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CN117357659B - Construction and application of boric acid modified polyalanine-based nano prodrug - Google Patents

Construction and application of boric acid modified polyalanine-based nano prodrug Download PDF

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CN117357659B
CN117357659B CN202311648173.XA CN202311648173A CN117357659B CN 117357659 B CN117357659 B CN 117357659B CN 202311648173 A CN202311648173 A CN 202311648173A CN 117357659 B CN117357659 B CN 117357659B
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quercetin
boric acid
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CN117357659A (en
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王勇
洪科泽
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Jinan University
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Abstract

The invention belongs to the fields of polymer chemistry and biomedical engineering, and discloses construction and application of a boric acid modified phenylalanine-based nano prodrug. The boric acid modified phenylalanine-based nano prodrug consists of a polymer shown in a formula (I) and quercetin and R837 loaded by the polymer; the structure of the polymer of formula (I) is shown below, wherein m is 45; the value range of n is 15-18. The polymer of the formula (I) realizes high-efficiency load of quercetin, improves the overall hydrophobicity of a hydrophobic section of a polymer prodrug system, and is beneficial to the subsequent self-assembly of R837; the polymer of the formula (I) is loaded with quercetin and R837 to obtain the boric acid modified polyphenyl alanine nanometer prodrug which can down regulate the high expression PD-L1 receptor on the surface of tumor cells and further activate tumor immunity, and the quercetin and R837 have synergistic effect on improving the microenvironment of liver cancer tumors.

Description

Construction and application of boric acid modified polyalanine-based nano prodrug
Technical Field
The invention belongs to the fields of polymer chemistry and biomedical engineering, and particularly relates to construction and application of a boric acid modified polyalanine-based nano prodrug.
Background
Currently, drugs for systemic treatment of hepatocellular carcinoma mainly include chemotherapeutics such as docetaxel, sorafenib, apa Qu Ni, and lamatinib, and the use of such chemotherapeutics is often accompanied by serious adverse reactions, and the therapeutic effect is unsatisfactory.
The tumor microenvironment of hepatocellular carcinoma generally comprises liver cancer cells and surrounding fibroblasts, immune cells, vascular endothelial cells, extracellular matrix, cytokines, etc. In general, a great number of activated tumor-associated fibroblasts (TAFs) exist in the liver tumor microenvironment, and the tumor-associated fibroblasts secrete a great number of collagens and other matrixes to change the surrounding cellular environment of the tumor so as to prevent the delivery of medicines and infiltration of effector T cells, promote angiogenesis to help the growth of the tumor to happen, and play a certain role even in resisting and transferring the medicines; meanwhile, a large number of mature and hindered dendritic cells directly block antigen recognition, so that immune tolerance is caused, and hepatocellular carcinoma is a highly vascularized tumor, and has high Vascular Endothelial Growth Factor (VEGF) level and a programmed death receptor ligand (PD-L1) with high micro-environment expression, so that immune suppression of the hepatocellular carcinoma is further aggravated.
PD-L1 (Programmed Cell Death Ligand), also known as CD274, is a protein that is normally present on the surface of tumor cells, immune cells, and certain normal cells. In liver cancer (hepatocellular carcinoma), PD-L1 is usually expressed on the surface of liver cancer cells, while also being present on other cells in the tumor microenvironment of liver cancer. It is well known that the key role of PD-L1 is its interaction with the PD-1 (Programmed Cell Death Protein 1) receptor. This interaction inhibits the activity of the immune system, and in particular, it impedes the normal activation of T cells, making them ineffective in attacking liver cancer cells. This mechanism helps liver cancer evade monitoring and destruction of the immune system, thereby promoting tumor growth and spread. In addition, overexpression of PD-L1 can lead to the formation of immunosuppression in liver cancer. Such immunosuppression is manifested by inhibiting activation of immune cells, promoting impairment of immune cell function and inducing production of regulatory T cells. These factors act together to create an immunosuppressive liver cancer microenvironment. Quercetin (Quercetin) is a natural plant compound, which belongs to one of flavonoid compounds and is widely found in many plants. Studies have shown that quercetin not only can reverse the activation state of tumor-associated fibroblasts, but also can down-regulate the apoptosis receptor ligand (PD-L1) on the surface of tumor cells, thereby improving the microenvironment of tumor immunosuppression. In addition, R837 is an immunomodulatory drug approved by the united states Food and Drug Administration (FDA): TLR7 receptor agonists are reported to have great potential in stimulating dendritic cell maturation presenting tumor antigens to activate tumor immunotherapy. Therefore, the combination of the quercetin and the R837 can greatly improve the tumor immunosuppression microenvironment and enhance the curative effect of the liver cancer immunotherapy.
In recent years, the development of polymer nano-carriers has great potential in reducing toxic and side effects of medicaments and medicament delivery, and has profound significance in improving the solubility of hydrophobic medicaments, targeting by surface modification, specific microenvironment controlled release and biodegradability. However, conventional drug-loaded nanosystems by hydrophobic interaction are generally very limited in drug-loading efficiency, which is an important reason for impeding further application of nano-drugs. The tumor microenvironment sensitive bond linkage is an effective way for improving the drug loading, but most of the systems are complex to synthesize and limited in application. Therefore, the exploration of a polymer-drug nano system which is simple and easy to prepare and accurately regulates and controls release is an important scientific problem.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary object of the present invention is to provide a boric acid modified polyalanine-based nano-prodrug.
The boric acid modified phenylalanine-based nano prodrug of the invention improves the drug loading effect, improves the liver cancer immunosuppression microenvironment and realizes cooperative immunotherapy.
The invention also aims to provide a preparation method of the boric acid modified polyalanine-based nano prodrug.
The invention also aims to provide an application of the boric acid modified polyalanine-based nano prodrug in preparing liver cancer drugs.
The aim of the invention is achieved by the following scheme:
a boric acid modified polyphenyl alanine nanometer prodrug is composed of a polymer of a formula (I) and quercetin loaded by the polymer, and R837; the structure of the polymer of formula (I) is as follows:
wherein m is 45; the value range of n is 15-18.
The preparation method of the boric acid modified polyphenyl alanine based nano prodrug comprises the following steps:
s1, catalyzing cyclization of boron phenylalanine and triphosgene by using epoxypropane to obtain boron phenylalanine carboxyl cyclic anhydride;
s2, using polyethylene glycol-amino as a macromolecular initiator, catalyzing by 1, 3-tetramethylguanidine, and initiating boron phenylalanine anhydride to carry out ring-opening polymerization to obtain a polymer shown in the formula (I);
s3, loading quercetin and R837 on the polymer in the formula (I) to obtain the boric acid modified polyalanine nanometer prodrug.
The preparation method of the boric acid modified polyphenyl alanine based nano prodrug specifically comprises the following steps:
s1, mixing and stirring boron phenylalanine, epoxypropane and a solvent, adding triphosgene, reacting, filtering, concentrating and precipitating the obtained product, and recrystallizing the solid to obtain boron phenylalanine carboxyl cyclic anhydride;
s2, mixing and stirring polyethylene glycol-amino, 1, 3-tetramethyl guanidine and a solvent, adding the boron phenylalanine carboxyl cyclic anhydride obtained in the step S1, reacting, precipitating, and drying the solid to obtain a polymer of the formula (I);
s3, blending the polymer of the formula (I) obtained in the step S2, quercetin and R837 with a solvent, and adding water to obtain the boric acid modified polyalanine-based nano prodrug.
The solvent in the step S1 is tetrahydrofuran.
The solvent dosage in the step S1 is as follows: so that 1g of borophenylalanine is contained in every 100mL of solvent.
The molar ratio of the borophenylalanine to the propylene oxide in the step S1 is 1:10.
The molar ratio of the borophenylalanine to the triphosgene in the step S1 is 2:1.
the stirring time in the step S1 is 10-15min; the reaction was carried out at room temperature for 12h.
The precipitation in the step S1 is to add the concentrated solution into petroleum ether to obtain the precipitate.
The molar ratio of the polyethylene glycol-amino group to the 1, 3-tetramethylguanidine in the step S2 is 1:1.
the solvent in the step S2 is tetrahydrofuran.
The solvent dosage in the step S2 is as follows: so that the polyethylene glycol-amino, 1, 3-tetramethyl guanidine and boron phenylalanine carboxyl ring anhydride are completely dissolved.
The molar ratio of the polyethylene glycol-amino group to the borophenylalanine carboxyl cyclic anhydride in the step S2 is 1:15.
the stirring time in the step S2 is 10-15min; the reaction is carried out for 6-8 hours at room temperature.
The precipitation in the step S2 is to add the reacted solution into glacial diethyl ether to obtain the precipitate.
The mass ratio of the polymer of the formula (I), the quercetin and the R837 in the step S3 is 20:2-6:2.
And the solvent in the step S3 is at least one of dimethyl sulfoxide and methanol.
The dosage ratio of the polymer of the formula (I) to the solvent in the step S3 is as follows: 10mg:1mL.
The ratio of polymer of formula (I) to water used in step S3 was 1 mg/1 mL.
The blending in step S3 is specifically vortex for 1min at room temperature.
After blending in step S3, the resulting mixture was dialyzed against water for 3-5 days to remove the solvent and free drug.
The application of the boric acid modified polyalanine nanometer prodrug in preparing liver cancer drugs.
Compared with the prior art, the method has the following advantages:
the invention optimizes the synthesis process of the borophenylalanine cyclic anhydride, so that the borophenylalanine cyclic anhydride is simple and easy to prepare, and the polymerization process of the polyborophenylalanine is optimized, so that the polymerization time of the polyborophenylalanine is greatly reduced. Polyethylene glycol introduced into the polymer has good biocompatibility, can form a hydration layer on the surface of the polymer carrier, effectively shields the adsorption of negatively charged protein in the body to the polymer carrier, and avoids the clearance of a reticuloendothelial system to the nano-drug, thereby improving the stability of the nano-drug in vivo; the overall charge is weak and negative, so that longer in vivo circulation time can be realized, the bonding effect of phenylboronic acid and quercetin can realize high-efficiency load of quercetin, and meanwhile, the overall hydrophobicity of the hydrophobic section of the polymer prodrug system is improved, so that the subsequent self-assembly is facilitated; phenylboronic acid is a multi-responsive functional group that is capable of responding to both local high Reactive Oxygen Species (ROS) and pH in the microenvironment. The invention provides a polymer capable of being bonded with 1,2 or 1,3 alcohol or phenolic hydroxyl, which improves drug loading efficiency through chemical bond bonding. Aiming at improving the microenvironment of liver cancer tumor, quercetin with 1,2 or 1,3 phenolic hydroxyl groups can reverse the activation state of tumor-related fibroblasts, inhibit the generation of cell interstitials and simultaneously down regulate the PD-L1 receptor with high expression on the surface of tumor cells; the immune agonist R837 can induce the mature hindered dendritic cells which are infiltrated in a large amount by the liver cancer microenvironment to mature, promote the occurrence of tumor immunogenicity death, release tumor antigens, further activate tumor immunity and synergistically improve the tumor immunosuppression microenvironment, thereby achieving the aim of efficiently treating the liver cancer, and the quercetin and the R837 have a synergistic effect on improving the liver cancer tumor microenvironment.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of BPA-NCA obtained in example 1.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of PEG-PBPA obtained in example 1.
FIG. 3 is a graph showing the particle size of the nano-drug obtained in example 2 after loading quercetin and R837.
FIG. 4 is a transmission electron microscope image of the nano-drug loaded with quercetin and R837 obtained in example 2.
FIG. 5 is a schematic of PEG-PBPA mixed with quercetin and an ultraviolet absorbance graph.
FIG. 6 is a flow cytometry graph of a nano-drug to improve liver tumor microenvironment.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The reagents used in the examples are commercially available as usual unless otherwise specified.
Petroleum ether: boiling point of 60-90 DEG C
Example 1: synthesis of Polymer of formula (I)
（I）；
S1, synthesizing boron phenylalanine carboxyl cyclic anhydride:
is prepared by cyclizing boron phenylalanine and triphosgene, and the reaction and the process thereof are as follows:
1g of boron phenylalanine (mw: 209.01) was added to a reaction flask, 100mL of Tetrahydrofuran (THF) was added thereto and stirred, 2.77g of Propylene Oxide (PO) (mw: 58.08) was added to the above-mentioned reaction system and stirred for 10 minutes to mix uniformly, then 0.7g of triphosgene (BTA) (mw: 296.73) was weighed and added thereto and stirred at room temperature for 12 hours. Filtering the residual unreacted substances after the reaction is finished, concentrating the filtrate, adding petroleum ether for precipitation, and recrystallizing to obtain boron phenylalanine carboxyl cyclic anhydride (BPA-NCA).
The nuclear magnetic hydrogen spectrum of BPA-NCA is shown in FIG. 1. 1 H NMR (300 MHz, DMSO-d6,δ): 9.09 (s, 1H,-CONH-), 8.03 (s, 2H, -B(OH) 2 ), 7.71 and 7.14 (d, J = 7.6 Hz, 4H, -C 6 H 4 -), 4.79 (t, J = 5.2 Hz, 1H, -COCHNH-), 3.03 (d, J = 5.2 Hz, 2H, -C 6 H 4 CH 2 -).
S2 polyethylene glycol-polyborophenylalanine (PEG-PBPA)
Using polyethylene glycol-amino as macromolecular initiator to initiate ring-opening polymerization of boron phenylalanine anhydride so as to obtain the invented polymer
0.5 g PEG-NH was taken 2 (m=45, mw: 2000) was added to the reaction flask, dried under vacuum at 80℃for 4. 4 h, dissolved 0.028g of 1, 3-Tetramethylguanidine (TMG) (mw: 115.18) and stirred for 10 minutes, dissolved 0.88. 0.88 g boron phenylalanine cyclic anhydride (mw: 235.00) in THF and then added to the reaction flask, and the reaction flask was supplementedAnhydrous THF was charged to 25 mL, after 7h reaction at room temperature, the precipitate was precipitated in 200 mL iced diethyl ether, placed in a refrigerator overnight at 4 ℃, the precipitate was collected by centrifugation and dried under vacuum to give polyethylene glycol-polyborophenylalanine (PEG-PBPA, m=45, n=15).
The PEG-PBPA nuclear magnetic hydrogen spectrum is shown in figure 2. 1 H NMR (300 MHz, DMSO-d6, δ): 7.68 and 7.22(-C 6 H 4 B(OH) 2 ), 2.96-2.78 (-COCHNH-), 3.51 (-OCH 2 CH 2 O-), 4.49 (-C 6 H 4 CH 2 -).
Example 2: synthesis of boric acid modified polyalanine based nano prodrug
Polymer (I) 20mg prepared in example 1 was taken and dissolved in 2 mL DMSO along with 2mg quercetin and 2mg R837, slowly added to 20mL deionized water with vigorous stirring, then dialyzed in deionized water for 3 days to remove DMSO, and concentrated to obtain the boric acid modified phenylalanine-based nano-prodrug.
FIG. 3 is a graph of DLS particle size for a nano-prodrug having a particle size of 52nm; fig. 4 is a transmission electron microscope image visually verifying the particle size characterization described above.
Test example 1: load effect of quercetin
Taking 20mg PEG-PBPA prepared in example 1 and 6 mg quercetin respectively dissolved in 2 mL DMSO and 3 mL DMSO to obtain PEG-PBPA solution and quercetin solution, adding 1mL quercetin solution into the PEG-PBPA solution, and observing color change; the polymer solution, quercetin solution and polymer mixed solution were spectroscopically scanned using ultraviolet spectrophotometry, respectively.
As shown in fig. 5, both the uv results and the schematic diagrams indicate that the mixed solution has a significant uv absorption red shift compared to the quercetin solution, demonstrating that BPA forms a dynamic borate ester bond with quercetin.
Test example 2: quercetin drug loading rate of PEG-PBPA
Taking polymer (I) 20mg prepared in example 1, respectively dissolving 2mg, 3 mg, 4mg and 6 mg quercetin together in 2mg of DMSO, slowly adding into 20mL deionized water while vigorously stirring, and then dialyzing in deionized water for 3 days to remove DMSO and free drugs, thereby obtaining samples 1-4 to be tested. And obtaining the drug loading rate and the encapsulation rate corresponding to different dosage through ultraviolet spectrum scanning analysis.
TABLE 1 statistical table of drug loading rates
Test example 3: construction of mouse in-situ liver tumor model and nano-drug treatment
C57BL/6J mice are selected as modeling objects, and each mouse is positioned at the left liver She Jiechong 2.2x10 6 Slight bulge of abdomen is obviously observed after about 5 days of Hepa1-6 liver tumor cells, and the tail vein injection nanometer medicine treatment can be carried out after touching hard lumps (Control: injection of normal saline, Q-NP: quercetin-loaded group, 20mg of polymer (I) and 2mg of quercetin are prepared, I-NP: R837-loaded group, 20mg of polymer (I) and 2mg of R837 are prepared, QI-NP: double medicine co-loaded group, 20mg of polymer (I), 2mg of quercetin and 2mg of R837 are prepared, the administration concentration of mice is that quercetin is 10mg/kg, R837 is 4mg/kg., the administration time is sequentially 3 rd, 6 th, 9 th and 12 th day, the first day is 0 day. On day 15 of treatment, mice were sacrificed and tumor tissue was collected for flow analysis. Depending on the tumor sphere size, an appropriate amount of tissue digests (digests containing 200. Mu.g/mL DNase I,1 mg/mL collagenase I,0.1 mg/mL hyaluronidase dissolved in DMEM medium) were added, digested with shaking at 37℃for 0.5h, the digested cells were screened through a 70 μm sieve, centrifuged at 4℃at 2000 r for 5min, and the cell pellet was washed once with FACS (PBS+2% FBS) and subsequently used for flow-through staining analysis. The staining channel of the dendritic cells was set as: CD11c + CD80 + CD86 + ；IFNγ + The T cell staining channel was set up as: CD3 + CD4 - CD8 + IFNγ + . 600uL FACS was resuspended on-line after staining was complete. The analysis results are shown in FIG. 6.
Quercetin with 1,2 or 1,3 phenolic hydroxyl groups can reverse the activation state of tumor-associated fibroblasts, inhibit interstitial generation, and simultaneously down regulate the PD-L1 receptor with high expression on the surface of tumor cells; immunity excitationThe activator R837 can induce the maturation of mature hindered dendritic cells which are infiltrated in a large amount in the liver cancer microenvironment, promote the occurrence of immunogenic death, release tumor antigens and further activate tumor immunity. In FIG. 6, CD80 and CD86 are typical marker surface antigens for mature DCs; CD8 is a cytotoxic T cell marker surface antigen, IFN-gamma is an inflammatory-inducing factor secreted by the relevant cells. As shown in FIG. 6, the Control group showed very few mature DCs and CD8 + IFNγ + Infiltration of T cells at 20.8% and 3.12% respectively, which indicates that liver tumor is in immunosuppressive microenvironment; Q-NP and I-NP both increase DCs and CD8 to some extent + IFNγ + Infiltration of T cells; whereas QI-NP is capable of converting DCs and CD8 + IFNγ + The T cells are greatly improved to 59.7% and 47.6%. Single drug pair DCs and CD8 + IFNγ + Compared with the two medicines, the infiltration of T is reduced by 12.75 percent and 28.6 percent on average, so that the quercetin and R837 have the function of synergistically improving the liver tumor immunosuppression microenvironment, thereby achieving the purpose of efficiently treating the liver tumor.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (1)

1. The boric acid modified polyphenyl alanine nanometer prodrug is characterized by comprising a polymer of a formula (I) and quercetin and R837 loaded by the polymer;
the structure of the polymer of formula (I) is as follows:
wherein m is 45; the value range of n is 15-18;
the boric acid modified polyphenyl alanine based nano prodrug is prepared by the following steps:
s1, catalyzing cyclization of boron phenylalanine and triphosgene by using epoxypropane to obtain boron phenylalanine carboxyl cyclic anhydride;
s2, using polyethylene glycol-amino as a macromolecular initiator, catalyzing by 1, 3-tetramethylguanidine, and initiating boron phenylalanine anhydride to carry out ring-opening polymerization to obtain a polymer shown in the formula (I);
s3, loading quercetin and R837 on a polymer in the formula (I) to obtain a boric acid modified polyalanine-based nano prodrug;
the boric acid modified polyphenyl alanine based nano prodrug is used for preparing liver cancer drugs.
CN202311648173.XA 2023-12-05 2023-12-05 Construction and application of boric acid modified polyalanine-based nano prodrug Active CN117357659B (en)

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CN114917360A (en) * 2022-05-18 2022-08-19 暨南大学 Construction method and application of small-particle-size nano system for co-delivery of small-molecule drugs and siRNA
CN115417889A (en) * 2022-06-15 2022-12-02 苏州大学 L-4-dihydroxyborophenylalanine-N-carboxylic acid internal anhydride monomer and polyamino acid as well as preparation method and application thereof

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CN114917360A (en) * 2022-05-18 2022-08-19 暨南大学 Construction method and application of small-particle-size nano system for co-delivery of small-molecule drugs and siRNA
CN115417889A (en) * 2022-06-15 2022-12-02 苏州大学 L-4-dihydroxyborophenylalanine-N-carboxylic acid internal anhydride monomer and polyamino acid as well as preparation method and application thereof

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