CN113698556A - Preparation method of redox-sensitive targeted drug-loaded polymer - Google Patents
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- A61K47/6929—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
- A61K47/6931—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
- 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
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- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses a preparation method of a redox-sensitive targeted drug-loaded polymer, which comprises the step of adding (NH) poly (ethylene glycol) methacrylate and hydroxypropyl methacrylate4)2S2O8‑NaHSO3The compound system is initiated to react at 35-40 ℃ to prepare a product poly (mPEG-co-HM), then the product poly (mPEG-co-HM) -S-S-COOH is obtained by esterification reaction with 3, 3' -dithiodipropionic anhydride at room temperature, and the product poly (mPEG-co-HM) -S-S-DOX is obtained by amidation reaction with doxorubicin hydrochloride at room temperature. The redox-sensitive targeted drug-loaded polymer prepared by the invention has the particle size of 160.5 +/-4.7 nm and good stability. Stability experiments for a blank poly (mPEG-co-HM) -S-S-DOX NPs solution showed that: the NPs solutions with FBS added at 4 deg.C, 25 deg.C, 37 deg.C and at 37 deg.C all showed good stability.
Description
Technical Field
The invention belongs to the technical field of redox-sensitive targeted nano drug delivery systems, and particularly relates to a preparation method of a redox-sensitive targeted drug-loaded polymer.
Background
The concept of polymer drug conjugation was proposed by Helmut Ringsdorf in 1975. The Ringsdorf model consists of three parts: (1) a solubilizer or hydrophilic fragment to ensure water solubility; (2) drugs are typically bonded to a polymeric backbone through a linker; (3) targeting moieties are provided for transport to specific biological targets. The polymer prodrug conjugate is a product which is continuously developed in the nano-medicine field of cancer diagnosis and treatment in recent years, and the main advantage of using the polymer drug conjugate is that the chemical and physical properties of the polymer can be adjusted to improve the curative effect of the drug and reduce the toxicity of the drug. The stimulus responsiveness provides controlled release of the prodrug, avoiding adverse side effects, organ damage and toxicity caused by fluctuations associated with periodic dosing. The polymers self-assemble to form micelles, wherein the conjugated side chains of the anticancer drugs form a hydrophobic micelle core, and the hydrophilic segments form a micelle shell. Many studies have begun to attach anticancer drugs to hydrophobic polymer backbones.
Polyethylene glycol (PEG) is one of the most commonly used hydrophilic polymers approved by the FDA. The polyethylene glycol is used in a manner dependent on its molar mass. Polyethylene glycol of molar mass 20kDa to 50kDa was used for coupling of low molar mass drugs to slow renal clearance. Polyethylene glycols with molar masses of 1kDa to 5kDa are commonly used for coupling of large doses of drugs. In the second case, polyethylene glycol reduces non-specific interactions with blood. The hydrated polyethylene glycol shell reduces aggregation and the drug is shielded or bound by the polyethylene glycol, thereby increasing stability and prolonging the blood circulation time of the drug.
The redox sensitive chemical bond plays a very important role in a redox sensitive targeted nano drug delivery system, and is similar to the switch of the drug delivery system, and can influence the release of drugs. Disulfide bonds (-S-S-), monothio bonds (-S-), diselenide bonds (-Se-Se-), and single selenide bonds (-Se-), are common redox sensitive bonds. There are two methods of introducing redox sensitive bonds: one is to use a material containing a redox-sensitive bond to prepare a nanocarrier, and the other is to use a prodrug containing a redox-sensitive bond. The nano-carrier containing the redox sensitive bond structure can be obtained by using the redox sensitive material to prepare the nano-carrier, and when the sensitive bond is broken under a proper condition, the drug is released from a drug delivery system.
The disulfide bond (-S-S-) formed by the oxidation of two sulfydryl (-SH) groups is a valuable functional group in various chemical and biological preparations and has strong reactivity or biological activity. Disulfide bonds are suitable as cleavable linkers because they can accommodate various types of chemicals, such as cytotoxic agents and tumor targeting molecules, and can be cleaved by thiols that are abundant in cells. Because disulfide is biodegradable in the presence of thiols or in a reducing environment, disulfide-based biofunctionalization techniques have been widely applied in the development of chemical sensors, pro-drugs, polymer hydrogels, nanomaterial carriers and other agents. The disulfide bond drug delivery method has great advantages in biocompatibility, blood flow stability, disulfide reductase or metabolic thiol cleavage, and the like, and shows higher therapeutic effects. The disulfide bond coupling of the drug molecules and the carrier enables the drug molecules to be efficiently transferred to the action site; thus, the drug can accumulate to a higher concentration in the target tissue. However, many drugs and drug carriers do not contain disulfide functionality, require modification, and require the addition of disulfide bonds between the drug and the carrier. In recent years, disulfide bonds have been extensively studied as part of targeted drug conjugates, which can accumulate in target regions where drug activity is desired and release the drug upon selective cellular internalization.
Disclosure of Invention
The invention solves the technical problem of providing a preparation method of a redox-sensitive targeted drug-loaded polymer, which comprises the steps of esterifying hydroxyl at the tail end of poly (mPEG-co-HM) by DTDPA to obtain carboxylated poly (mPEG-co-HM) -S-S-COOH containing a disulfide bond, and carrying out amidation reaction with DOX/HCl to obtain a poly (mPEG-co-HM) -S-S-DOX prodrug.
The invention adopts the following technical scheme for solving the technical problems, and the preparation method of the redox sensitive targeted drug-loaded polymer is characterized by comprising the following specific processes: firstly, poly (ethylene glycol) methacrylate (mPEG MA)360) With Hydroxypropyl Methacrylate (HM) in (NH)4)2S2O8-NaHSO3The method comprises the steps of preparing a product poly (mPEG-co-HM) by reaction at 35-40 ℃ under initiation in a composite system, carrying out esterification reaction on the poly (mPEG-co-HM) and 3, 3' -dithiodipropionic anhydride (DTDPA) at room temperature to obtain poly (mPEG-co-HM) -S-S-COOH, and carrying out amidation reaction on the poly (mPEG-co-HM) -S-S-COOH and doxorubicin hydrochloride (DOX/HCl) at room temperature to obtain a final product, namely the redox sensitive drug-loaded targeting polymer poly (mPEG-co-HM) -S-S-DOX.
The preparation method of the redox-sensitive targeted drug-loaded polymer is characterized by comprising the following specific steps of:
step S1: in a dry reaction tube mPEG MA3600.3g and HM 0.9g were dissolved in 6mL of anhydrous ethanol to obtain a solution A, and (NH) was placed in a dry reaction tube4)2S2O8 0.045g、NaHSO3Dissolving 0.045g of the mixed system in 6mL of purified water to obtain a solution B, adding the solution B into the solution A, mixing, vacuumizing, filling nitrogen for 3 times, reacting the mixed system at 37 ℃ for 10 hours, dialyzing in the purified water for 48 hours after the reaction is finished, and freeze-drying the product for 24 hours to obtain a poly (mPEG-co-HM);
step S2: sequentially adding 0.5g of poly (mPEG-co-HM), 0.045g of 3,3 '-dithiodipropionic anhydride and 0.021g of N, N' -Dicyclohexylcarbodiimide (DCC) into a dry reaction tube, then adding 3mL of anhydrous DMF, finally adding 0.013g of 4-Dimethylaminopyridine (DMAP), vacuumizing, filling nitrogen for 3 times, stirring at room temperature for reaction for 48 hours, dialyzing the reaction solution in purified water for 48 hours after the reaction is finished, and then freeze-drying the product for 24 hours to obtain a white target product poly (mPEG-co-HM) -S-S-COOH;
step S3: adding 0.3g of poly (mPEG-co-HM) -S-S-COOH, 0.06g of DCC and 0.045g of N-hydroxysuccinimide (NHS) into a dry reaction tube in sequence, adding 9mL of anhydrous DMSO for dissolving, reacting at room temperature for 1h under the protection of nitrogen to obtain a solution C, adding 0.075g of doxorubicin hydrochloride and 90 mu L of triethylamine into a dry reaction tube under the condition of keeping out of the light, adding 6mL of anhydrous DMSO for dissolving, reacting for 1h at room temperature to obtain a solution D, adding the solution D into the solution C, vacuumizing, filling nitrogen, repeating for 3 times, stirring and reacting for 48h at room temperature under the condition of keeping out of the light, dialyzing the reaction solution in DMF for 24h under the condition of keeping out of the light after the reaction is finished, then dialyzing in distilled water for 48h, and freeze-drying the product for 24h to finally obtain a dark red target product poly (mPEG-co-HM) -S-S-DOX.
Further limiting, under the dark condition, dissolving 10mg of poly (mPEG-co-HM) -S-S-DOX in 2mL of trifluoroethanol, dropwise adding the solution into 10mL of deionized water under the magnetic stirring condition, and stirring the mixed system at room temperature to volatilize and remove the organic solvent to finally obtain the poly (mPEG-co-HM) -S-S-DOX nanoparticles.
Compared with the prior art, the invention has the following advantages and beneficial effects: the invention obtains a dark red solid poly (mPEG-co-HM) -S-S-DOX as a final product through reaction1The results in H NMR, FTIR, TGA, DSC and the nanoparticle plot indicate that poly (mPEG-co-HM) -S-S-DOX was successfully prepared. Analysis data of a Malvern Zatasizer Nano ZS90 particle size analyzer show that the particle size of poly (mPEG-co-HM) -S-S-DOX is 160.5 +/-4.7 nm, and the stability is good. Stability experiments of poly (mPEG-co-HM) -S-S-DOX NPs solutions show that: the NPs solutions with FBS added at 4 deg.C, 25 deg.C, 37 deg.C and at 37 deg.C all showed good stability.
Drawings
FIG. 1 shows poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, and poly (mPEG-co-HM) -S-S-DOX polymers1HNMR spectrogram.
FIG. 2 shows DOX, HM, mPEGMA360、poly(mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, poly (mPEG-co-HM) -S-S-DOX.
FIG. 3 is a DSC plot of poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, poly (mPEG-co-HM) -S-S-DOX, and poly (mPEG-co-HM) -S-S-COOH + DOX.
FIG. 4 is a photograph of samples of poly (mPEG-co-HM) -S-S-COOH and poly (mPEG-co-HM) -S-S-DOX, with poly (mPEG-co-HM) -S-S-COOH on the left and poly (mPEG-co-HM) -S-S-DOX on the right.
FIG. 5 is a poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-DOX TGA spectrum.
FIG. 6 is the Tyndall effect of poly (mPEG-co-HM) -S-S-DOX NPs solution, with poly (mPEG-co-HM) -S-S-DOX NPs solution to the left; the right side is deionized water.
FIG. 7 is a measurement of particle size for poly (mPEG-co-HM) -S-S-DOX NPs solution.
FIG. 8 is the stability of poly (mPEG-co-HM) -S-S-DOX NPs solutions under different conditions, corresponding to the conditions: 25 ℃ (fig. 8A), 4 ℃ (fig. 8B), 37 ℃ (fig. 8C) and 37 ℃ + FBS (fig. 8D).
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Examples
Synthesis of poly (mPEG-co-HM) -S-S-DOX Polymer:
the poly (mPEG-co-HM) -S-S-DOX polymer is synthesized by polymerization, esterification and amidation reactions, and the method comprises the following specific steps:
step S1: in a dry reaction tube mPEG MA3600.3g (0.83mmol) and HM 0.9g (6.25mmol) were dissolved in 6mL of anhydrous ethanol to obtain a solution A, and (NH) was placed in a dry reaction tube4)2S2O8 0.045g、NaHSO3Dissolving 0.045g in 6mL of purified water to obtain solution B, adding the solution B into the solution A, mixing, vacuumizing, filling nitrogen for 3 times, reacting the mixed system at 37 ℃ for 10h, dialyzing in purified water for 48h after the reaction is finished, and thenFreeze-drying the product for 24h to obtain a product poly (mPEG-co-HM);
step S2: sequentially adding 0.5g (0.1mmol) of poly (mPEG-co-HM), 0.045g (0.23mmol) of 3,3 '-dithiodipropionic anhydride and 0.021g (0.10mmol) of N, N' -Dicyclohexylcarbodiimide (DCC) into a dried reaction tube, then adding 3mL of anhydrous DMF, finally adding 0.013g (0.11mmol) of 4-Dimethylaminopyridine (DMAP), vacuumizing, charging nitrogen for 3 times, stirring at room temperature for reaction for 48h, dialyzing the reaction solution in purified water for 48h after the reaction is finished, and then freeze-drying the product for 24h to obtain a white target product poly (mPEG-co-HM) -S-S-COOH;
step S3: adding 0.3g (0.06mmol) of poly (mPEG-co-HM) -S-S-COOH, 0.06g of DCC and 0.045g of N-hydroxysuccinimide (NHS) into a dry reaction tube in sequence, adding 9mL of anhydrous DMSO for dissolving, reacting at room temperature for 1h under the protection of nitrogen to obtain a solution C, adding 0.075g (0.13mmol) of doxorubicin hydrochloride and 90 mu L of triethylamine into a dry reaction tube under the condition of keeping out of the light, adding 6mL of anhydrous DMSO for dissolving, reacting at room temperature for 1h to obtain a solution D, adding the solution D into the solution C, vacuumizing, charging nitrogen, repeating for 3 times, stirring and reacting for 48h at room temperature under the condition of keeping out of the light, dialyzing the reaction solution in DMF for 24h under the condition of keeping out of the light after the reaction is finished, then dialyzing in distilled water for 48h, and freeze-drying the product for 24h to finally obtain a dark red target product poly (mPEG-co-HM) -S-S-DOX.
Characterization of the polymer poly (mPEG-co-HM) -S-S-DOX:
infrared spectrograms for mPEG, HM, poly (mPEG-co-HM), DTDPA, poly (mPEG-co-HM) -S-S-COOH, DOX, and poly (mPEG-co-HM) -S-S-DOX were determined using Presitge infrared spectroscopy (Shimadzu, Japan) adjusted scans.
Using Bruker 400MHZNuclear magnetic resonance spectroscopy (germany) with deuterated chloroform (CDCL)3) The poly (mPEG-co-HM) -S-S-COOH spectrum was measured for the solvent poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-DOX, and deuterated dimethyl sulfoxide (DMSO-d6) for the solvent.
The patterns of poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH and poly (mPEG-co-HM) -S-S-DOX were measured by adjusting the reaction gas to 50mL/min, the shielding gas to 20mL/min, and the reaction temperature to 25 ℃ to 800 ℃ using a TGA-DSCII thermogravimetric analyzer (Switzerland).
Poly (mPEG-co-HM) -S-S-DOX and poly (mPEG-co-HM) -S-S-COOH and DOX physical mixing patterns were measured using a DSC-60Plus differential thermal scanner (Japan) with a reaction temperature set at 25 ℃ to 300 ℃.
Preparation of blank poly (mPEG-co-HM) -S-S-DOX Nanoparticles (NPs):
poly (mPEG-co-HM) -S-S-DOX NPs are prepared by using a solvent volatilization method, wherein poly (mPEG-co-HM) -S-S-DOX (10mg) is dissolved in trifluoroethanol (2mL) under the condition of keeping out of the light, then slowly dropwise added into deionized water (10mL) under the condition of magnetic stirring (380rpm/min), the solution is stirred for 12h at room temperature to volatilize and remove an organic solvent, and finally poly (mPEG-co-HM) -S-S-DOX nanoparticles (1mg/mL) are obtained, and the Tyndall effect is verified by using a laser pen.
Blank poly (mPEG-co-HM) -S-S-DOX NPs particle size determination:
the prepared poly (mPEG-co-HM) -S-S-DOX NPs solution was used to determine the particle size and particle size distribution of the NPs using a ZS-90 Malvern laser diffraction particle sizer (Malvern Instruments Ltd, Malvern, UK). The measurement temperature was constant at 25 ℃ and the instrument was equilibrated for 2min before measurement. Each sample was assayed in duplicate 3 times.
Blank poly (mPEG-co-HM) -S-S-DOX NPs stability:
preparing poly (mPEG-co-HM) -S-S-DOX NPs solution (1mg/mL), then placing the solution at 4 ℃, 25 ℃ and 37 ℃, respectively, taking 2mL of the solution at 0h, 2h, 4h, 6h, 8h, 10h, 24h, 48h and 72h, respectively, and measuring the particle size and the particle size distribution.
To examine the effect of bovine serum albumin on the stability of poly (mPEG-co-HM) -S-S-DOX NPs, a solution of poly (mPEG-co-HM) -S-S-DOX NPs (1mg/mL) and a solution of poly (mPEG-co-HM) -S-S-DOX NPs (1mg/mL) containing 10 wt% fetal bovine serum albumin (FBS) were prepared as described above, and then placed at 37 ℃ for 2mL at 0h, 2h, 4h, 6h, 8h, 10h, 24h, 48h, and 72h, respectively, to measure the particle size and the particle size distribution.
Synthesis and characterization of poly (mPEG-co-HM) -S-S-DOX:
in this experiment, DTDPA was synthesized according to previous studies. And poly (mPEG-co-HM)) the-S-S-DOX copolymer is synthesized by polymerization, esterification and amidation reactions. Can be arranged in1All characteristic peaks of poly (mPEG-co-HM) -S-S-DOX are observed in an H NMR spectrum (figure 1), wherein the poly (mPEG-co-HM), the poly (mPEG-co-HM) -S-S-COOH and a and b peaks are shown in the figure, which proves that the poly (mPEG-co-HM) -S-S-COOH polymer as a first step product is successfully synthesized, and the poly (mPEG-co-HM) -S-S-DOX as a final product is successfully prepared. FIG. 2 shows mPEG MA360(mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, DOX and poly (mPEG-co-HM) -S-S-DOX infrared spectra at 3424cm in spectrum-1A characteristic absorption peak for-OH was observed; at HM and mPEG MA3601627cm-1A characteristic absorption peak was observed at-C ═ C-, but at poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, poly (mPEG-co-HM) -S-S-DOX 1627cm-1No carbon-carbon double bond, and demonstrates HM and mPEG MA360Polymerization was successfully carried out.
Differential Scanning Calorimetry (DSC) results analysis:
FIG. 3 is a DSC plot of poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-COOH, poly (mPEG-co-HM) -S-S-DOX, and poly (mPEG-co-HM) -S-S-COOH + DOX. The melting point of the crystals of doxorubicin hydrochloride (DOX/HCl) was 205 ℃. This endothermic peak was also observed in the DSC dashed line for a physical mixture of the polymer poly (mPEG-co-HM) -S-S-COOH and DOX/HCl. This indicates that: the physical mixture of the polymer poly (mPEG-co-HM) -S-S-COOH with DOX/HCl did not effectively inhibit drug crystallization of DOX. DOX is still present in the mixture in crystalline form. In contrast, the melting point peak of doxorubicin was not found in the DSC curve of poly (mPEG-co-HM) -S-S-DOX polymer, indicating that DOX is molecularly bonded to the polymer, thereby effectively inhibiting the crystallization of DOX. FIG. 4 is a plot of poly (mPEG-co-HM) -S-S-COOH and poly (mPEG-co-HM) -S-S-DOX polymers changing color from white to dark red. The polymer poly (mPEG-co-HM) -S-S-DOX was demonstrated to be successfully prepared.
Thermogravimetric analysis (TGA) results analysis:
FIG. 5 is a TGA plot of poly (mPEG-co-HM), poly (mPEG-co-HM) -S-S-DOX. As can be seen from the figure: poly (mPEG-co-HM) starts to pyrolyze from 330 ℃ and ends up with a mass loss of about 26%. This is caused by the intramolecular ester bond undergoing pyrolysis. In contrast, poly (mPEG-co-HM) -S-S-DOX pyrolyzed from 330 ℃ with a final mass loss of over 65%. The reasons for this phenomenon are: the pyrolysis of the ester bond and the amido bond in the molecule is caused together. Therefore, the mass loss of poly (mPEG-co-HM) -S-S-DOX is significantly higher than that of poly (mPEG-co-HM). These data also laterally confirm that DOX is successfully bonded to poly (mPEG-co-HM) -S-S-DOX polymer.
Synthesis of poly (mPEG-co-HM) -S-S-DOX NPs and Tyndall effect:
this time, a solvent evaporation method was used to prepare poly (mPEG-co-HM) -S-S-DOX NPs solution. Deionized water and poly (mPEG-co-HM) -S-S-DOX NPs solution were placed in four smooth cuvettes, respectively, and irradiated with a laser pen, and the results showed: deionized water as a control group, no tyndall effect; the poly (mPEG-co-HM) -S-S-DOX NPs solution has a bright path (figure 6), the Tyndall effect is the property of the colloidal solution, and the successful preparation of the poly (mPEG-co-HM-S-S-DOX) NPs solution is initially demonstrated
Analysis of the particle size distribution result of poly (mPEG-co-HM) -S-S-DOX NPs:
a solution of poly (mPEG-co-HM) -S-S-DOX NPs was prepared. The particle size of the poly (mPEG-co-HM) -S-S-DOX NPs solution was measured using a ZS-90 Malvern laser diffraction particle sizer, and it was shown to be 160.5. + -. 4.7nm (FIG. 7), and the PDI value was 0.16. + -. 0.009. The results show that the poly (mPEG-co-HM) -S-S-DOX NPs solution has proper particle size and uniform distribution.
Analysis of the stability determination result of poly (mPEG-co-HM) -S-S-DOX NPs:
to examine the stability of poly (mPEG-co-HM) -S-S-DOX NPs solutions at 4 deg.C, 25 deg.C and 37 deg.C. Sampling is carried out for 0h, 2h, 4h, 6h, 8h, 10h, 24h, 48h and 72h respectively, and particle size measurement is carried out. FIG. 8A is a graph of the stability of poly (mPEG-co-HM) -S-S-DOX NPs at 25 ℃ showing: after 72h of measurement, the particle size of the NPs is maintained at about 180 nm. Meanwhile, PDI is maintained at about 0.18, which indicates that poly (mPEG-co-HM) -S-S-DOX NPs have good stability at 25 ℃. FIG. 8B shows the stability of poly (mPEG-co-HM) -S-S-DOX NPs at 4 deg.C, and the particle size of poly (mPEG-co-HM) -S-S-DOX NPs was maintained at about 170nm after 72h measurement. Meanwhile, PDI is maintained at about 0.20; the poly (mPEG-co-HM) -S-S-DOX NPs have good stability at 4 ℃. FIG. 8C shows the stability of poly (mPEG-co-HM) -S-S-DOX NPs at 37 deg.C, and the particle size of the NPs was maintained at about 160nm after 72h measurement. Meanwhile, PDI is maintained at about 0.16; the poly (mPEG-co-HM) -S-S-DOX NPs have good stability at 37 ℃. FIG. 8D shows the stability of NPs at 37 ℃ with 10 wt% bovine serum albumin, and the particle size of poly (mPEG-co-HM) -S-S-DOX NPs was maintained at around 140nm as measured for 72 h. Meanwhile, PDI is maintained at about 0.34; it is shown that poly (mPEG-co-HM) -S-S-DOX NPs obtained by adding 10 wt% bovine serum albumin have good stability at 37 ℃. In summary, poly (mPEG-co-HM) -S-S-DOX NPs show good stability at 4 deg.C, 25 deg.C and 37 deg.C, and in the presence of FBS.
The invention makes use of mPEG MA360Copolymerizing with HM, reacting with DTDPA under proper conditions, and bonding DOX to obtain poly (mPEG-co-HM) -S-S-DOX, and subjecting the structure to FTIR,1H NMR, TGA, DSC and NPs solutions, ultimately demonstrating the successful preparation of poly (mPEG-co-HM) -S-S-DOX. The product is prepared into poly (mPEG-co-HM) -S-S-DOX NPs solution by a solvent volatilization method, and the successful preparation of the nanoparticles is verified by the Tyndall phenomenon. Analysis of the particle size measurement results of the nanoparticle solution shows that the poly (mPEG-co-HM) -S-S-DOX NPs solution has proper particle size and uniform particle size distribution (the average particle size is 160.5 +/-4.7 nm, and the average particle size distribution PDI is 0.16 +/-0.009). Stability experiments for a blank poly (mPEG-co-HM) -S-S-DOX NPs solution showed that: the solution of poly (mPEG-co-HM) -S-S-DOX NPs added with FBS at 4 ℃, 25 ℃, 37 ℃ and 37 ℃ shows good stability, and the result is a reference for subsequent research of the inventor.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
Claims (3)
1. A preparation method of a redox sensitive targeted drug-loaded polymer is characterized by comprising the following specific steps: firstly, poly (ethylene glycol) methacrylate (mPEG MA)360) With Hydroxypropyl Methacrylate (HM) in (NH)4)2S2O8-NaHSO3The method comprises the steps of preparing a product poly (mPEG-co-HM) by reaction at 35-40 ℃ under initiation in a composite system, carrying out esterification reaction on the poly (mPEG-co-HM) and 3, 3' -dithiodipropionic anhydride (DTDPA) at room temperature to obtain poly (mPEG-co-HM) -S-S-COOH, and carrying out amidation reaction on the poly (mPEG-co-HM) -S-S-COOH and doxorubicin hydrochloride (DOX/HCl) at room temperature to obtain a final product, namely the redox sensitive drug-loaded targeting polymer poly (mPEG-co-HM) -S-S-DOX.
2. The preparation method of the redox-sensitive targeted drug-loaded polymer according to claim 1, characterized by comprising the following specific steps:
step S1: in a dry reaction tube mPEG MA3600.3g and HM 0.9g were dissolved in 6mL of anhydrous ethanol to obtain a solution A, and (NH) was placed in a dry reaction tube4)2S2O8 0.045g、NaHSO3Dissolving 0.045g of the mixed system in 6mL of purified water to obtain a solution B, adding the solution B into the solution A, mixing, vacuumizing, filling nitrogen for 3 times, reacting the mixed system at 37 ℃ for 10 hours, dialyzing in the purified water for 48 hours after the reaction is finished, and freeze-drying the product for 24 hours to obtain a poly (mPEG-co-HM);
step S2: sequentially adding 0.5g of poly (mPEG-co-HM), 0.045g of 3,3 '-dithiodipropionic anhydride and 0.021g of N, N' -Dicyclohexylcarbodiimide (DCC) into a dry reaction tube, then adding 3mL of anhydrous DMF, finally adding 0.013g of 4-Dimethylaminopyridine (DMAP), vacuumizing, filling nitrogen for 3 times, stirring at room temperature for reaction for 48 hours, dialyzing the reaction solution in purified water for 48 hours after the reaction is finished, and then freeze-drying the product for 24 hours to obtain a white target product poly (mPEG-co-HM) -S-S-COOH;
step S3: adding 0.3g of poly (mPEG-co-HM) -S-S-COOH, 0.06g of DCC and 0.045g of N-hydroxysuccinimide (NHS) into a dry reaction tube in sequence, adding 9mL of anhydrous DMSO for dissolving, reacting at room temperature for 1h under the protection of nitrogen to obtain a solution C, adding 0.075g of doxorubicin hydrochloride and 90 mu L of triethylamine into a dry reaction tube under the condition of keeping out of the light, adding 6mL of anhydrous DMSO for dissolving, reacting for 1h at room temperature to obtain a solution D, adding the solution D into the solution C, vacuumizing, filling nitrogen, repeating for 3 times, stirring and reacting for 48h at room temperature under the condition of keeping out of the light, dialyzing the reaction solution in DMF for 24h under the condition of keeping out of the light after the reaction is finished, then dialyzing in distilled water for 48h, and freeze-drying the product for 24h to finally obtain a dark red target product poly (mPEG-co-HM) -S-S-DOX.
3. The preparation method of the redox-sensitive targeted drug-loaded polymer according to claim 1 or 2, characterized in that: under the condition of keeping out of the sun, 10mg of poly (mPEG-co-HM) -S-S-DOX is dissolved in 2mL of trifluoroethanol, then dropwise added into 10mL of deionized water under the condition of magnetic stirring, and then the mixed system is stirred at room temperature to volatilize and remove the organic solvent, so that the poly (mPEG-co-HM) -S-S-DOX nanoparticles are finally obtained.
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