CN115120739A - RGD peptide modified tumor targeting polymer and preparation method and application thereof - Google Patents

RGD peptide modified tumor targeting polymer and preparation method and application thereof Download PDF

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CN115120739A
CN115120739A CN202110313377.2A CN202110313377A CN115120739A CN 115120739 A CN115120739 A CN 115120739A CN 202110313377 A CN202110313377 A CN 202110313377A CN 115120739 A CN115120739 A CN 115120739A
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fmoc
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黄伟
高钟镐
齐玲玲
刘超
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
    • C08G65/3348—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing nitrogen in addition to sulfur

Abstract

The invention belongs to the field of nano biomedical materials, and relates to an RGD peptide modified tumor targeting polymer, and a preparation method and application thereof. The RGD peptide-polyethylene glycol-fluorene methoxy carbonyl acyl (RGD-PEG-Fmoc) for actively targeting the tumor realizes tumor targeting through the interaction of the RGD peptide and an integrin receptor on the surface of a tumor cell, and can be used as a drug delivery carrier to construct a tumor targeted drug release system. The RGD-PEG-Fmoc/MMC nano-drug delivery system is constructed by encapsulating mitomycin C (MMC) by a self-assembly method, the preparation method is simple, convenient and economical, and the obtained nano-micelle has small particle size, round and round particles, uniform size and better development prospect.

Description

RGD peptide modified tumor targeting polymer and preparation method and application thereof
Technical Field
The invention belongs to the field of nano biomedical materials, relates to a polypeptide-modified tumor targeted delivery system and application thereof, and particularly relates to a tumor targeted delivery system formed by connecting RGD cyclopeptide c (Arg-Gly-Asp-d-Phe-Cys) containing an arginine-glycine-aspartic acid sequence and a polyethylene glycol-fluorene methoxycarbonyl group and application thereof.
Background
Bladder cancer is the most common malignant tumor in urinary system, and seriously threatens human health. The clinical treatment is mainly surgical treatment, and is assisted by chemotherapy and radiotherapy combined treatment. At present, the clinical treatment medicines for bladder cancer mainly comprise cytotoxic antitumor medicines, and the medicines have the treatment effect and bring serious toxic and side effects. Nanosudministration systems have been shown to significantly improve the in vivo performance of various anticancer drugs by altering their physicochemical properties, pharmacokinetic and distribution profiles.
The block polymer micelle is one of the important fields of the research of a pharmacy nano system, and the micelle has the advantages of easy preparation, small size and the like. Micelles can reduce their elimination rate in the circulatory system and enhance their enrichment in solid tumors by epr (enhanced permeability and retention effect), also known as enhanced osmotic retention effect. Most current micellar systems consist of two distinct domains, one hydrophilic and the other hydrophobic, with drug loading being dependent only on the interaction of their hydrophobic domains with drugs of low water solubility. These systems, while effective for highly hydrophobic/lipophilic drugs, are not ideal for moderately hydrophobic drugs due to limited drug-carrier compatibility. Recent studies have found that drug loading and formulation stability can be improved by introducing a mechanism of carrier/drug interaction.
Mitomycin C (Mitomycin C, MMC) is an antibiotic chemotherapeutic drug extracted from streptomyces, has a chemical structure with three active groups of benzoquinone, acetyiimino and carbamyl, has a similar action to an alkylating agent, depolymerizes DNA, forms cross-linking with a DNA chain, and antagonizes DNA replication. Also has inhibitory effect on RNA and protein synthesis at high concentration. Is mainly used for treating various solid tumors and is the first choice medicament for clinically treating bladder cancer.
The polyethylene glycol-fluorene methoxy carbonyl acyl group consists of a PEG hydrophilic chain segment and an Fmoc motif, and PEG has good water solubility, biocompatibility and biodegradability and is widely applied to scientific research. 9-fluorenylmethoxycarbonyl (Fmoc), a functional group commonly used for amino acid protection, can significantly improve drug loading and formulation stability through other mechanisms of interaction with drugs, such as pi-pi stacking and hydrogen bonding interactions.
Active targeting molecules are primarily a class of molecules that are capable of specifically binding to a receptor. The nano drug delivery system modified by specific target molecules delivers drugs to bladder tumor cells through receptor-mediated endocytosis, so that the nano drug delivery system can be more effectively adhered to the surface of the cancerated urothelial cells, and more accurate drug delivery is realized.
The RGD peptide is a sequence polypeptide containing three amino acids of Arg-Gly-Asp and is divided into a linear peptide and a cyclic peptide. They are the minimal recognition short peptide sequences of many extracellular matrix proteins (such as fibronectin, vitronectin, laminin and the like). RGD peptide as tumor cell surface over-expressed alpha v beta 3 The integrin receptor has obvious targeting property and plays an important role in regulating tumor growth, metastasis and angiogenesis. The research shows that the RGD cyclic peptide has better bioactivity and higher stability than RGD linear peptide.
Disclosure of Invention
The invention adopts cyclic peptide with the structural formula of cyclo (Arg-Gly-Asp-d-Phe-Cys), namely c (RGDFc). The RGD-PEG-Fmoc drug delivery system is constructed by modifying PEG-Fmoc with (RGDfc), so that a low-water-solubility drug can be encapsulated to enter a tumor part, and the targeted delivery of the drug is realized.
One of the technical problems to be solved by the invention is to compound RGD peptide as a molecule for realizing targeting with a long-circulating carrier material polyethylene glycol-fluorene methoxy carbonyl acyl group, and provide a novel, high-efficiency and low-toxicity multifunctional delivery carrier, and a preparation method and application thereof.
The second technical problem to be solved is to provide a preparation method and application of a carrier material polyethylene glycol-fluorene methoxy carbonyl acyl polymer.
In order to solve the technical problem, the invention can be realized by the following technical scheme:
the invention provides a polyethylene glycol-fluorene methoxy carbonyl acyl polymer modified by RGD peptide, which has the following molecular formula (I):
Figure BDA0002990180450000021
in the formula, the polymerization degree n of the polyethylene glycol is 5 to 450, preferably 8 to 230, more preferably 18 to 115, more preferably 25 to 55, even more preferably 30 to 50, most preferably 40 to 45, and even more preferably 45. Alpha v beta of RGD peptide through overexpression with tumor cell surface 3 The integrin is specifically combined to realize the tumor targeting effect of bladder cancer, the structural formula is cyclo (Arg-Gly-Asp-d-Phe-Cys), and the molecular formula is shown in the formula (II).
Figure BDA0002990180450000031
In the invention, amino is introduced into one end of a polyethylene glycol molecular chain to react with carboxyl of Fmoc, and PEG-Fmoc is generated through esterification reaction, wherein the reaction formula is shown as figure 1; the other end of the polyethylene glycol reacts with sulfhydryl of cysteine on the RGD peptide by introducing maleimide group, and the reaction formula is shown in figure 2.
The preferred preparation method is as follows:
(1) dissolving Fmoc-Lys (Boc) -OH, DMAP and EDCI in anhydrous tetrahydrofuran, activating for 1h under the protection of nitrogen, and adding MAL-PEG-NH 2 And reacting for 48 hours under the protection of nitrogen.
(2) After the reaction is finished, filtering and washing the precipitate by using cold methyl tert-butyl ether, and drying at 40 ℃ in vacuum to constant weight to obtain MAL-PEG-Fmoc powder.
(3) Dissolving c (RGDFc) and MAL-PEG-Fmoc in Hepes buffer solution with pH of 8, and stirring at normal temperature under the protection of nitrogen for reaction for 16 h.
(4) Dialysis is performed using a dialysis bag having a molecular weight cut-off of 200-. Dialyzing in deionized water for 24 h.
(5) And (4) freeze-drying the liquid to obtain freeze-dried powder for later use, thus obtaining the RGD peptide-polyethylene glycol-fluorenylmethoxycarbonyl (RGD-PEG-Fmoc) modified by the RGD peptide.
Wherein, in the reaction formulas of the attached figures 1 and 2, MAL-PEG-NH 2 Is maleimide-polyethylene glycol-amino, Fmoc-Lys (Boc) -OH is Nalpha-fluorenylmethoxycarbonyl-Nepsilon-tert-butoxycarbonyl-L-lysine, DMAP is 4-dimethylaminopyridine, EDCI is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, Hepes is 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid.
In step (1), MAL-PEG-NH 2 Molar ratio to Fmoc-Lys (Boc) -OH 1: 3-1: 5, preferably 1: 4, MAL-PEG-NH 2 EDCI/DMAP molar ratio of 1: 3-5: 0.2, preferably 1: 4: 0.2, the molar ratio of MAL-PEG-Fmoc to c (RGDfc) in step (3) is 1: 1.2-1.8, preferably 1: 1.5.
the invention combines the PEG polymer connected with the fluorenylmethoxycarbonyl with the RGD peptide with high expression of tumor cell surface receptors to prepare the long-circulating material with active targeting function, and the long-circulating material can be directly used or used for drug carriers, gene carriers and the like through other ways.
In another aspect of the present invention, an application of the RGD peptide-polyethylene glycol-fluorenylmethoxycarbonyl polymer as a drug delivery carrier is provided.
The polymer is prepared into a nanometer preparation by encapsulating active drugs and is used for treating diseases. In one embodiment, the drug entrapped in the polymeric micelle is mitomycin C, which is provided by the following process steps:
(1) weighing PEG-Fmoc and RGD-PEG-Fmoc, dissolving in 5mL of methanol, adding mitomycin C, and magnetically stirring at normal temperature for 30 minutes;
(2) the solvent is removed by rotary evaporation, and 5mL of deionized water is added for hydration for 30 min. And (4) passing through a 0.22-micron aqueous filter membrane to integrate the particle size, thus obtaining the RGD-PEG-Fmoc/MMC nano micelle.
Wherein in the step (1), the molar ratio of RGD-PEG-Fmoc to PEG-Fmoc is 1: 5,1: 10,1: 20,1: 50; most preferably 1: 10; the molar ratio of the support material to the MMC is 0.5: 1,1: 1,2.5: 1, most preferably 2.5: 1; in the step (2), the hydration time can be 15min, 30min, 45min, 60min, and most preferably 30 min.
The RGD peptide-polyethylene glycol-fluorene methoxy carbonyl acyl polymer provided by the invention can be used as an in vitro delivery carrier. The specific method and the steps are as follows:
(1) hemolysis test
Taking fresh orbital blood of a C57BL/6 mouse, rapidly stirring with a bamboo stick to remove fibrin in the blood, adding 0.9% physiological saline, centrifuging at 2000rpm for 10min in a centrifuge, and repeating the steps until the supernatant does not show red. The resulting red blood cells were diluted with 0.9% physiological saline to a 2% (v/v) suspension of red blood cells.
Taking the freeze-dried powder of the nano-micelle, diluting the freeze-dried powder of the nano-micelle with 0.9% of normal saline to be solutions with the concentrations of 0.10, 0.25, 0.5, 0.75 and 1.0mg/ml respectively, adding an equal volume of erythrocyte suspension, and setting deionized water as a positive control and the normal saline as a negative control. After shaking, incubation was carried out at 37 ℃ for 1h, followed by centrifugation at 2000rpm for 10min, and absorbance (A) was measured at 575nm in the supernatant taken to calculate the degree of hemolysis. Each set was provided with 3 secondary holes.
Degree of hemolysis (%) ═ a Sample (I) -A Negative of )/(A Positive for -A Negative of )×100％
(2) Inhibition of cell proliferation
The lyophilized preparation of the material is diluted with a culture solution to a solution of a predetermined concentration. Each concentration was set with 6 duplicate wells, and a control and zero-adjusted group were set. Proper amount of MB49 cells in logarithmic growth phase were inoculated into 96-well plates, and after further culture for 24h, the medium was replaced with fresh culture medium containing different drug concentrations, and further culture was continued for 24h or 48h, respectively. 200. mu.l of the culture medium containing 20. mu.l of CCK-8 reagent was added to each well. Incubation was continued for 2h and absorbance was measured at 450nm and 650nm as the reference wavelength. Cell viability was calculated by the formula:
cell viability (%) (OD) Experiment of -OD Zero setting )/(OD Control of -OD Zero setting )]×100％
(3)Cell migration assay
MB49 cells in log phase at 40X 10 4 The density of each well is inoculated on a 6-well plate and placed at 37 ℃ with 5% CO 2 And (5) performing static culture in an incubator for 24 hours. Discard old culture medium, use 200. mu.l aseptic gun head to make it verticalScratch, rinse 3 times with PBS. Adding fresh culture solution containing MMC solution or MMC RDG-PMs at 1 μ g/ml, setting culture solution without medicine as blank control group, photographing at 0h, 12h and 24h under an inverted microscope, and observing the healing condition of scratch of each group.
The beneficial technical effects are as follows:
1. the polymer prepared by the invention has good biocompatibility and a function of long blood circulation, and can obviously reduce the toxic and side effects of the encapsulated drug.
2. The polymer prepared by the invention adopts RGD peptide with tumor targeting to modify PEG-Fmoc, and can realize targeted delivery of tumor parts.
3. The polymer prepared by the invention can encapsulate some medicines (such as mitomycin C) with poor water solubility, and the solubility and bioavailability of the medicines are improved, so that the polymer has a better anti-tumor effect.
Drawings
FIG. 1: MAL-PEG-NH 2 And Fmoc-Lys (Boc) -OH
FIG. 2: reaction formula of PEG-Fmoc and c (RGDfc)
FIG. 3: of PEG-Fmoc 1 H-NMR spectrum
FIG. 4 is a schematic view of: of RGD-PEG-Fmoc 1 H-NMR spectrum
FIG. 5: MALDI-TOF-MS (matrix-assisted laser Desorption-time of flight-Mass Spectrometry) spectrum of PEG-Fmoc (polyethylene glycol-Fmoc)
FIG. 6: MALDI-TOF-MS (matrix-assisted laser Desorption/ionization time of flight-Mass Spectrometry) spectrum of RGD-PEG-Fmoc (arginine-Glycine-transferase-protein)
FIG. 7: dynamic Light Scattering (DLS) particle size distribution diagram of PEG-Fmoc/MMC nano-micelle
FIG. 8: dynamic Light Scattering (DLS) particle size distribution diagram of RGD-PEG-Fmoc/MMC nano-micelle
FIG. 9: transmission electron microscope image of mitomycin C-entrapped nano-micelle
FIG. 10: blank micelle proliferation inhibition result of mouse bladder cancer cell MB49
FIG. 11: result graph of proliferation inhibition result of mitomycin-entrapped C nano micelle on mouse bladder cancer cell MB49
FIG. 12: laser confocal microscope for observing cell entry condition of coumarin 6 nano-micelle
FIG. 13: graph showing migration capability results of mitomycin-entrapped C nano-micelle on mouse bladder cancer cell MB49
Detailed Description
Example 1: synthesis of PEG-Fmoc
93.7mg of Fmoc-Lys (Boc) -OH, 1.2mg of DMAP and 38.3mg of EDCI were weighed and dissolved in 5mL of anhydrous tetrahydrofuran, activated for 1 hour under the protection of nitrogen, and then 100mg of MAL-PEG-NH were added 2 And reacting for 48 hours under the protection of nitrogen. After the reaction is finished, filtering and washing the precipitate by using cold methyl tert-butyl ether, and drying at 40 ℃ in vacuum to constant weight to obtain PEG-Fmoc powder.
Example 2: synthesis of RGD-PEG-Fmoc
50mg of PEG-Fmoc and 20mg of c (RGDfc) were weighed out and stirred in 10ml of Hepes buffer solution with pH 8 under nitrogen protection for 16h at room temperature, and the reaction formula is shown in FIG. 2. After the reaction is finished, putting the mixture into a dialysis bag with the molecular weight cutoff of 2500 deionized water for dialysis for 24 hours. The liquid is frozen and dried to obtain RGD-PEG-Fmoc powder.
Dissolving dried PEG-Fmoc and RGD-PEG-Fmoc powder in CDCl 3 Nuclear magnetic resonance hydrogen spectrum of 400MHz ( 1 H-NMR) instrument, the results are shown in FIGS. 3 and 4. Of PEG-Fmoc 1 H-NMR As shown in FIG. 3, the proton peak of PEG appeared at 3.5-3.8ppm and the proton peak of Fmoc appeared at 7.2-7.8ppm, confirming the successful synthesis of PEG-Fmoc. Of RGD-PEG-Fmoc 1 H-NMR is shown in FIG. 4, in which the proton peak at 6.7ppm of maleimide disappears, indicating that the successful reaction of maleimide and thiol groups synthesizes RGD-PEG-Fmoc.
Matrix assisted laser desorption tandem time of flight mass spectrometry (MALDI-TOF-MS) was used to determine the molecular weights of PEG-Fmoc and RGD-PEG-Fmoc, and the results are shown in FIGS. 5 and 6. The molecular weight of PEG-Fmoc is 2634Da, the molecular weight of RGD-PEG-Fmoc is 2958Da, and the theoretical values are consistent, so that the PEG-Fmoc and RGD-PEG-Fmoc are successfully synthesized.
Example 3: preparation of polymer-encapsulated mitomycin C nano micelle
Weighing 10 mu mol of PEG-Fmoc (9 mu mol of PEG-Fmoc and 1 mu mol of RGD-PEG-Fmoc), dissolving in 5mL of methanol, adding 10 mu mol of mitomycin C, magnetically stirring at normal temperature for 30 minutes, removing the solvent by rotary evaporation, adding 5mL of deionized water, and hydrating for 30 minutes. And (4) passing through a 0.22 mu m microporous filter membrane to integrate the particle size to obtain PEG-Fmoc/MMC PMs and RGD-PEG-Fmoc/MMC PMs. And measuring the micelle particle size by using a Malvern nanometer particle size analyzer.
The particle size distribution diagram of PEG-Fmoc/MMC PMs is shown in FIG. 7, the particle size is 125.2nm, and the PDI is 0.159.
The particle size distribution diagram of RGD-PEG-Fmoc/MMC PMs is shown in figure 8. The particle size was 105.1nm, and PDI was 0.129.
The morphology of PEG-Fmoc/MMC PMs and PEG-Fmoc-RGD/MMC PMs was observed by transmission electron microscopy and the results are shown in FIG. 9. The transmission electron microscope result shows that the PEG-Fmoc/MMC PMs and the PEG-Fmoc-RGD/MMC PMs are spherical under the electron microscope visual field, the particles are round and uniform, and the particle size is consistent with the result measured by a Malvern particle size analyzer.
Example 4: hemolysis experiment of mitomycin C-entrapped nano-micelle
Taking fresh blood of eye socket of a C57BL black mouse, rapidly stirring with a bamboo stick to remove fibrin in the blood, adding 0.9% physiological saline, centrifuging at 2000rpm for 10min in a centrifuge, and repeating the steps until the supernatant does not show red. The resulting red blood cells were diluted with 0.9% physiological saline to a 2% (v/v) suspension of red blood cells.
Taking the freeze-dried powder of the nano micelle, diluting the freeze-dried powder of the nano micelle into solutions with the concentrations of 0.1, 0.25, 0.5, 0.75 and 1.0mg/ml by using 0.9 percent of physiological saline, adding the red blood cell suspension with the same volume, and setting deionized water as a positive control and the physiological saline as a negative control. After shaking, incubation was carried out at 37 ℃ for 1h, followed by centrifugation at 2000rpm for 10min, and absorbance (A) was measured at 575nm in the supernatant taken to calculate the degree of hemolysis. Each set was provided with 3 secondary holes.
Degree of hemolysis (%) ═ a Sample(s) -A Negative of )/(A Positive for -A Negative of )×100％
Figure BDA0002990180450000071
As can be seen from the table, the haemolysis phenomenon does not exist when the concentrations of the mitomycin C of the MMC PMs and the MMC RGD-PMs are respectively less than 5% and the haemolysis phenomenon does not exist when the concentrations of the mitomycin C are respectively 0.10, 0.25, 0.5, 0.75 and 1.0mg/ml, and the micelle has good safety and biocompatibility.
Experimental example 1: blank micelle cell proliferation inhibition
MB49 cells were seeded at 6000 cells/well in a 96-well plate and cultured at 37 ℃ under 5% CO2 for 24 hours. And (3) sucking out the old culture solution, adding blank micelles with MMC concentration of 0.0001, 0.001, 0.01, 0.1, 1, 5 and 10 mu g/mL, setting up a control group and a zero-setting group, sucking out the medium after further culture for 24h or 48h, adding 200 mu L of CCK-8 culture solution containing 20 mu L into each well, further incubating for 2h, measuring the OD value at 450mm by using a microplate reader, and taking 650nm as a reference wavelength.
Cell viability (%) - (OD) Experiment hole -OD Zero setting hole )/(OD Control well -OD Zero setting hole )×100％
The results are shown in fig. 10, the cell viability of the blank micelles is higher than 80% at different concentrations relative to mitomycin C, which indicates that the polymer is less toxic and has good safety.
Experimental example 2: CCK-8 method for investigating inhibitory capacity of mitomycin C nano-micelle on MB49 cell proliferation
MB49 cells were seeded at 6000 cells/well in 96-well plates at 37 ℃ with 5% CO 2 And standing and culturing for 24 hours under the condition. And (3) absorbing the old culture solution, adding nano micelles with MMC concentration of 0.0001, 0.001, 0.01, 0.1, 1, 5, 10 and 50 mu g/ml, establishing a control group and a zero-setting group, absorbing the medium after continuously culturing for 24h or 48h, adding 200 mu l of CCK-8 culture solution containing 20 mu l into each hole, continuously culturing for 2h, measuring the OD value at 450mm by using a microplate reader, and taking 650nm as a reference wavelength.
Cell viability (%) ═ (OD) Experiment hole -OD Zero setting hole )/(OD Control well -OD Zero setting hole )×100％
As a result, as shown in fig. 11, the nanomicelles were time-dependent and dose-dependent on the inhibition of the proliferation of MB49 cells, but no significant difference was observed in the inhibition of cell proliferation between the groups of mitomycin C nanomicelles modified with or without RGD peptide.
Experimental example 3: laser confocal microscope for observing cell entry condition of coumarin 6 nano micelle
MB49 cells were cultured at 20X 10 4 The cells/ml were seeded in 12-well plates with a slide on the bottom at 37 ℃ in 5% CO 2 And standing and culturing for 24 hours under the condition. Old culture solution was aspirated, washed 3 times with cold PBS, and coumarin 6(C6) solution and coumarin 6 nano-micelles at a concentration of 1. mu.g/ml were added. And (3) after standing and culturing for 2h, washing with cold PBS for 3 times, fixing with 4% paraformaldehyde for 10min, taking out the cell slide, reversing the cell slide onto a glass slide on which an anti-fluorescence attenuation sealing tablet is dripped, and observing the cell entry condition of the nano micelle under a laser confocal microscope.
As a result, as shown in fig. 12, after incubating the cells with the C6 solution, the C6 nanomicelle, and the C6 targeting micelle for 2h, no significant green fluorescence was observed in the C6 solution group, indicating almost no uptake. The distinct green fluorescence observed in the micellar group indicates that the micellated C6 is more easily taken up by cells, but the uptake amount is not significantly different.
Experimental example 4: examination of mitomycin C nano-micelle capability of inhibiting MB49 cell migration
MB49 cells at log phase at 40X 10 4 The density of each well is inoculated on a 6-well plate and placed at 37 ℃ with 5% CO 2 And (5) performing static culture in an incubator for 24 hours. The old culture was discarded, vertically scratched with a 200. mu.l sterile tip, and washed 3 times with PBS. Adding fresh culture solution containing MMC solution or MMC RDG-PMs of 1 microgram/ml, setting culture solution without medicine as blank control group, respectively placing under an inverted microscope for photographing at 0h, 12h and 24h, and observing the healing condition of the scratch of each group.
As shown in fig. 13, in the group incubated with MMC, the cells in the scratch grow slowly, and dead cells appear at the edge of the scratch, and the scratch is still clearly visible, while the scratch of the control group gradually heals. After 24h incubation, the width of the scratch was in order from wide to narrow for MMC-targeted nanomicelle > MMC solution > control. These results indicate that MMC can effectively inhibit scratch healing and growth migration of MB49 cells, and that micellized MMC can be more taken up by MB49 cells, thereby inhibiting cell growth.

Claims (8)

1. An RGD peptide-polyethylene glycol-fluorene methoxy carbonyl acyl polymer shown in formula (I) is characterized in that the structure composition is as follows: one end of a polyethylene glycol molecular chain is connected with c (RGDFc) peptide through sulfydryl of maleimide group and cysteine, the other end of the molecular chain is connected with N-alpha-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-L-lysine through the reaction of amino and carboxyl, the polymerization degree N is selected from 8-230,
Figure FDA0002990180440000011
2. the polymer of claim 1, wherein the c (RGDFc) peptide has the sequence c (Arg-Gly-Asp-d-Phe-Cys) and the formula (II)
Figure FDA0002990180440000012
3. The polymer according to claim 1, wherein the polymerization degree n of the polyethylene glycol is preferably 8 to 230, more preferably 18 to 115, more preferably 25 to 55, even more preferably 30 to 50, most preferably 40 to 45, and even most preferably 45.
4. A process for the preparation of the polymer of claim 1:
(1) dissolving Nalpha-fluorenylmethoxycarbonyl-Nepsilon-tert-butyloxycarbonyl-L-lysine, 4-dimethylaminopyridine and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in anhydrous tetrahydrofuran, activating for 1h under the protection of nitrogen, and adding MAL-PEG-NH 2 Reacting for 48 hours under the protection of nitrogen;
(2) filtering, washing the precipitate with cold methyl tert-butyl ether, and vacuum drying at 40 deg.C to constant weight to obtain MAL-PEG-Fmoc powder;
(3) dissolving c (RGDFc) and MAL-PEG-Fmoc in Hepes buffer solution with pH of 8, and stirring at normal temperature under the protection of nitrogen for reaction for 16 h;
(4) dialyzing in deionized water for 24 hr by using a dialysis bag with certain molecular weight cut-off;
(5) and (4) freeze-drying the liquid to obtain freeze-dried powder for later use, thus obtaining the RGD-PEG-Fmoc modified by the RGD peptide.
5. The method of claim 4, wherein the MAL-PEG-NH is present 2 Molar ratio to Fmoc-Lys (Boc) -OH 1: 3-1: 5, MAL-PEG-NH 2 The mol ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to 4-dimethylaminopyridine is 1: 3-5: 0.2, the molar ratio of MAL-PEG-Fmoc to c (RGDfc) is 1: 1.2-1.8.
6. The use of an RGD peptide-polyethylene glycol-fluorenylmethoxycarbonyl polymer as claimed in claim 1 as a carrier in the delivery of a drug.
7. The use according to claim 6, wherein the polymer is formulated as a nano-formulation by entrapment of the active drug for disease treatment.
8. The use according to claim 7, wherein the active drug entrapped in the nanoformulation is selected from mitomycin C.
CN202110313377.2A 2021-03-24 2021-03-24 RGD peptide modified tumor targeting polymer and preparation method and application thereof Pending CN115120739A (en)

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