CN114832114B

CN114832114B - ATRA-PBAE prodrug copolymer, and preparation method and application thereof

Info

Publication number: CN114832114B
Application number: CN202210488407.8A
Authority: CN
Inventors: 李伟男; 孙佳琳; 王艳宏
Original assignee: Heilongjiang University of Chinese Medicine; Heilongjiang Vocational College for Nationalities
Current assignee: Heilongjiang University of Chinese Medicine; Heilongjiang Vocational College for Nationalities
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2024-01-30
Anticipated expiration: 2042-05-06
Also published as: CN114832114A

Abstract

The invention discloses an ATRA-PBAE prodrug copolymer, a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides an ATRA-PBAE prodrug copolymer, which is synthesized into a PBAE copolymer with pH sensitivity through Michael addition reaction; the pH-responsive ATRA prodrug copolymer is then synthesized by an esterification reaction. The prodrug compound disclosed by the invention has obvious effects on preventing and treating tumors, and the drug-loaded nanoparticle is SchB/ATRA-PBAE, is obtained by coating SchB with ATRA-PBAE prodrug copolymer, and has obvious effects on preventing and/or treating tumors, especially breast tumors.

Description

ATRA-PBAE prodrug copolymer, and preparation method and application thereof

Technical Field

The invention relates to an ATRA-PBAE prodrug copolymer and a drug nanoparticle of an entrapped SchB, belonging to the technical field of drugs.

Background

At present, malignant tumors become common diseases and frequently-occurring diseases of various countries in the world, and the morbidity and mortality of the malignant tumors are in a trend of rising year by year. Among them, breast Cancer (BC) has been the most common cancer worldwide over lung cancer, the first leading killer to threat to female health. Although the survival rate of patients in most high-income countries has exceeded 80%, the survival rate in low-income countries is still low. The current breast cancer treatment still faces a number of problems, such as: the accumulation of the drug in tumor tissues is low, drug resistance exists, and the recurrence and metastasis are easy. Many antitumor small molecule chemotherapeutics in clinic are less bioselective due to systemic distribution after administration, have low drug concentration in tumor tissues, and tumor cells have MDR (multidrug resistance), so that the treatment effect is not ideal. In addition, the physical and chemical properties such as solubility of the drug also greatly influence the curative effect of the drug.

Today, breast cancer is treated only for differentiated tumor cells, and those remaining heterogeneous tumor cells dormant for a period of time can self-renew to form new tumors, resulting in recurrence of breast cancer cells. Therefore, the treatment of breast cancer is not only to eliminate common tumor cells, but also to target and kill heterogeneous tumor cells, so that the breast cancer is treated as fundamentally as possible. The discovery and theoretical suggestion of tumor stem cells (Cancer stem cells, CSCs) provides a more specific goal and direction for tumor treatment. CSCs theory and research suggest that CSCs are a subset of cells with specific expression, have complex signal transduction pathways and high-expression drug-resistant proteins, and are the root cause of tumor cell MDR phenomenon. Therefore, how to construct a drug delivery system that can target tumor cells and CSCs simultaneously is important.

ATRA is a natural derivative of vitamin a, mainly comprising a hydrophobic end, a conjugated side chain and a hydrophilic end, and has photosensitivity and is easy to be rapidly metabolized. The antitumor activity of ATRA has been of great interest, which is an anti-leukemia (acute promyelocytic leukemia, APL) drug. In APLA treatment, ATRA inhibits APL stem cell growth mainly by modulating the PmL-RAR- α pathway. However, the target of ATRA is still not determined, and in order to solve the problem, wei et al developed a high throughput screening (High throughput screening, HTS) for compounds targeting active Pin1, and found that ATRA mainly formed a salt bridge by simulating the psER/Thr-Pro gene sequence in Pin1 substrate, so that the hydroxyl group in the structure of ATRA was combined with Pin1 catalytic residues, and finally selectively inhibited and degraded active Pin1 in cancer cells, and exerted effective anticancer activity on APL and triple-negative breast cancer, TNBC.

In recent years, although ATRA has remarkable anti-tumor effect, clinical application is limited due to the problems of low oral bioavailability, poor water solubility, short half-life and the like. Thus, in order to effectively solve the above problems, a stable delivery system is required to transport ATRA. The nano-drug delivery system increases the stability of the drug and can lead the drug to reach lesion sites such as tumors in a targeting way. Li and the like successfully prepare a dacarbazine-carried and ATRA lipid nano-preparation (lipid nanoformulations, LNF), and the result shows that the LNF effectively inhibits the proliferation of melanoma cells, induces the apoptosis of tumor cells and inhibits the cell cycle process and the cell migration. Kamel develops an inhalable double-targeting mixed lipid nano-core-protein shell complex, effectively realizes local delivery of ATRA and genistein, and provides a potential approach for local lung cancer treatment. In addition, to enhance the water solubility and oral absorption availability of ATRA, ATRA is encapsulated into liposomes, solid lipid nanoparticles, and PLLA/PEG-PLLA nanoparticles.

However, the conventional physical method for encapsulating the nano-preparation has the problems of low loading rate, burst release, leakage and the like, and further application of the nano-preparation is limited. In contrast, the method for synthesizing the prodrug by utilizing the original drug and the nano technology can greatly improve the drug loading rate and better control the drug release. Zhen et al synthesized amphiphilic ATRA-PEG conjugate by esterification reaction with ATRA as model drug, and self-assembled by emulsion-solvent evaporation method to form nano micelle. The results show that the oral availability of the prodrug nano-formulation is improved by 2.0 times compared with ATRA, and the membrane permeability is enhanced. Zhu et al coupled ATRA with Pluronic F127 by esterification and self-assembled to form nano-micelles, which significantly increased cytotoxicity compared to free ATRA; the combination with cisplatin can synergistically enhance the cytotoxic effect of cisplatin, and effectively inhibit the growth of breast tumors in vivo. ATRA and Glycerophosphorylcholine (GPC) successfully synthesize a dual ATRA-PC prodrug, which can be assembled into uniform-particle-size, negatively-surface-charged liposomes, and is stable in a neutral environment, with sustained release of ATRA under weakly acidic conditions. And prodrugs exhibit cytotoxicity comparable to free drug, longer blood residence time and greater bioavailability. In conclusion, the prodrug self-assembled nanoparticle has certain pharmaceutical advantages including good stability, high tumor accumulation, long in vivo circulation time, strong cytotoxicity and the like, and has extremely strong anti-tumor effect. In addition, ATRA also has MDR phenomenon in clinical application, which is the biggest trouble for clinical application.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: to overcome the disadvantages of low ATRA bioavailability, poor water solubility and short in vivo biological half-life, an ATRA-PBAE prodrug copolymer is provided. Meanwhile, in order to solve the problem of tumor drug resistance, a SchB/ATRA-PBAE prodrug nanoparticle is also provided, which is obtained by encapsulating SchB with ATRA-PBAE prodrug copolymer.

In order to solve the technical problems, the invention provides a prodrug compound which is an ATRA-PBAE prodrug copolymer. The preparation method of the prodrug compound is that PBAE copolymer with pH sensitivity is synthesized by Michael addition reaction; the pH-responsive ATRA prodrug copolymer is then synthesized by an esterification reaction.

The method specifically comprises the following steps:

(1) Synthesis of poly (beta-amino esters)

PBAE synthesis 400mg of 5-amino-1-pentanol and 640mg of 1, 4-butanediol diacrylate are precisely weighed by Michael addition reaction in a molar ratio of 1.2:1, dissolved in methylene dichloride and magnetically stirred at 55 ℃ for 48 hours, and after the reaction is finished, the reaction product is purified according to the following process: the product was placed in a 10mL EP tube, 5mL diethyl ether was added, vortexed for 5min, centrifuged (5600 rpm,5 min), the top solution was discarded, and repeated 3 times. And (3) drying in vacuum to remove the organic solvent to obtain a product, and storing at 4 ℃ for later use.

(2) Synthesis of poly (beta-amino ester) -all-trans retinoic acid prodrugs

ATRA-PBAE synthesis ATRA containing carboxyl groups was completely dissolved in dichloromethane by esterification, EDC 10mg and DMAP 5mg were precisely weighed and dissolved in dichloromethane, and slowly added dropwise to a dichloromethane solution of ATRA; stirring at 20deg.C for half an hour; in addition, a previously prepared dichloro solution of PBAE was added to the above solution; the mixture was stirred in the dark at 20℃for 48h; excess ATRA was removed by precipitation in ice-cold ether; to completely remove free EDC HCl and DMAP, the solution was transferred to a dialysis membrane and dialyzed against ultrapure water; purifying for 3 times for 24 hours; finally, the organic solvent is removed by vacuum drying and stored at 4 ℃.

Meanwhile, the invention also provides application of the nano particles formed by the prodrug polymer-entrapped drug in preparing drugs for preventing and/or treating tumors. Furthermore, the tumor mainly refers to tumor stem cells, especially breast tumor stem cells.

The invention provides a prodrug nanoparticle medicine, which is a preparation intermediate prepared by taking a prodrug compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Furthermore, the ATRA-PBAE provided by the invention can also be used for encapsulating a certain medicine. Furthermore, the invention provides an ATRA-PBAE-entrapped SchB nanoparticle, which is a SchB/ATRA-PBAE nanoparticle and is obtained by entrapping SchB by an ATRA-PBAE prodrug copolymer.

Meanwhile, the invention also provides application of the SchB/ATRA-PBAE nanoparticles in preparing medicines for preventing and/or treating tumors. The tumor mainly refers to tumor stem cells, in particular breast tumor stem cells.

The invention designs a novel pH response type drug delivery system for synchronously delivering All-trans retinoic acid (ATRA) and schisandrin B (gamma-Schizandrin B, schB) to target breast tumor cells and stem cells thereof. The basic idea is that poly (beta-amino esters) (PBAE) with pH sensitivity is taken as a carrier to construct ATRA prodrug nano particles with pH sensitive release function aiming at breast tumor cells and breast tumor stem cells (Breast cancer stem cell, BCSCs), the problems of poor ATRA water solubility, short biological half-life and the like are improved by utilizing a prodrug technology, and the killing effect on the breast tumor cells and the stem cells thereof is realized by means of the pH sensitivity and the mediated lysosome escape function of the ATRA prodrug nano particles; on the basis, an ATRA prodrug nanoparticle (SchB/ATRA-PBAE Nanoparticles, schB/ATRA-PBAE NPs) carrying SchB with the function of reversing multi-drug resistance (multidrug resistance, MDR) is constructed, so that the synergistic delivery of the Pin1 inhibitor ATRA and the P-gp inhibitor SchB is realized, the MDR function of BCSCs can be reversed, the treatment effect of ATRA on resisting the BCSCs is further improved, and the aim of synchronously killing tumor cells and the BCSCs is fulfilled.

The beneficial effects of the invention are as follows:

the invention selects all-trans retinoic acid and schisandrin B as model drugs. Constructing an ATRA prodrug nano drug delivery system carrying SchB. Firstly, ATRA-PBAE prodrug nano-particles with pH sensitive characteristics are constructed by ATRA and PBAE, so that the defects of short half-life period of ATRA in vivo, poor water solubility, low plasma drug concentration and the like are overcome. Secondly, the SchB serving as a P-gp inhibitor can effectively overcome the phenomenon that ATRA is excreted by BCSCs, so that the ATRA effect is maximized, and a new idea is provided for constructing a novel anti-tumor targeted drug delivery system. After reaching the focus part, the nanoparticle smoothly enters the inside of the cell through an endocytic way, and the escape of the medicine in a lysosome is realized, so that the targeted killing of tumor cells and CSCs at the focus position is realized. Meanwhile, the toxicity and uptake conditions of the double drug-loaded nanoparticles on MCF-7 cells and MS cells are studied deeply, and the action mechanism of MDR is reversed, so that a reference is provided for the related study of reversing the MDR action of the CSCs.

Drawings

The following describes the embodiments of the present invention in further detail with reference to the drawings.

FIG. 1-1 is a polymer synthesis route (a) PBAE synthesis route, (b) ATRA-PBAE synthesis route.

FIGS. 1-2 are FT-IR characterization of (a) ATRA, (b) PBAE, (c) ATRA-PBAE.

FIGS. 1-3 are nuclear magnetic patterns of 5-amino-1-pentanol (a) and PBAE (b).

FIGS. 1-4 are nuclear magnetic resonance spectra of ATRA (a) and ATRA-PBAE (b).

FIGS. 1-5 are titration curves for ATRA-PBAE.

FIG. 2-1 is an ATRA-PBAE prodrug nanoparticle morphology (a) versus transmission electron microscopy (b).

Fig. 2-2 is an ATRA release profile in ATRA-PBAE polymers at different pH conditions, n=3.

FIG. 3-1 is a photomicrograph (x 100 times) of MCF-7 (left) and MS cells (right).

FIG. 3-2 shows cell viability of MCF-7 cells after 48h incubation with ATRA-PBAE-prodrug copolymer, free ATRA (a) and different concentrations of blank PBAE copolymer (b) at different pH conditions.

FIGS. 3-3 are graphs showing the inhibitory effect of free ATRA and ATRA-PBAE prodrug nanoparticles on MS cells at different pH conditions.

FIGS. 3-4 are CLSM observations of uptake of Cy5.5-PBAE nanoparticles by MCF-7 cells.

Fig. 3-5 are flow cytometry assays for fluorescence intensity of MCF-7 cells on cy5.5-PBAE nanoparticle uptake (n=3, < p < 0.05). FIGS. 3-6 are graphs showing cell uptake after incubation of Cy5.5-PBAE with MS cells for 0.5h, 2h and 4h at different pH conditions under CLSM.

Figures 3-7 are flow cytometry assays for fluorescence intensity of MS cells uptake of cy5.5-PBAE nanoparticles (n=3, < p < 0.05).

Figures 3-8 are relative uptake ((n=3, ×p < 0.05) of cy5.5-PBAE NPs by MCF-7 cells (a) and MS cells (b) after inhibitor incubation.

FIGS. 3-9 are CLSM observations of uptake of Cy5.5-PBAE NPs by MCF-7 cells and MS cells.

FIG. 4-1 is a transmission electron microscope image (scale: 200 nm) of (a) the appearance of SchB/ATRA-PBAE NPs (A: water, B: ATRA-PBAE NPs, C: schB/ATRA-PBAE NPs; B) the appearance of SchB/ATRA-PBAE NPs.

FIG. 4-2 is a graph of particle size of ATRA-PBAE NPs.

FIGS. 4-3 are particle size diagrams of SchB/ATRA-PBAE NPs.

Fig. 4-4 are SchB in vitro cumulative release rate curves (n=3).

Fig. 5-1 is the MS cell inhibitory effect of SchB-loaded nanoparticles at different pH conditions (n=3).

FIG. 5-2 is a fluorescence microscope of uptake of C6/ATRA-PBAE NPs by MS at 4h under different pH conditions.

Fig. 5-3 are flow cytometry assays for fluorescence intensity of MS uptake of C6/ATRA-PBAE nanoparticles (n=3, < p < 0.05).

FIGS. 5-4 show the effect of different formulations on the P-gp expression in MS cells.

FIGS. 5-5 are total protein standard curves.

Detailed Description

Example 1

This example provides the synthesis and characterization of ATRA-PBAE, with the following specific procedures:

1. instrument and reagent

The experimental apparatus and reagents and materials used in this example are commercially available products.

2. Experimental method

2.1 Synthesis of Poly (beta-amino esters)

PBAE synthesis is via michael addition reactions. 400mg of 5-amino-1-pentanol and 640mg of 1, 4-butanediol diacrylate were precisely weighed in a molar ratio of 1.2:1, dissolved in methylene chloride and magnetically stirred for 48h at 55 ℃. After the reaction, the reaction product was purified according to the following procedure: the product was placed in a 10mL EP tube, 5mL diethyl ether was added, vortexed for 5min, centrifuged (5600 rpm,5 min), the top solution was discarded, and repeated 3 times. And (3) drying in vacuum to remove the organic solvent to obtain a product, and storing at 4 ℃ for later use.

2.2 Synthesis of Poly (beta-amino ester) -all-trans retinoic acid prodrugs

ATRA-PBAE synthesis is by esterification. The ATRA containing carboxyl groups was completely dissolved in dichloromethane. 10mg of EDC and 5mg of DMAP are weighed out precisely, dissolved in methylene chloride and added slowly dropwise to a methylene chloride solution of ATRA. Stirred at 20℃for half an hour. In addition, a previously prepared dichloro solution of PBAE was added to the above solution. The mixture was stirred in the dark at 20℃for 48h. Excess ATRA was removed by precipitation in ice-cold ether. To completely remove free EDC HCl and DMAP, the solution was transferred to a dialysis membrane and dialyzed against ultrapure water. The purification was performed 3 times for 24 hours. Finally, the organic solvent is removed by vacuum drying and stored at 4 ℃.

2.3 Characterization of ATRA-PBAE prodrugs

2.3.1 FT-IR

PBAE, ATRA, PBAE-ATRA was characterized using FT-IR techniques. Appropriate amounts of ATRA, PBAE and PBAE-ATRA solids were weighed, mixed with dry potassium bromide powder, ground, tabletted and tested using an infrared spectrometer.

2.3.2 ¹ H-NMR

Structural characterization of ATRA, PBAE and ATRA-PBAE by 1H-NMR technique, respectively dissolving appropriate amounts of ATRA, PBAE and ATRA-PBAE in CDCl ₃ In a solvent, a nuclear magnetic spectrometer and Tetramethylsilane (TMS) are used as internal standards to determine the molecular structure.

3. Experimental results

3.1 Synthesis of prodrug ATRA-PBAE

In this example, a pH-responsive PBAE was used, which was soluble in a weak acid environment. The synthesis mechanism of PBAE is Michael addition reaction, and the synthetic route is shown in FIG. 1-1 (a). And 5-amino-1-pentanol and 1, 4-butanediol diacrylate are selected for addition reaction to synthesize the acrylate-terminated PBAE. In order to avoid overhigh temperature and volatilization of dichloromethane in the experimental process, which further influences experimental results, a condensing device is adopted. After 48h of reaction, the polymer obtained was a clear, yellowish, viscous liquid with a yield of 90.8%.

The synthesis of ATRA-PBAE employs an esterification reaction. ATRA (containing a carboxyl group in the structure) reacts with EDC to form O-acylisourea, which is more reactive than carboxylic acid, and is attacked by alcohol to yield the corresponding ester. The medicine ATRA is a light-resistant compound, and the whole light-resistant process is ensured. After 48h a yellow viscous liquid was obtained in 48.2% yield. The synthetic route is shown in FIG. 1-1 (b).

3.2 characterization of prodrug ATRA-PBAE

3.2.1 FT-IR characterization

As shown in FIGS. 1-2 (a), 3045cm ^-1 And 2926cm ^-1 Corresponding to the C-H olefins and aliphatic stretching vibrations in the ATRA structure. 1683cm ^-1 A c=o stretching vibration peak of all-trans retinoic acid occurs. 1598cm ^-1 The strong band appearing here is related to the stretching vibration of the c=c olefinic bond. At 1182cm ^-1 The peak at which is related to the tensile vibration of C-O. The FT-IR spectrum of the PBAE is shown in FIGS. 1-2 (b). In the infrared spectrum of PBAE, at a wavenumber of 3057cm ^-1 There, a=c—h stretching vibration absorption peak appears at 2941cm ^-1 、2852cm ^-1 、1458cm ^-1 And 1392cm ^-1 At this point, an absorption peak of methyl group appears. At a wave number of 1730cm ^-1 The new ester-bonded carbonyl group showed a stretching vibration absorption peak, and in the infrared spectrum of PBAE, at a wave number of 1650cm ^-1 About, there was no absorption peak, indicating that the primary amino group did not appear, and further indicating that the terminal amino group of 5-amino-1-pentanol was linked to an olefinic group on 1, 4-butanediol diacrylate, indicating that the PBAE synthesis was successful. In FIGS. 1-2 (c), the structural difference between the ATRA-PBAE polymer and the PBAE copolymer cannot be judged from the infrared spectrogram.

3.2.2 ¹ Characterization by H-NMR

FIGS. 1-3 are nuclear magnetic resonance spectra of 5-amino-1-pentanol and PBAE. In the nuclear magnetic resonance spectrum of the 5-amino-1-amyl alcohol, each hydrogen proton peak of the 5-amino-1-amyl alcohol belongs to the following steps: delta 6:1.81ppm (-NH) ₂ )，δ5：2.69ppm(-CH ₂ -NH ₂ )，δ2-4：1.42-1.59ppm(-CH ₂ -CH ₂ -CH ₂ -)，δ1：3.61ppm(-CH ₂ -OH)。

In the PBAE nuclear magnetic resonance spectrum, each hydrogen proton peak of PBAE is attributed as follows: delta: 6.14-5.82ppm (=CH) ₂ )，δ2：6.41ppm(-CH＝CH ₂ )，δ3,6：4.10ppm(-O-CH ₂ -)，δ13：3.65ppm(-CH ₂ -OH)，δ8：2.80ppm(-CH ₂ -N-CH ₂ -)，δ7,9：2.47ppm(-N-CH ₂ -，-CH ₂ -CO-O-)，δ4,5：1.78ppm(-CH2-CH2-)，δ10,12：1.56ppm(-CH ₂ -CH ₂ (-), δ11:1.42ppm (- -CH2- -). In the PBAE nuclear magnetic resonance spectrum, the original appearance in the 5-amino-1-amyl alcohol nuclear magnetic resonance spectrumPrimary amine bond (-NH) with chemical shift of about 1.81ppm ₂ ) And disappeared, thereby forming a tertiary amine bond. Thus, this result may indicate successful synthesis of PBAE.

FIGS. 1-4 are ATRA, PBAE, ATRA-PBAE nuclear magnetic resonance spectra. In the ATRA nuclear magnetic resonance spectrum, each hydrogen proton peak of ATRA is attributed as follows: δ2,3:7.05ppm (-ch=ch-), δ6:6.30ppm (-ch=), δ4,5:6.19ppm (=ch-), δ1:5.82ppm (-CO-ch=), δ13:2.39ppm (-CH) ₃ )，δ14：2.03ppm(-CH ₃ )，δ15：1.74ppm(-CH ₃ )，δ9,10,11：1.65-1.49ppm(-CH ₂ (-), delta 16,17:1.04ppm (CH 3-C-CH 3). The assignment of each hydrogen proton peak of PBAE has been described. The individual hydrogen proton peaks of the ATRA-PBAE were attributed as follows: delta 15,16:6.30-6.22ppm (-ch=ch-), δ 17,18,19:6.14-6.02ppm (-ch=c-), delta 14:5.78ppm (o=c—ch=), δ 3,6,13:4.10ppm (-O-CH) ₂ -)，δ8：2.80ppm(-N-CH ₂ -)，δ7,9,23：2.47ppm(-CH ₂ -CO-O-，-N-CH ₂ -)，δ24 1.98ppm(＝C-CH3)，δ4,5,25：1.78ppm(-CH ₂ -CH ₂ -)，δ10,12：1.57ppm(-CH2-)，δ11：1.45ppm(-CH2-)，δ20,21,22：1.34-1.25ppm(-CH ₂ --CH ₂ -CH ₂ -CH ₂ (-), delta 26,27:1.04ppm (CH 3-C-CH 3). Comparing the nuclear magnetic resonance patterns of the three copolymers of ATRA, PBAE, ATRA-PBAE, the peak-to-peak ratio of the ATRA and PBAE polymers can be found in the ATRA-PBAE nuclear magnetic resonance patterns. Wherein, the peak of the two copolymers of PBAE and ATRA-PBAE is compared, and the displacement in the PBAE nuclear magnetic resonance spectrum is delta 133.62ppm (-CH) ₂ The peak of-OH) is significantly shifted in the ATRA-PBAE nuclear magnetic resonance spectrum (δ13.10ppm) because the carboxyl structure of ATRA undergoes an esterification reaction with the hydroxyl group of PBAE to form an ester bond, and the methylene shift linked to-O-is significantly increased. This result demonstrates the successful synthesis of the ATRA-PBAE copolymer.

This example uses a Michael addition reaction to synthesize a PBAE polymer and an esterification reaction to synthesize an ATRA-PBAE prodrug polymer.

Adopts FT-IR, ¹ The H-NMR performed structural characterization on PBAE and ATRA-PBAE, demonstrating successful synthesis of PBAE and ATRA-PBAE. In addition, the prodrug polymer had a pKa of 6.55 as measured by acid-base titration,as the pH decreases, the particle size increases and comprehensive analysis indicates that the ATRA-PBAE prodrugs are pH sensitive.

Example 2

This example provides a formulation evaluation of ATRA-PBAE prodrug nanoparticles based on successful synthesis of the ATRA-PBAE prodrug polymer of example 1.

1. Instrument and materials

2. Experimental part

2.1 selection of conditions for high Performance liquid chromatography

Chromatographic column: diamond C18 (4.6X105 mm,5 μm); mobile phase: methanol-water-glacial acetic acid (90:9.5:0.5);

Column temperature: 25 ℃; flow rate: 0.8mL/min; detection wavelength: 340nm; sample injection amount: 10 mu L.

2.2 Preparation of ATRA-PBAE prodrug nanoparticles

And preparing ATRA-PBAE prodrug nanoparticles by adopting a precipitation method. Precisely weighing 10mg of ATRA-PBAE polymer and dissolving with 1mL of acetone; and under the action of magnetic stirring, the prodrug acetone solution is slowly dripped into the poloxamer 188 water solution containing 1 percent. Stirring is continued at room temperature until acetone is completely volatilized, and the prodrug nanoparticle aqueous solution with light blue opalescence is obtained through a filtering membrane.

2.3 Evaluation of ATRA-PBAE prodrug nanoparticle formulations

2.3.1 Determination of ATRA-PBAE prodrug nanoparticle drug loading

The drug loading of ATRA in the polymer was determined by nuclear magnetic peak area method. Three ATRA-PBAE prodrug copolymers of different grafting ratios 1:0.5, 1:1, 1:2 were prepared as described in example 1, and the grafting ratio of ATRA was calculated by nuclear magnetic resonance peak area integration. for-CH in ATRA structure in ATRA-PBAE nuclear magnetic diagram ₃ -C-CH ₃ -and-N-CH in PBAE structures ₂ The area of the peak was integrated and the ATRA Grafting ratio (Grafting rate) was calculated according to the following formula.

2.3.2 particle size distribution

The particle size and particle size distribution of the ATRA-PBAE prodrug nanoparticles at 3 grafting rates were determined using a malvern particle size analyzer. A small amount of nanoparticle solution is sucked and added dropwise into a cuvette, a small amount of deionized water is added to dilute the sample until the height of the sample pool is 1cm, the balance is 120s, the measurement is performed in parallel for 3 times, and the average particle size and the corresponding dispersion coefficient of the nanoparticles are recorded.

2.3.3 morphological observations

The invention adopts a transmission electron microscope (Transmission Electron Microscope, TEM) to observe the morphology of the prodrug nanoparticles.

2.34 In vitro release investigation of ATRA-PBAE prodrug nanoparticles

To investigate the pH sensitive release behavior of ATRA-PBAE prodrug nanoparticles, the ATRA-PBAE prodrug nanoparticle in vitro release experiments were performed in two different pH environments. According to literature, the release medium was selected to be 3% ethanol in PBS. 5mL of the prodrug nanoparticle solution was measured and placed in a dialysis bag with a molecular weight cutoff of 2700. The dialysis bag was immersed in 100mL of PBS with pH 5.8 or 7.4 in 3% ethanol. The mixture was vibrated horizontally at 100rpm for 120 hours at 37.+ -. 0.5 ℃ in a water bath shaker. At certain time intervals, 1mL of release medium is sucked up, a corresponding volume of fresh release medium is replenished, the concentration of the drug in the release medium is measured according to chromatographic conditions, the cumulative release amount and the release percentage are calculated according to the following formula, and a release curve is drawn.

In the above formula: e (E) _r Cumulative amount of ATRA released,%;

V _e replacement volume of PBS, mL;

C _i -the concentration of drug released upon the ith sampling, μg/mL;

V ₀ -release medium volume, mL;

C _n drug release on nth samplingConcentration of the substance, μg/mL;

m _drug -ATRA content in the drug-loaded nanoparticle, mg.

3. Results and discussion

3.1 Evaluation of ATRA-PBAE prodrug nanoparticle formulations

3.1.1 ATRA-PBAE prodrug nanoparticle drug-loading rate determination

ATRA and PBAE are synthesized into 3 ATRA-PBAE with different grafting rates according to different mole ratios, and the ATRA-PBAE is prepared from ¹ Peak area of-CH-of ATRA and-N-CH in PBAE structure in H-NMR spectrum ₂ The ratio of the peak areas allows the calculation of the ATRA grafting. From the results, the grafting ratio gradually increased as the ATRA ratio increased. When the molar ratio is 1:0.5, the ATRA has relatively small quantity of-COOH on the structure, can react with excessive-OH, and has low grafting rate due to the existence of residual PBAE in the system; when the ratio is 1:1, the quantity of-COOH participating in the reaction is increased to continuously react with the quantity of-OH on the PBAE, so that the grafting rate is increased; when the ratio reaches 1:2, the grafting ratio is at a maximum. The grafting ratio of the ATRA-PBAE prodrug copolymer is shown in Table 1.

TABLE 1 grafting ratio of ATRA-PBAE prodrug copolymer

3.1.2 particle size distribution

Considering the particle size distribution of the prodrug nanoparticles under 3 different ratios, when the molar ratio is 1:0.5, the particle size of the ATRA prodrug nanoparticles is the largest, probably because the ATRA content capable of reacting with PBAE in the system is less, the formed chemical structure is loose, and the particle size of the nanoparticles is larger; the lower particle size at a 1:1 ratio may be due to the greater compaction and stability of the ATRA-PBAE chemical structure formed after reaction with PBAE as the ATRA content increases in the overall system; at a ratio of 1:2, the particle size of the entire nanosystem is larger than that at 1:1, probably as the ATRA content in the system solution increases, the amount of reaction with PBAE also increases, and the ATRA-PBAE chemical structure also becomes larger. Since the particle size of the coated SchB is also increased in the subsequent experiments, the particle size of the ATRA-PBAE nanoparticle is controlled to be about 100nm in order to control the particle size of the coated SchB. The results of the combination of grafting and particle size distribution are shown in tables 1 and 2, with a molar ratio of 1:1 being chosen as the optimum ratio of PBAE to ATRA.

TABLE 2 particle size distribution of ATRA-PBAE nanoparticles

3.1.2 morphological observations

As can be seen from fig. 2-1 (a), the ATRA-PBAE prodrug nanoparticle solution appears blue opalescent, translucent in appearance. The prodrug nanoparticles have a particle size of about 100nm as measured by the previous particle size distribution, which is observed herein using a transmission electron microscope for better visualization of the prodrug nanoparticles. As shown in fig. 2-1 (b), the prodrug nanoparticles were uniformly spherical and had a size of about 100 nm.

3.2.3 ATRA-PBAE prodrug nanoparticle in vitro release experiment

The in vitro release behavior of ATRA prodrug nanoparticles under different pH conditions was examined by dialysis. The results are shown in fig. 2-2, where the free drug ATRA is released completely within 12h, while the prodrug nanoparticles significantly prolonged the release rate of ATRA. At pH5.5, the release rate of ATRA was significantly faster than at pH 7.4. The release of ATRA reached 60% at pH5.5 within 72 hours, indicating a faster release at weak ATRA acid. Whereas drug release at ph7.4 was less than 40%, indicating that the ATRA prodrug nanoparticles were relatively intact under physiological conditions. The release profile shows that the release behavior of ATRA-PBAE prodrug nanoparticles is pH sensitive, and at lower pH values, the ester linkage between the drug and the polymer may be readily cleaved by hydrolysis in the lysosome acidic environment, releasing free ATRA from NPs. The pH sensitive system can release the combined medicine at the tumor part, and the overall treatment effect is improved.

Example 3

This example is a study of the effect of ATRA-PBAE prodrug nanoparticles on tumor-heterogeneous cells

CSCs are a heterogeneous cell of tumor, have high self-renewal capacity and differentiation potential, closely match with tumorigenesis, recurrent metastasis and drug resistance, and are the main cause of breast tumor recurrence. Pin1 is involved in a plurality of mechanism pathways of breast cancer tumors, and Pin1 is not only overexpressed in breast cancer cells, but also a key regulatory factor of BCSCs. Therefore, the activity of Pin1 is regulated, and the growth or inhibition of breast tumor cells can be effectively controlled.

The present example further investigated the proliferation inhibition effect and cell uptake of ATRA prodrug nanoparticles on MCF-7 cells and MS cells, and examined the two cell uptake mechanisms and drug delivery in cells, based on successful preparation of ATRA-PBAE nanoparticles.

1. Instrument and materials

2. Experimental method

2.1MCF-7 culture

MCF-7 cell culture: and taking out the MCF-7 cells which are frozen in advance, carrying out water bath at 37 ℃ and rapidly dissolving within 1min, so as to avoid damage to the cells caused by slow melting of extracellular ice crystals. The frozen stock solution was added to a flask in 10 volumes of 10% fbs-containing medium, after which the thawed cells were added, and the cells were gently blown into single cell suspension using a pipette, and the flask was placed in a 37 ℃ incubator. Cell status was observed and culture medium was changed every other day.

2.2 Culture of MS cells

Preparing a serum-free culture solution, precisely weighing 200mg of bovine serum albumin, dissolving in 50mL of DMEM-F12 culture solution, adding 0.1mL of EGF solution (10 mug/mL), 0.1mL of bFGF solution (10 mug/mL), 0.5mL of double antibody and 0.5mL of insulin, and filtering with a 0.22 mu m filter membrane to obtain the serum-free culture solution.

MS cell culture: MCF-7 cells in logarithmic growth phase are taken, washed twice by PBS, digested with 2mL pancreatin, added with complete culture solution after the cells are rounded, gently blown for several times, and centrifuged at 1500rpm for 6min. The supernatant was discarded, 5mL of serum-free medium was added, and after gently pipetting several times, the cells were still treated under the same centrifugation conditions. Finally, the step of obtaining the product,the supernatant was discarded and the cells were resuspended in culture flasks with 4mL of serum-free medium. At 37 ℃,5% CO ₂ Culturing under the condition, observing the balling condition under a microscope, and carrying out passage every 5 days.

2.3 ATRA-PBAE prodrug nanoparticle antitumor activity

To investigate the antitumor activity of the prodrug ATRA-PBAE copolymer, we selected MCF-7 cells and MS cells in the log phase and used the MTT method to determine the cytotoxicity of the prodrug ATRA-PBAE copolymer. Cells in logarithmic growth phase are digested with pancreatin to form single cell suspension, and the single cell suspension is respectively treated with 7×10 ³ The individual/wells were seeded in 96-well plates. After overnight incubation, the culture broth was discarded and the concentration of free ATRA solution (0.1, 025, 0.5, 1, 2.5, 5, 10, 20, 40, 80 and 100 μg/mL), the concentration of prodrug polymer solution (0.1, 025, 0.5, 1, 2.5, 5, 10, 20, 40, 80 and 100 μg/mL), the concentration of PBAE polymer solution (0.1, 1, 5, 25, 50, 100, 200, 400 and 800 μg/mL) were added, respectively, with 3 multiplex wells being provided for each concentration, with blank wells being provided. After 48h incubation in an incubator, 20. Mu.l MTT (5 mg/mL) solution was added, after 4h incubation 150. Mu.l DMSO solvent was added, and shaking was performed for 10min to determine the OD at 490 nm. The cell viability was used to infer the magnitude of drug toxicity, which was calculated as follows:

wherein OD _test Absorbance values for the experimental group; OD (optical density) _blank Absorbance values for the blank group; OD (optical density) _control Absorbance values for the control group.

Data processing was performed using GraphPad prism.8.0.2 software and half maximal inhibitory concentration was determined (Half inhibition concentration, IC ₅₀ ). And the drug resistance index was calculated according to the following formula

2.4 qualitative and quantitative assessment of cellular uptake

This example uses a laser confocal microscope to observe endocytosis of the prodrug copolymer in the cell. Since ATRA does not have fluorescent properties, cy5.5 dye with carboxyl structure is selected to replace ATRA, and cy5.5-PBAE carrier material is synthesized according to the method and steps for synthesizing prodrug. Taking MCF-7/MS cells in logarithmic growth phase, and digesting with pancreatin to obtain single cell suspension at a ratio of 1×10 ⁵ The medium was discarded after incubation overnight at 37℃in 6-well plates containing coverslips at density. Then, 2mL of Cy5.5-PBAE solution diluted with serum-free culture solution was added to each well, and incubated for 0.5h, 2h and 4h, respectively, after the incubation was completed, cell uptake was stopped with cold PBS, and the cells were fixed with paraformaldehyde by washing 3 times with PBS, and the coverslips were taken out and photographed under CLSM to observe the cell interior. And the cells were resuspended in 1mL cold PBS and the intracellular fluorescence intensity was measured using a flow cytometer.

2.5 investigation of the cellular uptake pathways

MCF/MS cells in logarithmic growth phase are taken, digested by pancreatin and prepared into single cell suspension by using complete culture medium, and inoculated into 12-well plates. After 24h incubation in incubator, old broth was discarded, washed twice with PBS, and 500. Mu.l of serum-free DMEM high-sugar broth was added to each well and incubated for 15min. After that, 500 μl of uptake inhibitor was added, respectively: sucrose (154 mg/mL), nystatin (10. Mu.g/mL), colchicine (10. Mu.g/mL), 3 duplicate wells were placed for each inhibitor well. After 1h incubation, C6 nanoparticle-loaded solution was added to each well, after 2h incubation, the culture was discarded, PBS was washed and digested with pancreatin, the cells were collected, and the fluorescence intensity of the cells under different inhibitors was measured using a flow cytometer.

2.6 lysosomal escape

MCF-7 cells and MS cells at 10, respectively ⁵ cells/well were seeded in 6-well plates containing coverslips and after overnight incubation, cells were washed 3 times with PBS. Then, 2mLATRA-PBAE NPs solution was added, incubated for 0.5h, 1h, 2h, old medium was discarded, and washed 3 times with cold PBS. Adding 200 mu LLyso-tracker Green lysosome fluorescent probe to mark intracellular lysosomes, incubating for 30min, washing cells with PBS, andcells were fixed with 4% paraformaldehyde and placed under CLSM to observe drug transport in MCF-7 cells and MS cells.

1. Experimental results and discussion

3.1 MCF-7 cell and breast cancer stem cell culture

Microscopic pictures of MCF-7 cells and MS cells are shown in fig. 3-1. MCF-7 cells are cells which grow on the wall, are polygonal, grow rapidly and can be passaged about every 2-3 days. Serum-free and serum-free culture is a common method for enriching tumor stem cells. MCF-7 cells are digested into single cell suspension by pancreatin, and are continuously cultured in a culture solution containing EGF solution, bFGF solution, double antibody and insulin. On the next day, most cells grew in suspension, and some cell spheres were observed at days 5-7 as the incubation time increased. And subject groups in early experimental studies, antigen of MS cells has been identified, and the results indicate that MCF-7 stem cells exhibit a cd4+/CD 24-phenotype, which is consistent with the MS cell phenotype reported in the literature.

3.2 ATRA-PBAE prodrug nanoparticle antitumor activity

3.2.1 Inhibition of MCF-7 cells by ATRA-PBAE prodrug nanoparticles

The free ATRA is selected as a positive control, the MTT method is adopted to examine the inhibition capability of ATRA-PBAE prodrugs on MCF-7 cells, and the cell survival rate of ATRA-PBAE nanoparticle groups containing different ATRA concentrations under different pH conditions after being incubated with MCF-7 cells for 48 hours is examined. It can be seen from FIGS. 3-2 (a) that ATRA-PBAE nanoparticles did not exhibit toxic effects at pH 7.4, probably because ATRA-PBAE did not break, and ATRA did not exert cytotoxic effects. Whereas at pH 5.5, the ATRA-PBAE prodrug nanoparticle group had significant cell inhibition (p<0.05 And its IC) ₅₀ (11.43. Mu.g/mL) was slightly higher than the free ATRA group (IC ₅₀ ：8.302μg/mL，p<0.05 The above results not only demonstrate that the prodrug nanoparticles have an inhibitory effect on MCF-7 cell proliferation, but also that the prodrug nanoparticles have pH sensitivity. IC of prodrug nanoparticles ₅₀ Slightly higher than the free group because ATRA takes a period of time to release from the prodrug nanoparticle and becauseThe pH sensitivity of the nanoparticle is lower than that of the nanoparticle under the conditions of pH 7.4 and pH 5.5.

In addition, it was verified that the ATRA-PBAE prodrug copolymer was because ATRA only exerted tumor cell inhibiting properties, not because of the carrier polymer. Therefore, this example also examined the inhibition of MCF-7 cells by PBAE vector material. As shown in FIG. 3-2 (b), MCF-7 cells still had higher survival rates at concentrations of up to 800. Mu.g/mL of PBAE copolymer, indicating that PBAE had better biocompatibility and inhibited cell mass. As can be seen from comparison of FIGS. 3-2 (a) (b), at pH 5.5, MCF-7 cells cultured with ATRA-PBAE prodrug polymer had a viability of 54.41% at ATRA concentration of 10.00. Mu.g/mL, corresponding to a prodrug polymer concentration of about 100. Mu.g/mL, whereas PBAE treated cells had a viability of over 85%, indicating that the proliferation inhibition of MCF-7 cells by ATRA-PBAE prodrug was due primarily to ATRA toxicity in the prodrug, but not polymer toxicity. From the above results, it is demonstrated that ATRA-containing prodrug nanoparticles have a strong anticancer effect, and that ATRA prodrugs have a relatively lower toxicity than free ATRA, because ATRA-PBAE prodrug nanoparticles require a certain time to release after entering tumor cells by endocytosis.

3.2.2 Inhibition of MS cells by ATRA-PBAE prodrug nanoparticles

ATRA is a powerful CSCs differentiating agent, which has been widely studied in the treatment of CSCs. The invention examines the inhibition effect of ATRA-PBAE prodrug nanoparticles on MS cells, and the cell survival rate of free ATRA and ATRA-PBAE (pH 7.4 and pH 5.5) after 48h incubation with MS cells is shown in figures 3-3. From the results in fig. 3-3, it can be seen that the free ATRA and ATRA-PBAE nanoparticles have about inhibitory effect on MS cells, and that the inhibition capacity increases with increasing drug concentration, and is dose dependent. From fig. 3-3, it can be seen that the cell inhibition rate of the ATRA-PBAE prodrug nanoparticles is significantly reduced (p < 0.0.5) compared to the free ATRA group at ph 7.4; whereas at pH 5.5, the ATRA-PBAE group was not significantly different from the free ATRA group; the reason is probably that after the ATRA-PBAE prodrug nano-particles enter cells, the prodrug nano-particles are subjected to the influence of low pH environment in tumor cells, and depolymerization occurs under the prodrug nano-particles, so that the drug can be quickly released and acts, and further the ATRA-PBAE prodrug nano-particles are proved to have pH sensitivity.

Taken together, it is clear that both the free drug and the ATRA-PBAE have inhibitory effects on proliferation of both MCF-7 cells and MS cells. As can be seen from Table 3, MCF-7 cells and MS cells have an IC for free ATRA ₅₀ 8.302 + -0.44 μg/mL and 38.44 + -0.63 μg/mL, respectively, with a drug resistance index of 4.63; the MS cells were shown to be significantly resistant to free ATRA and ATRA-PBAE. The reason may be that P-gp in MS cells acts as a drug-efflux pump "resulting in lower intracellular drug concentrations and stronger drug resistance. The drug resistance index at pH 5.5 was significantly reduced compared to pH7.4, exhibiting comparable toxic effects to free ATRA. This may be because after ATRA-PBAE nanoparticles enter the cells, ATRA is released rapidly in lysosomal acidic environments, with a small fraction of the free ATRA entering the cytoplasm to play a role. However, most drugs still suffer from the effects of P-gp.

TABLE 3 IC of different ATRA preparations in MCF-7 cells and MS cells ₅₀ And RI

*p<0.05vs IC _50(MS)

3.3 cell uptake study

Observation of prodrug nanoparticle uptake by 3.3.1MCF-7 cells

The cellular uptake of prodrug nanoparticles by MCF-7 cells at different pH conditions was observed using CLSM (FIGS. 3-4). Although ATRA needs to be preserved in the dark, it does not have fluorescent properties in itself. Whereas cy5.5 belongs to the cyanine dye, which belongs to the liposoluble dye and has fluorescent properties. Therefore, this example selects Cy5.5 instead of ATRA to examine the uptake of prodrug nanoparticles in MCF-7 cells. The results are shown in figures 3-4, the fluorescence intensity is lower and the cell entry amount of the medicine is smaller at 0.5 h; at 2h, a slight increase in fluorescence intensity was observed in the cells, with some drug entering the nucleus; at 4h, the fluorescence intensity in the cells is obviously enhanced, and the amount of the drug entering the cells is obviously increased, which proves that the intake of the drug by the MCF-7 cells is obviously increased along with the extension of the incubation time, and the time-dependent behavior is realized. At the same time, the intracellular fluorescence intensity is significantly higher at pH 5.5 compared to pH 7.4, indicating that the nanoparticle is able to deliver the drug into the cytoplasm due to the change in pH environment. The analysis of the results is combined, and the pH sensitivity of the nanoparticle is proved to influence the MCF-7 cells to the drug intake.

The fluorescence intensity of MCF-7 cells on Cy5.5-PBAE nanoparticles uptake under different pH conditions was measured using a flow cytometer and is shown in FIGS. 3-5. Under the condition of pH 7.4, fluorescence uptake intensities of the Cy5.5-PBAE nanoparticles at 0.5h, 2h and 4h are respectively as follows: 66.53 + -1.83, 292.67 + -1.73, 400.33 + -2.08; and under the condition of pH 5.5, fluorescence uptake intensities of the Cy5.5-PBAE nanoparticles at 0.5h, 2h and 4h are respectively as follows: 289.33 + -2.52, 367.33 + -2.08, 466+ -2.66, which is significantly higher than the fluorescence intensity at pH 7.4 (p < 0.05). This result is consistent with the qualitative experimental results.

3.3.2 Uptake of ATRA-PBAE prodrug nanoparticle by MS cells

As shown in fig. 3-6, in MS cells, the intracellular fluorescence intensity increases with time; the fluorescence of the Cy5.5-PBAE group at pH 5.5 was significantly weaker than that at pH 7.4.

The uptake of the nanofabricated preparation by MS cells under different pH conditions is shown in FIGS. 3-7. In MS cells, the fluorescence intensity within the cells increases significantly with prolonged incubation time. At pH 7.4, at 0.5h, 2h and 4h, MS intracellular fluorescence intensities were 26.10 + -2.85, 46.38 + -2.26 and 116.85 + -1.61, respectively; and the intracellular fluorescence intensities at pH 5.5 are respectively: 46.92.+ -. 2.01, 75.77.+ -. 1.18 and 173.50.+ -. 3.65. In summary, the fluorescence intensity at pH 5.5 is significantly higher than pH 7.4, indicating that nanoparticle uptake is affected by microenvironment pH. The above results are consistent with those observed by a laser microscope. In addition, the fluorescence intensity of the MS cell preparation group is significantly lower compared with that of MCF-7 cells, indicating that the content of the intracellular drug is relatively small, which is probably related to the excretion of the P-gp on the tumor cell membrane, and is consistent with the cytotoxicity result.

3.4 cell uptake mechanism experiment

Currently, nanoparticlesThe cells are entered mainly by endocytosis. Endocytosis can be classified into clathrin-mediated endocytosis, caveolin-mediated endocytosis and megacytosis-mediated endocytosis according to the proteins involved. Clathrin-mediated endocytosis is a common mode of macromolecular transmembrane transport and can be carried out on adapter proteins, dynein and Ca ²⁺ The transport vesicles and vesicles formed under the action are subjected to intracellular digestion. The process of endocytosis mediated by the cellular proteins is in a cholesterol-sensitive pathway, and endocytosis can form membrane pocket vesicles, multiple vesicles and other forms into the golgi apparatus or endoplasmic reticulum. Macropolytics mediated endocytosis is a non-selective way to endocytose foreign substances and is a process by which irregular endocytosis vesicles are formed and taken up by nanoparticles and cell membranes independent of receptor binding to ligands.

In this example, sucrose, colchicine, and nystatin were mainly selected as endocytic inhibitors. Sucrose is a clathrin inhibitor. Colchicine is a giant pinocytosis pathway selection inhibitor that can bind to tubulin to depolymerize microtubules, mainly by inhibiting microtubule formation. Thereby inhibiting the transport of endocytic vesicles to the endosome. Nystatin is a cryptan-dependent endocytosis inhibitor that mainly acts to bind cholesterol. The effect of different endocytic inhibitors on cellular uptake was examined using a flow cytometer. As a result, as shown in FIGS. 3-8 (a), after 2 hours of incubation, the uptake of the cells of the nystatin-treated MCF-7 cells was reduced to 88.5% (p > 0.05) of the control group, and the uptake of Cy5.5-PBAE NPs was not via the cellular proteins. Indicating that when MCF-7 cells were treated with sucrose, colchicine and 88.55% (p < 0.05), the uptake decreased to 52.5%, 66.5% and 88.55%, respectively, indicating that clathrin and macropolytics mediated endocytosis was involved in uptake of drug-loaded nanoparticles by MCF-7 cells, wherein clathrin plays a major role.

Inhibitors the pathway of uptake of prodrug nanoparticles by MS cells was investigated. As shown in fig. 3-8 (b), the uptake of the 3 inhibitors after treatment of MS cells was reduced to 46.9%, 88.62% and 95.69% of the control group, respectively; the sucrose inhibitor group had a significant inhibitory effect on cellular uptake compared to the other inhibitor groups. The results indicate that the prodrug nanoparticles enter the cells mainly through clathrin-mediated endocytosis pathways.

3.5 lysosomal escape

The endocytosis of the nanoparticle into the cell is followed by reaching the lysosome, and the escape from the lysosome to reach the cytoplasm is a key problem. This example uses CLSM to observe the intracellular transport of prodrug nanoparticles in MCF-7 and MS. Since Cy5.5 is a red fluorescent dye, lyso-Tracker Green was used as a lysosome fluorescent probe. As shown in FIGS. 3-9, after incubation for 0.5h, weak yellow fluorescence (fluorescence color after the overlap of Lyso-Tracker Green and Cy5.5) was observed in both MCF-7 and MS cells, and Green fluorescence was unchanged, red fluorescence was gradually increased and yellow fluorescence after overlap was also increased with the lapse of time. However, MCF-7 and MS cells were found to have significantly weaker yellow fluorescence in the cells after incubation with chloroquine.

In summary, the nanoparticle enters the cell to transport the drug to the cytoplasm mainly through the lysosome, and the PBAE in the prodrug nanoparticle structure can be protonated in the acidic environment of the lysosome, so that the nanoparticle is depolymerized, and the lysosome is ruptured to release the drug. In addition, chloroquine treated MCF-7 and MS cells found that chloroquine primarily inhibited the acidic environment of lysosomes, rendering drug unreleasable, further indicating that nanoparticles reached the cell interior and were delivering drug to the cytoplasm via lysosomes.

(1) In the embodiment, MCF-7 cells and MS cells are used as models, and MTT method is used for examining the cell inhibition effect of ATRA-PBAE prodrug nanoparticles under different pH conditions. The results show that the ATRA-PBAE prodrug nanoparticles have inhibition effects on MCF-7 and MS cells, and are dose-dependent. In addition, MS cells have significant resistance to prodrug nanoparticles through evaluation of the drug resistance index.

(2) The fluorescence intensity of ATRA-PBAE prodrug nanoparticles in MCF-7 cells and MS cells is qualitatively and quantitatively analyzed by using CLSM and a flow cytometer. The results indicate that MCF-7 and MS cells are time dependent on prodrug nanoparticle uptake and that pH sensitivity of the prodrug nanoparticles affects cellular uptake.

(3) The experiment of the uptake mechanism shows that the uptake process of the prodrug nanoparticle by the MCF-7 cell is clathrin and megaloblastic-mediated endocytosis; prodrug nanoparticles enter MS cells primarily through clathrin-mediated endocytosis. After the medicine reaches the cell, the medicine mainly enters the lysosome, and the prodrug nano-particles can escape from the lysosome due to the unique pH of the prodrug nano-particles, and further play a role. Due to the timing and experimental scheduling, the mechanism of inhibiting Pin1 molecules with respect to ATRA-PBAE prodrug nanoparticles will be examined in subsequent studies.

Example 4

Schisandrin B (Schisandrin B, schB) mainly comes from one active substance in Chinese medicinal fructus Schisandrae, and has antiulcer, antagonistic, antioxidant, liver protecting and MDR reversing effects.

This example uses the ATRA-PBAE prodrug copolymer with pH sensitivity synthesized in the above example as a carrier, designs the prodrug nanoparticles loaded with SchB, and characterizes the preparation process and the pharmaceutics.

1. The preparation method comprises the following steps: 20mg of ATRA-PBAE prodrug polymer and 20% of SchB by weight of the carrier were precisely weighed, and 1mL of acetone was used to dissolve the polymer and the drug to prepare an organic phase. Slowly dripping the organic solution into 15mL of 0.5% poloxamer water solution at 1000rpm, continuously stirring at room temperature until acetone is completely volatilized, and filtering with a 0.22 μm filter membrane to obtain nanoparticle colloid solution with blue opalescence.

Evaluation of SchB/ATRA-PBAE NPs pharmaceutics

2.1 morphological observations

Appearance morphology observation is carried out on the SchB/ATRA-PBAE NPs prepared by the precipitation method. In order to obtain a clearer nanoparticle form, a Transmission Electron Microscope (TEM) is adopted in the embodiment to obtain a nanoparticle form, and the specific operation method is as follows: a small amount of the prepared nanoparticle solution is sucked and dripped on a copper mesh, redundant liquid is sucked by filter paper after 3min, and 200 mu L of 2% phosphotungstic acid is added for dyeing. After drying at room temperature, the nanoparticle morphology was observed using a transmission electron microscope and photographed.

2.2 particle size distribution

The particle size and particle size distribution of SchB/ATRA-PBAE NPs were determined using a Markov particle size analyzer. A small amount of nanoparticle solution is sucked and added dropwise into a cuvette, a small amount of deionized water is added to dilute the sample until the height of the sample pool is 1cm, the balance is 120s, the measurement is performed in parallel for 3 times, and the average particle size and the corresponding dispersion coefficient of the nanoparticles are recorded.

2.3 determination of in vitro Release behavior

SchB/ATRA-PBAE NPs prepared using the optimal recipe were measured precisely and placed in dialysis bags of molecular weight 3500 and then transferred to PBS buffer containing 50mL of 30% methanol at different pH values. The samples were taken at regular time intervals with shaking in a water bath at 37℃while replenishing the same volume of fresh release medium. The sample taken out was passed through a 0.45 μm filter and the drug concentration in the sample was measured by sampling under the following chromatographic conditions. And the cumulative release amount and the release percentage were calculated according to the following formulas, and a cumulative release curve was drawn.

In the above formula: e (E) _r Cumulative amount of SchB released,%;

V _e replacement volume of PBS, mL;

C _i -the concentration of drug released upon the ith sampling, μg/mL;

V ₀ -release medium volume, mL;

C _n -the concentration of drug released upon nth sampling, μg/mL;

m _drug SchB content in the drug-loaded nanoparticle, mg.

Chromatographic conditions: chromatographic column: diamond C18 column (250X 4.6mm,5 μm); mobile phase: methanol: water=85:15 (v/v); column temperature: 25 ℃; flow rate: 0.8mL/min; detection wavelength: 220nm; sample injection amount: 10 mu L.

Evaluation of SchB/ATRA-PBAE NPs pharmaceutics

3.1 morphological observations

As shown in FIG. 4-1, the SchB/ATRA-PBAE NPs exhibited a semitransparent blue opalescent solution in appearance, which was more clearly observed in comparison to water (FIG. 4-1 (a)), while the overall solution clarity was reduced in comparison to the blank nanoparticles, possibly associated with the addition of the drug. The graph is a transmission electron microscope graph of SchB/ATRA-PBAE NPs, and the graph shows that the nano particles are in a sphere-like shape, are uniformly distributed, and have the size of about 135 nm.

3.2 Particle size distribution of SchB/ATRA-PBAE NPs

Particle size results for blank prodrug nanoparticles and SchB/ATRA-PBAE NPs are shown in FIGS. 4-2 and 4-3. The particle diameter of the blank nanoparticle is 102.33+/-1.72 nm, and the PDI is 0.283+/-0.025; the particle size of SchB/ATRA-PBAE NPs is 134.97 + -0.25 nm and PDI is 0.151+ -0.026. The particle size of the nano particles is increased due to the loading of the medicine, and the result is consistent with the micro morphology result of the nano particles.

3.3 in vitro Release assay

The release profile of SchB/ATRA-PBAE NPs in PBS buffer solutions of different pH (pH 7.4, pH 5.5) is shown in FIGS. 4-4. From the results, the free SchB solution was released completely from the dialysis bag into the medium solution at 12h, indicating that the dialysis bag did not limit the release of SchB. Compared with the prior art, the release speed of the drug-loaded nanoparticle is relatively slow, and the drug release time is obviously prolonged. The release behavior of the antitumor drug delivery system in different pH environments is very important, the pH value in normal blood and tissues is 7.35-7.45, the pH value in tumor tissues is 6.5-7.0, and the internal environment of a lysosome is about 5.5. Therefore, in this example, 7.4, 6.8, and 5.5 are selected as the pH environment of the drug delivery system. From the release profile, the release rate sequence of SchB is: pH 5.5>pH 6.8>pH 7.4. At pH5.5, schB is released significantly faster than the other two pH conditions. Within 48h, at pH5.5, release of SchB was achieved; at pH 6.8, release of SchB is achievable; at pH7.4, schB release was achieved. This is probably due to the protonation of the PBAE in the ATRA-PBAE prodrug carrier at low pH conditions, causing the nanocore to break and the drug to be released rapidly from the core. Whereas at ph7.4 SchB release rate was lower, which demonstrates that the nanoparticle maintained a relatively intact nanoparticle structure under normal physiological conditions. In addition, at pH 6.8, schB was not completely released, indicating that the nanoparticle structure in the tumor environment was not destroyed, ensuring entry of SchB into the cytoplasm.

Example 5

This example is a reverse MDR study of the effect of tumor stem cells on SchB/ATRA-PBAE nanoparticles prepared in example 4.

1. Instrument and materials

2. Experimental method

2.1 Culture of MS cells

Taking MCF-7 cells in logarithmic growth phase, digesting the MCF-7 cells into single cell suspension by 2ml of pancreatin, adding 3ml of culture solution containing 10% to stop digestion, lightly blowing for a plurality of times, and centrifuging at 1500rpm/min for 6min; removing supernatant, adding 3mL of serum-free culture solution, blowing off the precipitated cells, centrifuging at 1500rpm/min for 6min, removing culture solution, adding appropriate amount of serum-free DMEM-F12, culturing the resuspended cells, and standing at 37deg.C and 5% CO ₂ The culture was continued in the incubator of (2), and the condition of balling was observed under a microscope and passaged every 5 days.

2.2 investigation of MS toxicity by SchB/ATRA-PBAE NPs

The principle of the cytotoxicity experiment is examined by adopting a thiazole blue (MTT) colorimetric experiment method: MTT was reduced to blue-violet, water-insoluble Formazan (Formazan) by succinic acid dehydrogenase in live cell mitochondria, the blue-violet crystals were dissolved in DMSO, and the absorbance (OD) was measured at 490nm using a multifunctional microplate reader. Since the dead cells do not contain succinate dehydrogenase, the addition of MTT did not react. The formazan production is proportional to the number of cells over a range of cell numbers. The number of living cells was determined based on the OD value, and the cell viability was used to infer the magnitude of drug toxicity.

MCF-7/MS cells were seeded at a density of 7000/well in 96 wells at 5% CO ₂ Incubated at 37℃for 24h. The plates were removed, the broth was discarded, and the prepared SchB solution, ATRA+SchB mixed solution, and SchB/ATRA-PBAE NPs solution (pH 7.4 and 5.5) were added to 96 wells (whereinThe ATRA concentration corresponds to approximately 1, 5, 10, 20, 30 μg/mL; schB concentrations were approximately 2.5, 12.5, 25, 50, 75 μg/mL), 5 total concentrations per group, 3 duplicate wells per concentration were set, with blank wells set. The cell plates were then placed at 37℃in 5% CO ₂ Culturing under the condition. After 48h, 20. Mu.L of 5mg/mL MTT solution was added to each well, incubation was continued for 4h, the culture was terminated again, the medium was carefully aspirated off from the well, 150. Mu.L of DMSO solvent was added, and shaking at low speed for 10min was performed to allow the blue-violet crystals to dissolve well. And finally, measuring an absorbance value (A) at 490nm by using a multifunctional enzyme-labeled instrument, and calculating the survival rate of the cells according to a formula. Data processing was performed using GraphPad prism.8.0.2 software and half maximal inhibitory concentration (IC ₅₀ )。

And the inversion multiple is calculated according to the following formula

2.5 SchB/ATRA-PBAENPs cell uptake assay

Preparation of C6/ATRA-PBAE-carried nanoparticles: because SchB has no fluorescence characteristic, and is unfavorable for observing intracellular uptake, the embodiment selects a poorly water-soluble fluorescent probe Coumarin-6 (Coumarin-6, C-6) as a model drug. 5mg of C6 powder is precisely weighed out in a dark place, added with acetone to dissolve the C6 powder, and the mixture is fixed to a 10mL volumetric flask to prepare 0.5mg/mL of C6 stock solution. Then, according to the preparation method described in example 4, C6-loaded nanoparticles are prepared, and the C6-loaded nanoparticles are filtered through a 0.22 mu m microporous filter membrane, so that free C6 which is not loaded is removed, and a C6/ATRA-PBAE nanoparticle solution is obtained for later use.

Qualitative test of cellular uptake: MS cells in logarithmic growth phase were resuspended in complete medium (10% FBS) after pancreatin digestion at a density of 2.5X10 ⁴ Density was seeded in 6-well plates. Incubate overnight in incubator, take out the plates the next day, discard the medium, wash 2 times with PBS. C6/ATRA-PBAE nanoparticle solutions diluted in serum-free medium (pH adjusted to 7.4 and 5.5, respectively) were added and incubated at 37℃for 0.5h, 2h, 4h. After the incubation, cold PBS was added to stop cell uptake, PBS was washed twice, and the cells were observed under a fluorescence microscope. Finally, measuring by using a flow cytometerCell fluorescence intensities at different pH at different times were determined.

2.6 SchB/ATRA-PBAENPs reverse tumor MDR study

2.6.1 Determination of P-gp expression

Collecting the cells in logarithmic phase, digesting with pancreatin to obtain single cell suspension, inoculating the cells into 12-well plate with cell density of 1×10 ⁶ cells/wells. After 24h incubation in incubator, 2ml PBS was washed twice, followed by ATRA+SchB, ATRA-PBAENPs, schB/ATRA-PBAENPs, respectively. After incubation for 12h with the blank culture solution as control, cells were collected by centrifugation, washed twice with ice PBS and fixed with 4% paraformaldehyde for 30min. And then, continuously centrifuging the cells, washing the cells for 2 times by using PBS, respectively adding the rabbit anti-human P-gp antibody into each group, incubating the cells for 30 minutes at 4 ℃ in a dark place, centrifuging to remove unbound antibodies, adding FITC-labeled goat anti-rabbit IgG secondary antibodies diluted to a certain concentration, uniformly mixing, incubating the cells at 4 ℃ in a dark place for 30 minutes, centrifuging to remove the supernatant, washing the cells twice by using PBS, re-suspending the cells by using a little PBS, and detecting the expression of P-gp by using a flow cytometer.

2.6.2 intracellular protein content determination

And (3) selecting a BCA protein concentration determination kit to determine the protein concentration of MS cells. First, 25mg/mL of the protein standard solution was prepared as well, and diluted with a diluent to a concentration of 5mg/mL. Next, 5mg/mL of the protein standard solution was diluted to 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0 with the diluent. 200. Mu.l BCA working solution was added to each well and incubated at 37℃for 30min. And measuring the absorbance of the standard solution concentration at 562nm by using an enzyme-labeled instrument, and drawing a standard curve.

Collecting logarithmic growth MS cells, and digesting into single cell suspension by pancreatin at 5×10 per well ⁵ The cells were seeded at a density in 12-well plates, incubated for 24h, and washed 2 times with physiological saline (to prevent contamination of the samples with phosphorus). ATRA+SchB, ATRA-PBAE NPs, schB/ATRA-PBAE NPs were then added separately, with blank medium as control. After incubation in an incubator for 2h, cells were collected by pancreatin digestion and MS cells were resuspended in 0.3mL of physiological saline. Cells were disrupted by a cell disrupter. The MS cell suspension was diluted 3-fold with physiological saline and assayed. Calculation of samples using protein standard curvesProtein concentration of the product.

2.6.3 Effect of SchB/ATRA-PBAE NPs on intracellular ATPase (ATPase) Activity of MS

Selecting ultra-trace Na ⁺ -K ⁺ 、Ca ²⁺ -Mg ²⁺ The total ATPase assay kit measures the effect of SchB/ATRA-PBAE NPs on ATPase (ATPase) activity in MS. Firstly, preparing the required solution according to the specification, dividing each well of the cell suspension in-2.6.2' into a control group and Na ⁺ -K ⁺ -ATPase、Ca ²⁺ -Mg ²⁺ ATPase, group T-ATPase, and enzymatic reaction assays are performed according to the specification procedures. Taking the supernatant after the reaction to perform a phosphorus determination reaction, and measuring an absorbance value (A) at 636nm under a multifunctional enzyme-labeled instrument. Total ATPase (T-ATPase) activity (U/mgprot) = (assay tube OD value-control OD value)/(standard OD value-blank OD value) ×6×7.8/protein concentration of sample to be tested (mgprot/mL) according to the following formula.

3. Experimental results

3.1 examination of MS toxicity by SchB/ATRA-PBAE NPs

MTT method was used to examine the inhibition of MS cell proliferation by SchB+ATRA group and SchB/ATRA-PBAE NPs under different pH conditions. As can be seen from the results of FIG. 5-1, the free drug and the formulation group at different pH conditions showed an increase in MS cytotoxicity with increasing concentration of SchB, and thus a dose-dependent effect. At pH7.4, with free SchB group (IC ₅₀ : 7.31.+ -. 1.12. Mu.g/mL) compared to SchB-loaded nanoparticles (IC ₅₀ : 11.17+ -1.64 μg/mL) significantly decreased inhibitory effect (p)<0.05 Mainly because free drugs often enter the interior of cells in a diffuse manner and act rapidly; the SchB-loaded nano particles enter cells mainly through an endocytosis mode, and have slower acting speed than the free medicine, so that the nano particles have a certain slow release function, and have the potential of inhibiting tumor growth compared with the free medicine. At pH 5.5, the SchB nanoparticle-loaded cell inhibition was significantly enhanced compared to the nanoparticle at pH7.4 (p <0.05 Further, the nano-preparation can rapidly act in the acidic environment of tumor cells, and the nano-preparation has pH sensitivity.

In addition, since both ATRA and SchB in the nanoparticles exert antitumor activity, the MS cell inhibition effect of the drug-loaded nanoparticle group and the free atra+schb group was compared. From fig. 5-1, it can be seen that the cell inhibitory effect of the SchB-loaded nanoparticle group was significantly reduced (p < 0.05) at ph7.4 compared to the free atra+schb group under the same conditions, but the cell inhibitory effect of the SchB-loaded nanoparticle group was not as good as that of the free atra+schb group at ph 5.5.

The invention verifies the inhibition effect of ATRA-PBAE prodrug nano-particles on MS cells, but has larger drug resistance index, which indicates that ATRA has obvious tumor drug resistance. And the SchB is used as a P-gp inhibitor, and the effect of ATRA on inhibiting tumor cells is obviously improved after the SchB is combined with ATRA. From FIG. 5-1 and Table 4, it can be seen that MS cells have an IC for ATRA+SchB group (pH 7.4 and 5.5) ₅₀ Far lower than the ATRA group, the reversal fold was 3.44 (pH 7.4) and 3.77 (pH 5.5), demonstrating that the combination of SchB can overcome the effects of MDR, thereby increasing the antitumor activity of ATRA. In addition, schB/ATRA-PBAE NPs have strong MDR reversing effects on MS with reversing indexes of 2.77 and 3.68 respectively. The SchB-loaded nanoparticles reverse MDR more strongly at pH5.5 (p <0.05 Equivalent to the reversion effect of free drug group, this may be due to the rapid release of ATRA and SchB into the cytoplasm in lysosomal acidic environment (ph 5.5) after nanoparticle entry into the cell, on the one hand the massive presence of SchB reverses MDR effect to maximize ATRA effect, on the other hand it may be due to the fact that the nanoparticle can escape P-gp efflux through lysosomes and thus the nanoparticle has a stronger cytotoxic effect.

TABLE 4 IC of different ATRA preparations in MS cells ₅₀ RF (radio frequency)

*p<0.05vs Free ATRA；**p<0.05vs ATRA+SchB

3.2 Observation of uptake of C6/ATRA-PBAE NPs by MS cells

And observing the uptake condition of the C6/ATRA-PBAE nano particles by the MS cells under different pH conditions by adopting a fluorescence microscopy method. SchB has no fluorescence property, and can not observe the uptake condition of cells to medicines under a fluorescence microscope, C6 is an organic dye with extremely strong fluorescence property, and the lipid solubility of the organic dye is similar to that of SchB, so that the organic dye is used as a fluorescent marker instead of SchB in the embodiment. The results are shown in FIG. 5-2, and the fluorescence intensity of the MS intracellular drugs increases with the extension of the incubation time. After 2h incubation, MS cells are compared, and the fluorescence intensity of the carried C6 nanoparticle is stronger at the pH of 5.5 than at the pH of 7.4, which indicates that the MS cells are more sensitive to nanoparticle uptake at the pH of 5.5. The uptake of C6-loaded nanoparticles by MS cells was determined using a flow cytometer. As shown in fig. 5-3, the fluorescence intensities of the drug-loaded nanoparticles at ph7.4 were respectively: 25.97+ -5.59, 151.77 + -5.67, 255.21 + -7.85; the fluorescence intensity of the drug-loaded nanoparticles at pH5.5 is respectively: 97.97 + -4.32, 226.24 + -6.24, 349.57 + -8.27. At 0.5, 2, 4h, the average fluorescence intensity of C6/ATRA-PBAE NPs at pH5.5 was higher than that at pH7.4 (p < 0.05), which also further validated the verification fluorescence microscope results.

3.3 Effect of SchB/ATRA-PBAE-NPs on expression of drug resistant protein P-gp

The effect of SchB/ATRA-PBAE NPs on P-gp expression was examined using flow cytometry. As a result, as shown in FIGS. 5 to 4, after the MS cells were treated with ATRA-PBAE NPs, the expression level of P-gp was not significantly changed (P > 0.05) as compared with that of the blank group. Whereas the expression of P-gp was significantly reduced in MS cells after free ATRA+SchB treatment (P < 0.05). After MS cells are treated by SchB/ATRA-PBAE NPs group, compared with a blank group, the expression level of P-gp is remarkably reduced (P < 0.05); the expression level of P-gp after the free ATRA+SchB treatment is not quite different. The results show that: the reverse of multidrug resistance of MS cells by SchB/ATRA-PBAE NPs is possible by inhibiting P-gp expression levels.

3.4 intracellular protein content determination

Referring to FIGS. 5-5, the absorbance at 562nm is on the ordinate y with the standard protein concentration on the abscissa x, and the linear regression equation is: y=0.7297x+0.0917, r ² =0.9994, indicating that the protein is well-related in the concentration range of 0-1 mg/mL. The total protein concentration in MS cells was calculated to be 0.883mgprot/mL.

3.5 Effect of SchB/ATRA-PBAE NPs on intracellular ATPase (ATPase) Activity of MS

ATPases are mainly used for supplying energy for P-gp transport by hydrolyzing ATP, and the P-gp transport process requires the participation of ATPases. The drug can influence energy supply by inhibiting the activity of ATPase, thereby influencing the P-gp transport and achieving the effect of reversing MDR. Wherein Na is ⁺ -K ⁺ ATPase can promote ATP decomposition and maintain intracellular and extracellular Na by activating ATPase activity ⁺ 、K ⁺ Concentration equilibrium can form a resting potential. Galectin-3 can be prepared by regulating P-gp and inhibiting Na ⁺ -K ⁺ ATPase activity effects modulation of doxorubicin MDR effects in drug-resistant cells. Ca (Ca) ²⁺ -Mg ²⁺ ATPase drives intracellular and extracellular Ca by catalyzing ATP hydrolysis ²⁺ And Mg (magnesium) ²⁺ Transport, and maintain intracellular energy stability. When Na is ⁺ -K ⁺ ATPase and Ca ²⁺ -Mg ²⁺ When the activity of ATPase is reduced, na is discharged from the inner and outer rows of tumor cells ⁺ And Ca ²⁺ Reducing the abnormal transportation function of tumor cells, breaking the biological transportation balance, and interfering the ingestion of calcium ions by mitochondria, thereby affecting the abnormal energy metabolism in tumor cells.

This example essentially demonstrates SchB/ATRA-PBAENPs vs. Na ⁺ -K ⁺ -ATPase、Ca ²⁺ -Mg ²⁺ -influence of ATPase and total ATPase activity. As shown in Table 5, schB and SchB/ATRA-PBAE NPs were used to act on MS cells, respectively, and Na was used in the SchB group and SchB/ATRA-PBAE NPs group compared with the blank group ⁺ -K ⁺ -ATPase、Ca ²⁺ -Mg ²⁺ -significant decrease in viability of ATPase and total ATPase (p<0.05 Indicating that SchB/ATRA-PBAE NPs are probably produced by inhibition of Na ⁺ -K ⁺ -ATPase、Ca ²⁺ -Mg ²⁺ Viability of ATPase and total ATPase, leading to intracellular and extracellular Na ⁺ 、K ⁺ 、Ca ²⁺ And Mg (magnesium) ²⁺ The concentration disorder causes abnormal intracellular transport function, and the intracellular energy disorder blocks the energy source for P-gp transport, thereby inhibiting the drug function of tumor cell efflux and achieving the effect of reversing tumor MDR.

Table 5 influence of SchB/ATRA-PBAE NPs on ATPase in MS cells.

This example uses MTT method to examine the effect of pH sensitive SchB/ATRA-PBAE NPs on cytotoxicity of tumor heterogeneous cells and reversion of tumor MDR under different pH conditions. The results show that the free atra+schb group and the SchB/ATRA-PBAE NPs are more toxic than the free drug alone and both exhibit a dose-dependent cytotoxic effect, and that the SchB/ATRA-PBAE NPs are both more toxic than the free atra+schb group. The addition of SchB proved to increase the toxic effects of ATRA, reversing the effects of tumor cell MDR. With the change of pH value, the toxicity of the nano particles is different, and the reverse index is also changed, which shows that the SchB/ATRA-PBAE NPs not only have pH sensitivity, but also reverse tumor MDR effect.

MS cells were selected as drug resistant cell model to examine the effects of free SchB and SchB/ATRA-PBAE NPs in reversing tumor stem cell MDR. The effect of SchB and SchB/ATRA-PBAE NPs on P-gp expression was measured using a flow cytometer, and the results showed that the P-gp expression level on MS after SchB and SchB/ATRA-PBAE treatment was significantly reduced. Adopts ultra-trace Na ⁺ K ⁺ 、Ca ²⁺ Mg ² ⁺ The effect of SchB and SchB/ATRA-PBAE NPs on ATPase activity was examined in the total ATPase assay kit and the results indicate that SchB and SchB/ATRA-PBAE NPs can inhibit ATPase activity. By combining the results, the SchB/ATRA-PBAE NPs can reverse tumor MDR action by inhibiting P-gp expression quantity, and can also influence ATP synthesis by inhibiting ATPase activity, thereby reducing the effect of P-gp on drug excretion and further effectively realizing the effect of reversing tumor cell drug resistance.

It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that various changes, modifications, additions and substitutions can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A preparation method of drug-loaded nano-particles is characterized in that the drug-loaded nano-particles are obtained by coating SchB with ATRA-PBAE prodrug copolymer,

the ATRA-PBAE prodrug copolymer is synthesized by Michael addition reaction to obtain a PBAE copolymer with pH sensitivity, and then the pH-responsive ATRA prodrug copolymer is synthesized by esterification reaction; the preparation method specifically comprises the following steps:

(1) Synthesis of Poly (beta-amino ester) (PBAE)

PBAE synthesis 400 mg of 5-amino-1-pentanol and 640 mg of 1, 4-butanediol diacrylate are precisely weighed by Michael addition reaction in a molar ratio of 1.2:1, dissolved in methylene dichloride, magnetically stirred at 55 ℃ for 48 and h, and after the reaction is finished, the reaction product is purified according to the following process: placing the product in a 10 mL EP tube, adding 5 mL diethyl ether, swirling for 5 min, centrifuging at 5600 rpm for 5 min, discarding the upper layer solution, repeating for 3 times, vacuum drying to remove the organic solvent, and storing at 4deg.C for use;

(2) Synthesis of poly (beta-amino ester) -all-trans retinoic acid prodrugs

ATRA-PBAE synthesis ATRA containing carboxyl groups was completely dissolved in dichloromethane by esterification, EDC 10 mg and DMAP 5 mg were precisely weighed and dissolved in dichloromethane, and slowly added dropwise to a dichloromethane solution of ATRA; stirring for half an hour at 20 ℃; in addition, a previously prepared dichloro solution of PBAE was added to the above solution; the mixture was stirred 48 h in the dark at 20 ℃; excess ATRA was removed by precipitation in ice-cold ether; to completely remove free EDC HCl and DMAP, the solution was transferred to a dialysis membrane and dialyzed against ultrapure water; purging 3 times for 24 h; finally, removing the organic solvent by vacuum drying, and preserving at 4 ℃;

the molar ratio of PBAE to ATRA is 1:1;

the preparation method of the drug-loaded nanoparticle comprises the following steps:

accurately weighing 20 mg ATRA-PBAE prodrug copolymer and 20% carrier weight of SchB, dissolving the copolymer and the drug with 1 mL acetone to prepare an organic phase, slowly dripping the organic phase into 15 mL of 0.5% poloxamer aqueous solution at a rotating speed of 1000 rpm, continuously stirring at room temperature until acetone is completely volatilized, and filtering through a 0.22 μm filter membrane to obtain the nanoparticle colloid solution with blue opalescence.