) -1 ) With carbonyl (1717 cm) -1 ). Furthermore, the peak of primary amine in the spectrum of TZFH (3396 cm) -1 And 3291cm -1 ) The introduction of Fmoc-H-Tuftsin was demonstrated.
Example 2 construction of amino acid/polypeptide coordination polymer nanoparticles with different substrates loaded on inner and outer layers
First, precisely weighing the required Fmoc-H, dissolving in diluted hydrochloric acid to prepare 0.026M Fmoc-H solution, and preparing 0.026M Fmoc-H-Tuftsin aqueous solution, 1M Tris (hydroxymethyl) aminomethane (Tris) aqueous solution, 0.01M zinc nitrate hexahydrate aqueous solution, and 0.5mg/mL MTX Tris solution. mu.L of water, 98. mu.L of Fmoc-H solution, 2. mu.L of Fmoc-H-Tuftsin solution and 50. mu.L of zinc nitrate hexahydrate were mixed and stirred for 3 minutes under magnetic stirring, and then a Tris solution of MTX was added so that the final concentration of MTX was 0.18 mg/mL. Subsequently, 1M Tris solution is added dropwise to adjust the pH of the solution to be neutral, and TZFH-MTX is obtained. After the product was dialyzed, 22. mu.L of a 5mg/mL aqueous solution of potassium chloroplatinite was added to the system, and the mixture was stirred for 1 hour under ice bath conditions. And then adding sufficient sodium borohydride, vigorously stirring to react for 3h, standing overnight, and obtaining the amino acid/polypeptide coordination polymer nanoparticles (marked as TZFH-MTX-Pt) with the platinum nanoparticles loaded on the outer layer and the MTX loaded on the inner layer. Finally, the nanoparticles were purified by centrifugation and stored in a refrigerator at 4 ℃.
TEM representation:
the TZFH-MTX-Pt freshly prepared in example 2 is diluted by a certain multiple and dropped on a copper net, and the shape and size of the material are characterized on a transmission electron microscope after natural air drying.
The TEM image (FIG. 3) shows that TZFH-MTX-Pt has good spherical morphology and the size is about 58.42 nm. The outer layer of the TZFH can be seen as small dark dots, indicating that the TZFH supports platinum nanoparticles primarily in the form of a carrier.
UV-vis assay:
the absorption spectra of the freshly prepared TZFH-MTX and TZFH-MTX-Pt resuspension of example 2 were measured at 1mL each using UV-vis.
The results of the UV-vis spectra (FIG. 4) show that TZFH-MTX and TZFH-MTX-Pt contain characteristic absorption peaks for MTX (372 nm). TZFH-MTX-Pt also contains an absorption peak for platinum at 450nm-650 nm.
XRD characterization:
50mg of TZFH-MTX and 50mg of TZFH-MTX-Pt which are freshly prepared in example 2 are taken respectively, and after drying, the crystal form of the nanoparticles is analyzed by XRD.
XRD (FIG. 5) showed that TZFH-MTX is an amorphous structure and that TZFH-MTX-Pt presents two typical crystal planes (111) and (200) of platinum nanoparticles.
EDS characterization:
the TZFH-MTX-Pt freshly prepared in example 2 was diluted by a certain factor and dropped onto a copper mesh, and the element distribution of the material was characterized on a transmission electron microscope after air-drying.
EDS results (fig. 6) show that the TZFH-MTX-Pt surface has platinum and zinc elements with a large proportion of platinum elements, demonstrating successful loading of platinum nanoparticles.
XPS characterization:
50mg of TZFH-MTX-Pt freshly prepared in example 2 was taken, dried and analyzed by XPS.
XPS (figure 7) shows that TZFH-MTX-Pt has both a platinum 4f orbit and a zinc 2p orbit and contains C, N, O elements, which indicates that organic matter exists in the TZFH-MTX-Pt and two metal elements of platinum and zinc exist, and proves the load of the platinum nanoparticles on the outer layer of the TZFH.
The combination of the test results proves that the inner layer and the outer layer of the amino acid/polypeptide coordination polymer nanoparticle can successfully load different types of substrates.
Example 3 stability, drug Release ability and blood compatibility evaluation of MTX and Pt Supported amino acid/polypeptide coordination Polymer nanoparticles
Stability:
an appropriate amount of the freshly prepared TZFH-MTX-Pt from example 2 was centrifuged and resuspended in 0.01M PBS (pH 7.4), 0.01M PBS (pH 6.5), 0.01M PBS (pH 5.5), deionized water and 0.01M PBS containing 10% heat-inactivated fetal bovine serum (pH 7.4) and the hydrodynamic diameter was determined at different times.
Hydrodynamic diameter stability results (fig. 8) show that nanoparticles can maintain good particle size stability in three solutions of 0.01M PBS (pH 7.4), deionized water, and 10% hot fetal bovine serum. However, the nanoparticles showed a rapid increase in hydrodynamic diameter in 0.01M PBS (pH 6.5) and 0.01M PBS (pH 5.5) because the imidazole group was protonated with Zn in an acidic environment 2+ The coordination bonds are broken, resulting in disintegration of the nanoparticles.
And (3) drug release:
the freshly prepared TZFH-MTX-Pt from example 2 was taken in a dialysis bag of 7kD and placed in PBS (pH 7.4, 6.5, 5.5) at three different phs. Under the condition of constant-temperature stirring at 37 ℃, the release medium is taken out at different times, the MTX content is measured by UV-vis, and the cumulative release amount is calculated.
The drug release results (fig. 9) show that MTX loaded therein is released at an accelerated rate due to structural destruction of TZFH-MTX-Pt under acidic conditions of pH 5.5 or pH 6.5, and is released slowly in a solution of pH 7.4, indicating that TZFH-MTX-Pt has the potential to respond to drug release under low pH conditions in an inflammatory microenvironment and a cytolysome.
Blood compatibility:
appropriate amounts of TZFH-MTX-Pt freshly prepared in example 2 were taken to prepare solutions at concentrations of 22.5, 112.5, 225 and 450. mu.g/mL, respectively, and after incubation with rat red blood cells for 30min, the absorbance values were measured at 545nm using UV-vis.
The results of the blood compatibility experiment (fig. 10) show that the coefficient of hemolysis (< 1%) of the nanoparticles is still very low at a concentration far higher than that of the nanoparticles administered to animals, which proves that the nanoparticles have good blood compatibility.
Example 4 cellular uptake Studies of amino acid/polypeptide coordination Polymer nanoparticles
Preparation of Fluorescein Isothiocyanate (FITC) -loaded amino acid/polypeptide coordination polymer nanoparticles:
the procedure is as in example 2, replacing only MTX by 10. mu.L FITC (1 mM). The nanoparticles loaded with FITC were designated TZFH-FITC-Pt.
Cellular uptake imaging:
inoculating macrophage RAW264.7 into a confocal culture dish, wherein the inoculation density is 5 multiplied by 10 6 One dish and incubated overnight in a sterile incubator (37 ℃ C., saturated humidity). After 24h, the stock culture was replaced with fresh medium containing 100.0ng/mL Lipopolysaccharide (LPS) and co-cultured with the cells for 24 h. The medium was aspirated, DMEM complete medium containing TZFH-FITC-Pt and ZFH-FITC-Pt at 5. mu.g/mL FITC was added and incubated for 8 h. The medium was discarded, washed 3 times with PBS solution, and stained by adding 10. mu.g/mL DAPI and Lyso-Tracker Red. After 15min, the laser confocal dish was placed under a laser confocal microscope (CLSM) for imaging observation. In addition, RAW264.7 without LPS stimulation was used as a control and incubated with TZFH-FITC-Pt for 8h, and the same procedure was followed under CLSM for observation.
CLSM image shows that most green fluorescence is observed in RAW264.7 after LPS stimulation of 8h TZFH-FITC-Pt is incubated, while weak green fluorescence is observed in ZFH-FITC-Pt group, which proves that active targeting mediated by Tuftsin sequence enables amino acid/polypeptide coordination polymer nanoparticles to have good selectivity on inflammation RAW 264.7. In addition, little green fluorescence was observed in RAW264.7 without LPS stimulation, further illustrating the selectivity of TZFH-FITC-Pt for inflammatory RAW 264.7.
EXAMPLE 5 pharmacodynamic Studies of amino acid/polypeptide coordination Polymer nanoparticles
An adjuvant arthritis model was made in healthy Wistar rats. The rats were injected subcutaneously with 800 μ L of complete freund's adjuvant (10mg/mL) in the right hind paw, and when the clinical score reached 4, RA rats were randomly divided into 6 groups (n ═ 6), the day of molding was set as day 0, and the adjuvant-type arthritis rat molding was completed on day 12. From day 13, the rats of each group were injected with physiological saline, MTX, ZFH-Pt, ZFH-MTX-Pt and TZFH-MTX-Pt formulations, respectively, at a dose of 0.275mg/kg MTX. The paw thickness and the size of the clinical score of the rats were also recorded every other day.
The pharmacodynamic results are shown in figure 12 and table 1. The results show that TZFH-MTX-Pt most significantly inhibited arthritis progression in rats (paw thickness 4.85-5.78, clinical score 1). The body weight of the rats continued to rise over the 14 day treatment period (FIG. 12B), with the TZFH-MTX-Pt group in particular rising in body weight being most pronounced.
TABLE 1 arthritis Scoring of drug vs. rat arthritis model
The amino acid/polypeptide coordination polymer nanoparticle loaded with MTX and Pt synthesized by the invention has an anti-RA treatment effect in a rat body which is obviously superior to that of other preparation groups, and has no obvious toxic or side effect.
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Claims (10)
1. An amino acid/polypeptide coordination polymer, characterized in that said coordination polymer is formed by self-assembly of coordination center ions and ligands; wherein the coordination center ion is Zn 2+ 、Fe 3+/2+ 、Ca 2+ Or Cu 2+ One or more of the above; the ligand is 9-fluorenylmethyloxycarbonyl protected histidine, macrophage targeting peptide modified by 9-fluorenylmethyloxycarbonyl protected histidine derivatives, or a mixture of 9-fluorenylmethyloxycarbonyl protected histidine and 9-fluorenylmethyloxycarbonyl protected macrophage targeting peptide.
2. An amino acid/polypeptide coordination polymer according to claim 1, characterized in that the molar ratio of coordination center ion to ligand is 4-20: 1.
3. An amino acid/polypeptide coordination polymer according to claim 2, characterized in that the 9-fluorenylmethyloxycarbonyl protected histidine derivative is modified at the C-or N-terminus of the macrophage targeting peptide.
4. The amino acid/polypeptide coordination polymer of claim 3, wherein said ligand is one or more of Fmoc-H-DGR, Fmoc-H-RGD, Fmoc-H-Tuftsin, Tuftsin-H-Fmoc, Fmoc-H-M2pep, M2pep-H-Fmoc, VIP-H-Fmoc, Fmoc-H-VIP, Fmoc-H-TAT, or TAT-H-Fmoc.
5. The amino acid/polypeptide coordination polymer according to claim 1, characterized in that 9-fluorenylmethyloxycarbonyl protected histidine is mixed with 9-fluorenylmethyloxycarbonyl protected macrophage targeting peptide in a molar ratio of 24:0-12 to form a mixture.
6. The amino acid/polypeptide coordination polymer of any of claims 1-4, characterized in that said coordination polymer is a nanoparticle.
7. The method for preparing amino acid/polypeptide coordination polymer according to claim 1, characterized in that macrophage targeting peptide modified by histidine protected by 9-fluorenylmethyloxycarbonyl or/and 9-fluorenylmethyloxycarbonyl-histidine derivative and coordination center ion are mixed in acidic aqueous solution, the pH value of the reaction system is adjusted to be neutral under stirring, and the coordination metal ion and the ligand can be self-assembled to form nano-scale amino acid/polypeptide coordination polymer nanoparticles;
wherein the coordination center ion is Zn 2+ 、Fe 3+/2+ 、Ca 2+ Or Cu 2+ ；
The acidic condition is pH 1-4;
the neutral condition is pH 7-8;
the molar ratio of the 9-fluorenylmethyloxycarbonyl protected histidine to the macrophage targeting peptide modified by the histidine derivative is 24:0-12, and the molar ratio of the coordination center ion to the ligand is 4-20: 1.
8. Use of an amino acid/polypeptide coordination polymer according to any of claims 1-6 in the preparation of a drug delivery vehicle.
9. The use of claim 8, wherein said drug delivery vehicle carries a drug, a probe.
10. The use according to claim 9, wherein the carrier drug is methotrexate, platinum ion.
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