CN116585271A - A preparation method and application of mixed nanoparticles with bone targeting function - Google Patents

Abstract

The invention discloses a preparation method and application of a hybrid nanoparticle with a bone targeting function, which are characterized in that a DSPE-PEG-Asp8 polymer and a phenylboronic acid-hyaluronic acid copolymer are prepared, and then the DSPE-PEG-Asp8 polymer and the phenylboronic acid-hyaluronic acid copolymer are mixed to prepare a bone targeting nano delivery system with hydrogen peroxide responsiveness, wherein the nanoparticle can encapsulate a plurality of insoluble drug molecules, breaks through the traditional drug delivery mode of drug encapsulation by a carrier, has hydrogen peroxide responsiveness, can target and select bone tissues, and can realize rapid targeting release of encapsulated active drugs by utilizing the characteristic of high expression of active oxygen free radicals in tumor sites and inflammatory disease microenvironment through triggering of H2O2, normal cells are not damaged, and metabolites of the nanoparticle have good biocompatibility and can be used as a potential drug targeting delivery system.

Description

A preparation method and application of mixed nanoparticles with bone targeting function

technical field

The invention belongs to the technical field of drug delivery systems, and in particular relates to a preparation method and application of mixed nanoparticles with bone targeting function.

Background technique

Bone disease is a general term for bone and joint-related diseases, mainly including osteoporosis, fracture healing disorders, arthritis, and various acute and chronic neck, shoulder, waist, and leg pains. In addition to common bone diseases, bone is also a common metastasis site of solid tumors. The survival period of patients with bone metastasis is about 12-53 months. During this period, patients will also face the risk of bone complications, such as pathological fractures, high Calcemia etc. Although there have been some corresponding drug researches for various bone diseases and bone metastases, due to the high hardness of bone tissue, low blood flow, poor permeability, and special physiological and biochemical processes, almost all drugs for bone diseases exist. The pathway is difficult to effectively transport to the site of action and the curative effect is poor. Most drugs need to be administered systemically. In order to achieve an effective therapeutic concentration in bone tissue, the dosage must be increased, which not only reduces the therapeutic index of the drug, but also causes serious toxic side effects on other organs and tissues of the patient.

In recent years, micelles and nanoparticles have been widely used as drug carriers. With the development of nanotechnology, a variety of artificial nanocarriers have been developed for in vivo drug delivery. Since human tissue contains a nanoscale (1-100nm) layered structure, the size of nanocarriers is close to the size of human tissue structures, and the control of nanocarriers The size can make it easy to penetrate biological barriers in vivo, effectively improving the efficiency of drug delivery in vivo. Compared with traditional drugs, these nanocarriers have the following advantages: increase drug solubility, improve drug stability and blood circulation time, and change the systemic biodistribution of drugs, such as liposomes, polymers, polymeric peptides, calcium phosphate, and metal nanoparticles.

However, due to the lack of targeting of these traditional drug delivery systems, and the problems of liver accumulation and reticular endothelial system (Reticular endothelial system, RES) clearance, it will lead to low concentration of drug distribution in bone tissue. Therefore, PEG on the surface of liposomes Modification of bone tissue and OB targeting molecules to construct a bone-targeted drug delivery system can effectively improve the enrichment of drugs in bone tissue and enhance drug therapy and tumor suppression effects.

Currently, the common bone-targeting molecules are mainly oligopeptides and bisphosphonates. Among them, the main representatives of oligopeptides are eight-repeated aspartic acid octapeptide Asp8 and a short peptide (AspSerSer)6 combining aspartic acid and serine. Studies have shown that the short peptide sequence of Asp8 only interacts with hard tissues (bone and teeth) in vitro and in vivo (Kasugai et al., 2000; Yokogawa et al., 2001; Ouyang et al., 2009; Murphy et al. , 2007; Ogawa et al., 2013)). In fact, aspartic peptide sequences have also been used by several groups to target drugs to bone tissue. For example, bone accumulation of small molecular weight drugs, such as radiogallium-labeled bone imaging agents, has been reported in preclinical animal studies (Ogawa et al., 2013).

DSPE-PEG2000 is a pharmaceutical polymer material approved by the US FDA. It is non-toxic, non-immunogenic and non-antigenic. It is often used to encapsulate proteins, peptides and other drugs. The maleimide on the DSPE-PEG-MAL obtained by modifying PEG can be coupled with the sulfhydryl group on the aspartate octapeptide to synthesize a bone-targeted DSPE-PEG-Asp8 polymer. Although the modified Asp8 nanocarrier can play a bone-targeting therapeutic effect to a certain extent, its target is not entirely bone tumor cells, because Asp8 mainly targets bone structures rather than tumor cells.

ROS-responsive nanomaterials are able to respond to intracellular reactive oxygen species (ROS) and release pharmaceutical or diagnostic substances. These nanomaterials have been extensively studied for cancer therapy and diagnosis because cancer cells usually have higher ROS levels. Phenylboronic acid and its esters can be reacted selectively with hydrogen peroxide to form a boronic acid intermediate that is rapidly hydrolyzed to release the leaving group to form phenol and boronate or boronic acid. Boronic acids and their esters have no inherent toxicity concerns, and the boronic acid end products are considered nontoxic to humans. Furthermore, the selective reactivity of boronic acids and their esters to hydrogen peroxide provides a chemically specific, biocompatible reaction for detecting endogenous hydrogen peroxide production. This method was used as a highly selective fluorescent probe for hydrogen peroxide for cell imaging. These properties, coupled with their relative stability, make phenylboronic acids and their esters candidate triggering units for the development of potent ROS-activating prodrugs. Recently, Cohen's group developed hydrogen peroxide-activated MMPis using borate esters as H2O2-sensitive triggers (Major Jourden JL et al., 2010).

To address the need to transform single-ligand-modified nanocarriers into active tumor cell-targeting drug delivery systems, a synthetic DSPE-PEG-Asp8 polymer was combined with hydrogen peroxide-responsive phenylboronate-hyaluronic acid (HA) copolymers are co-prepared to obtain mixed nanoparticles, which can increase the tumor targeting of nanocarriers, selectively deliver drugs to tumor cells, and make them accumulate in tumor cell sites.

The inventors of the present invention therefore disclosed the preparation of a DSPE-PEG-Asp8/phenylboronate-HA bone-targeting hybrid nanoparticle.

Contents of the invention

The purpose of the present invention is to provide a preparation method of mixed nanoparticles with bone targeting function in order to overcome the shortcomings and deficiencies of the prior art.

For achieving the above object, the present invention provides following technical scheme: its steps are as follows:

(1) Get the aspartic acid eight repeat sequence and distearoylphosphatidylacetamide-polyethylene glycol-maleimide and mix them with a molar ratio of 1:1, dissolve them in PBS, and the pH of the PBS solution is 7.4, the sulfhydryl group of the eight repeat sequence of aspartic acid is coupled with the double bond of distearoylphosphatidylacetamide-polyethylene glycol-maleimide to form a thioether bond to generate a DSPE-PEG-Asp8 polymer;

(2) Take 4-hydroxymethylphenylboronic acid pinacol ester and hyaluronic acid, make it dissolve in DCC and DMAP under the condition of anhydrous dimethyl sulfoxide, 4-hydroxymethylphenylboronic acid pinacol ester The hydroxyl group of the product reacts with the carboxyl group of hyaluronic acid to produce phenylboronic acid-hyaluronic acid copolymer;

(3) Mixing the polymers prepared in step (1) and step (2) to prepare mixed nanoparticles with bone targeting function.

The further configuration of the present invention is: in step (1), the solution formed by being dissolved in PBS is stirred and reacted at room temperature for 24 hours, dialyzed with pure water for 48 hours, and finally the dialyzed solution is freeze-dried to obtain a white solid which is DSPE- PEG-Asp8 polymer.

The further setting of the present invention is: in step (2), hyaluronic acid, DCC and DMAP are dissolved in 45ml anhydrous dimethyl sulfoxide, the solution is stirred 30min under nitrogen protection to activate the hydroxyl group of hyaluronic acid, then Add 1.0g of 4-hydroxymethylphenylboronic acid pinacol ester, react overnight under nitrogen, 250rpm stirring, dialyze the reaction solution with pure water for 48h, to remove DMAP and other water-insoluble by-products, and freeze-dry the product; Then the product is dispersed in absolute ethanol, and unreacted HA is removed by ultrasonic waves; finally, the precipitated product is freeze-dried to obtain a white solid, which is phenylboronate-HA copolymer.

The further setting of the present invention is: DSPE-PEG-Asp8 polymer and phenylboronate-HA copolymer are dissolved in 10mL dimethyl sulfoxide, are added dropwise to 100mL PBS buffer solution under stirring, keep stirring for 30min, and avoid Lightly stirred for 4 hours, then dialyzed through a dialysis bag for 24 hours to remove the organic solvent, and finally freeze-dried and stored.

The invention discloses an application of the mixed nanoparticle prepared by the method in the preparation of bone disease medicine.

The further setting of the present invention is: the application of the mixed nanoparticle as a drug delivery carrier in the preparation of bone tumor medicine.

In summary, the beneficial effects of the present invention are: the mixed nanoparticles with bone targeting function introduced in the present invention, by preparing DSPE-PEG-Asp8 polymer and phenylboronic acid-hyaluronic acid copolymer, and then mixing the two A hydrogen peroxide-responsive bone-targeted nano-delivery system was prepared. The nanoparticles can carry some insoluble drug molecules, breaking through the traditional carrier-carried drug delivery mode, and having hydrogen peroxide responsiveness. It can target and select bone tissue, and use the characteristics of high expression of active oxygen free radicals in tumor sites and inflammatory disease microenvironments, and realize the rapid targeted release of encapsulated active drugs through H2O2 triggering without harming normal cells. And its metabolites have good biocompatibility, which can be used as a potential drug delivery system.

Description of drawings

Fig. 1 is the synthetic preparation flow chart of the mixed nanoparticle of embodiment 1 of the present invention;

Fig. 2 is the nuclear magnetic resonance spectrogram of the DSPE-PEG-Asp8 polymer that the embodiment of the present invention 1 makes;

Fig. 3 is the particle size distribution diagram of the mixed nanoparticles provided by Example 1 of the present invention;

Fig. 4 is the electron microscope topography diagram of the hybrid nanoparticle provided by Example 1 of the present invention;

Figure 5 is a PKM2-IN-1 HPLC peak spectrum;

Fig. 6 is the PKM2-IN-1 high performance liquid phase standard curve;

Fig. 7 is the drug release diagram of the mixed nanoparticles provided in Example 1 of the present invention.

Detailed ways

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further elaborated below in conjunction with specific embodiments and accompanying drawings, but the following embodiments are only preferred embodiments of the present invention, not all . Based on the examples in the implementation manners, other examples obtained by those skilled in the art without making creative efforts all belong to the protection scope of the present invention.

Specific embodiments of the present invention will be described below in conjunction with the accompanying drawings.

Preparation of nanoparticles:

As shown in Figure 1-A, the synthesis of DSPE-PEG-Asp8 polymer of the present invention:

The coupling between the sulfhydryl group at the n-terminal of the Asp8 peptide sequence and the double bond of the maleimide of DSPE-PEG-MAL generates a thioether bond, forming an Asp8-liposome polymer.

Asp8 and DSPE-PEG-Mal were mixed at a molar ratio of 1:1, dissolved in PBS (PH7.4), and stirred at room temperature for 24 hours. Then the reaction solution was dialyzed with pure water for 48 hours to remove unreacted polypeptide.

Finally, the dialyzed solution was freeze-dried to obtain a white solid, namely DSPE-PEG-Asp8 polymer.

As shown in Figure 1-B, the synthesis of phenylboronate-hyaluronic acid (HA) copolymer of the present invention:

(1) Esterification reaction between the hydroxyl group (-OH) of 4-hydroxymethylphenylboronic acid pinacol ester and the carboxyl group (-COOH) of hyaluronic acid to synthesize phenylboronic acid-hyaluronic acid copolymer.

(2) HA, DCC (N, N-dicyclohexylcarbodiimide) and DMAP (catalyst 4-dimethylaminopyridine) (HA:DCC:DMAP=1:3:0.3, mol:mol:mol) Dissolve in 45mL of anhydrous dimethyl sulfoxide, stir the solution for 30min under the protection of nitrogen to activate the hydroxyl group of HA, then add 1.0g of 4-hydroxymethylphenylboronic acid pinacol ester, in nitrogen, under the stirring of 250rpm The reaction was carried out overnight, and then the reaction solution was dialyzed with pure water for 48 hours to remove DMAP and other water-insoluble by-products, and the product was freeze-dried.

(3) Dispersed in absolute ethanol, with the help of ultrasonic waves, to remove unreacted HA.

(4) Finally, the precipitated product was freeze-dried to obtain a white solid, which was phenylboronate-HA copolymer.

The product synthesized by the esterification reaction was carried out in an environment of 60°C.

Dialyze the reaction solution against pure water and exchange frequently.

Impurities are removed from the dialyzed solution, resulting in higher purity polymers.

Preparation of DSPE-PEG-Asp8/phenylboronate-HA hybrid nanoparticles:

Dissolve DSPE-PEG-Asp8 polymer and phenylboronate-HA copolymer in 10 mL dimethyl sulfoxide (DMSO), add dropwise to 100 mL PBS buffer under stirring, and keep stirring for 30 min. Stir at room temperature in the dark for 4 hours, then dialyze through a dialysis bag (molecular weight cut-off = 3.5KD) for 24 hours to remove the organic solvent, and finally freeze-dry and store.

Characterization of Hybrid Nanoparticles:

The structure of DSPE-PEG-Asp8 polymer was characterized by 1H-NMR spectroscopy (AVANCE III 600MHz, Bruker, Switzerland) on a nuclear magnetic resonance spectrometer with D2O as solvent and TMS as internal standard. The 1HNMR spectrum of the non-conjugated polymer is shown in Fig. 2-A. The characteristic peak of hydrogen of the maleimide group appears at 6.7 ppm. The hydrogen peak near 1.5ppm belongs to the methyl group of lactide; and the peak near 4.8ppm belongs to the methene group of glycylurea. The hydrogen peak for poly(ethylene glycol) occurs between 3.3 ppm and 3.6 ppm. The 1HNMR spectrum of the peptide-conjugated polymer is shown in Figure 2-B. After coupling, the characteristic peak of maleimide at 6.7ppm was not detected, indicating that the maleimide functional group reacted with the peptide.

Take 20 mg of mixed nanoparticles in 0.5 mL of tetrahydrofuran, slowly drop them into 20 mL of distilled water, stir for 24 hours, and measure the particle size distribution, as shown in Figure 3, the average particle size is 140.6 nm. Take another little drop on the silicon wafer, after drying, spray gold, measure the electron microscope, the scanning electron microscope picture measured is shown in Figure 4, it can be seen that the morphology of the mixed nanoparticles is spherical.

Application of Hybrid Nanoparticles: Using PKM2-IN-1 as a Model Drug

Drug loading:

DSPE-PEG-Asp8 polymer, phenylboronate-HA copolymer and PKM2-IN-1 were dissolved in 10 mL dimethyl sulfoxide (DMSO), and added dropwise to 100 mL PBS buffer under stirring, and the stirring was maintained for 30 min. Stir in the dark at room temperature for 4 hours, then dialyze through a dialysis bag (molecular weight cut-off = 3.5KD) for 24 hours to remove organic solvents and free drugs, and finally freeze-dry and store.

Determination of encapsulation efficiency and drug loading:

PKM2-IN-1 Specificity Investigation of High Performance Liquid Chromatography (HPLC):

Accurately weigh 5.0 mg of PKM2-IN-1 reference substance, place it in a 50 mL measuring bottle, add methanol to dissolve and constant volume, and prepare a reference substance solution with a mass concentration of 100 μg/mL. Take 5.0mg of DSPE-PEG-Asp8/phenylboronate-HA mixed nanoparticles loaded with PKM2-IN-1, add methanol to dilute and set the volume to 10mL, after ultrasonic treatment for 3min to break the emulsion, pass through a 0.22μm microporous membrane Filter and take the filtrate to obtain the test solution. Take 5.0 mg of blank DSPE-PEG-Asp8/phenylboronate-HA mixed nanoparticles, add methanol to dilute and set the volume to 10 mL, after ultrasonic treatment for 3 minutes to break the emulsion, filter through a 0.22 μm microporous membrane, and take the filtrate, namely Negative control solution was obtained. Take 10 μL of the reference substance solution, the test solution, and the negative control solution and inject them into high-performance liquid chromatography (HPLC, Agilent, USA) for determination, and record the chromatogram. As shown in Figure 5, the retention time of PKM2-IN-1 is about 7.0min, and the negative control solution has no chromatographic peak at the corresponding time point; the separation between the analyte peak and the adjacent chromatographic peak is greater than 1.5, and Both the main peak and the impurity peak can achieve baseline separation, and the negative control has no interference to the determination.

The making of PKM2-IN-1 standard curve of high performance liquid chromatography (HPLC):

The PKM2-IN-1 reference substance solution was diluted to make 1, 2.5, 5, 10, 25, 50 μg/mL serial concentration solutions for the standard curve. 10 μL of the supernatant was filtered through a 0.22 μm filter and injected into high performance liquid chromatography (HPLC, Agilent, USA) for determination, and the peak area was recorded. The peak area A was linearly regressed with the concentration C of PKM2-IN-1, and the standard curve of the concentration of PKM2-IN-1 was obtained after calculation. See Figure 6.

Instrument conditions: the HPLC system is Agilent 1260 series, and the detection conditions are as follows: the analytical column is Eclipse XDB-C18 (5 μm, 4.6 mm×150 mm). The mobile phase consisted of acetonitrile-pure water (90:10; v/v). The flow rate was 1.0 mL/min. The column temperature was maintained at 25 °C, and the wavelength of the ultraviolet detector was set at 254 nm.

Determination of encapsulation efficiency and drug loading:

The DSPE-PEG-Asp8/Oxi-HA mixed nanoparticle solution loaded with PKM2-IN-1 was dialyzed for 24h through a dialysis bag (molecular weight cut-off=3.5KD), and the dialysis medium was measured by high performance liquid chromatography (HPLC, Agilent, USA) Free drug content (m1), calculate encapsulation efficiency and drug loading. The formulas are encapsulation efficiency=[(m2-m1)/m2]×100%, drug loading=[(m2-m1)/mtotal]×100%, wherein m2 is the total amount of drug delivery, and mtotal is the mixed Total amount of nanoparticle carrier and drug. The calculation results showed that the encapsulation rate was 77.73%, and the drug loading was 7.07%.

Drug release in vitro:

As shown in Figure 7, the in vitro release behavior of DSPE-PEG-Asp8/phenylboronate-HA mixed nanoparticles was studied in PBS buffer (pH=7.4) and PBS buffer containing H2O2 at 37 ° C, 100 rpm carried out under constant stirring conditions. The concentration of PKM2-IN-1 in the medium taken out at different time points was measured by high performance liquid chromatography (HPLC, Agilent, USA), and the drug release curve was drawn. Figure 5 shows the process of DSPE-PEG-Asp8/phenylboronate-HA hybrid nanoparticles bursting and releasing drugs in different environments. The in vitro release curve of PKM2-IN-1 indicated that there were great differences in drug release from DSPE-PEG-Asp8/phenylboronate-HA mixed nanoparticles in different environments (P<0.05). Within 80 hours, the release amount of the drug in the PBS buffer solution was 40%, while under the condition of PBS containing hydrogen peroxide, the release amount of the drug reached about 80%. It shows that the new hybrid nano-drug loading system can respond to H2O2 and dissociate, and has slow drug release under normal physiological conditions.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and those described in the above-mentioned embodiments and description are only preferred examples of the present invention, and are not intended to limit the present invention, without departing from the spirit and scope of the present invention. Under the premise, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the claimed invention, and the claimed protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. A preparation method with bone targeting function mixed nanoparticles, characterized in that, the steps are as follows: (1) Take the aspartic acid eight-repeat sequence and distearoylphosphatidylacetamide-polyethylene glycol-maleimide at a molar ratio of 1:1, mix them in PBS, and the pH of the PBS solution is 7.4, the sulfhydryl group of the eight repeat sequence of aspartic acid is coupled with the double bond of distearoylphosphatidylacetamide-polyethylene glycol-maleimide to form a thioether bond to generate a DSPE-PEG-Asp8 polymer; (2) Take 4-hydroxymethylphenylboronic acid pinacol ester and hyaluronic acid, under the condition that DCC and DMAP are dissolved in anhydrous dimethyl sulfoxide, 4-hydroxymethylphenylboronic acid pinacol ester The hydroxyl group of the product reacts with the carboxyl group of hyaluronic acid to produce phenylboronic acid-hyaluronic acid copolymer; (3) Mixing the polymers prepared in step (1) and step (2) to prepare mixed nanoparticles with bone targeting function. 2. The preparation method according to claim 1, characterized in that: in the step (1), the solution formed by dissolving in PBS was stirred and reacted at room temperature for 24 hours, dialyzed with pure water for 48 hours, and finally the dialyzed The solution was lyophilized to obtain a white solid which was DSPE-PEG-Asp8 polymer. 3. The preparation method according to claim 1, characterized in that: in the step (2), dissolve hyaluronic acid, DCC and DMAP in 45ml of anhydrous dimethyl sulfoxide, and put the solution under nitrogen protection Stir for 30 min to activate the hydroxyl group of hyaluronic acid, then add 1.0 g 4-hydroxymethylphenylboronic acid pinacol ester, react overnight under nitrogen, 250 rpm stirring, and dialyze the reaction solution with pure water for 48 h, To remove DMAP and other water-insoluble by-products, freeze-dry the product; then disperse the product in absolute ethanol, and use ultrasonic waves to remove unreacted HA; finally freeze-dry the precipitated product to obtain a white solid, which is phenylboron Ester-HA copolymer. 4. preparation method according to claim 3, is characterized in that: described DSPE-PEG-Asp8 polymkeric substance and phenylboronate-HA copolymer are dissolved in 10 mL dimethyl sulfoxide, are added dropwise to 100 mL of PBS buffer, stirred for 30 minutes, stirred at room temperature for 4 hours in the dark, then dialyzed through a dialysis bag for 24 hours to remove organic solvents, and finally stored in freeze-dry. 5. The application of the mixed nanoparticles prepared by the method according to any one of claims 1 to 4 in the preparation of bone disease medicine. 6. The application according to claim 5, characterized in that: the mixed nanoparticles are used as a drug delivery carrier in the preparation of bone tumor drugs.