Codelivery of Doxorubicin/PI3K Inhibitor Nanomicelle Linked with Phenylboronic Acid for Enhanced Cytotoxicity to Pancreatic Cancer

Zeng, Xuan; Fan, Xiaoxiao; Fu, Chunyan; Yang, Jialu; Tian, Jiahui; Peng, Qian; Qin, WeiGuo; Wu, Yi

doi:https://doi.org/10.1155/2022/8758356

Journal of Nanomaterials

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Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 8758356 | https://doi.org/10.1155/2022/8758356

Codelivery of Doxorubicin/PI3K Inhibitor Nanomicelle Linked with Phenylboronic Acid for Enhanced Cytotoxicity to Pancreatic Cancer

Xuan Zeng,^1,2Xiaoxiao Fan,¹Chunyan Fu,^1,2Jialu Yang,¹Jiahui Tian,^1,2Qian Peng,^1,2WeiGuo Qin ,¹and Yi Wu^1,2

Academic Editor: Abdelwahab Omri

Received08 Feb 2022

Revised24 Apr 2022

Accepted26 May 2022

Published23 Jun 2022

Abstract

Multidrug combination therapy for pancreatic cancer is widely applied in clinical practice. In this study, we used phenylboronic acid and polyethylene glycol as materials of nanomicelles, loaded with the PI3K/mTORC1 dual inhibitor PF04691502 (PF) to inhibit the resistance and metastasis of pancreatic cancer and increase the sensitivity of doxorubicin (DOX). We prepared the PPD nanoparticles (NPs) with a small PDI and a uniform morphology by controlling the DOX substitution degree (size of nm and zeta potential of mV). We determined the rates of PF and materials through the combination experiment of free drugs and the obtained [email protected] NPs (size of nm and zeta potential of mV). The drug loadings of DOX and PF in the nanomicelle were and , respectively. And the drug release in vitro was slow ( for DOX and for PF). The cell assay showed that the NPs had a good curative effect and migration on BxPC-3 cells, and it could be continuously taken up by cells. The [email protected] NPs displayed a dose-dependent cytotoxicity with less cell viability () and higher uptake in BxPC-3 cells compared with the free drug. The combined medication or [email protected] NPs reduced tumor metastasis, indicating that [email protected] NPs had the potential for clinical application.

1. Introduction

Pancreatic cancer is among the cancers with the highest mortality rate [1, 2]. There is a higher risk of drug resistance or surgery in the clinical treatment of pancreatic cancer, and meanwhile, a higher probability of transfer will lead to a poor prognosis. At present, small molecule chemotherapy drugs are widely used in the treatment of pancreatic cancer, such as doxorubicin (DOX) combined with sorafenib or 5-fluorouracil [3]. DOX has a strong broad-spectrum antitumor effect through inhibiting DNA replication and repair. In addition, DOX can increase reactive oxygen species in cells, exert a synergistic effect with photodynamic therapy and ferroptosis, and induce immunogenic cell death in tumors [4, 5]. Therefore, it is of great significance to investigate the combination therapy delivery system of DOX.

It is a serious problem that single DOX treatment usually leads to drug resistance or low efficacy, as well as tumor metastasis or recurrence after chemotherapy. The existing studies have shown that the drug resistance of pancreatic cancer is highly related to the stemness. Generally, the epithelial-mesenchymal transition (EMT) of cancer cells will lead to a reproduction of cancer stem cells [6–9]. Therefore, the inhibition of EMT-related pathways can improve the efficacy of traditional small molecule drugs in pancreatic cancer treatment. It has been verified that the PI3K/AKT/mTOR pathway plays a key role in the progression of cancer stem cells [10–13]. Besides, PI3K is also a proto-oncogene with the highest mutation rate in human tumors [14–16]. Hence, the inhibition of PI3K pathway can reduce the metastasis and recurrence of pancreatic cancer and improve the toxicity of DOX to pancreatic cancer. PF04691502 (PF) is one of PI3K/mTORC1 dual inhibitor, an inhibitor for PI3K (α/β/δ/γ), which can inhibit PI3K pathway from multiple targets [17]. Therefore, it is hoped that with the combination of two drugs, a better pancreatic cancer treatment effect can be achieved.

Inhibitors need to be effectively targeted to tumors to reduce side effects caused by the wide expression of PI3K [18, 19]. Nanodelivery system can significantly improve all the shortcomings mentioned above. Compared with traditional polymer nanoparticles, the micellar nanoparticles, with simple structure, stable properties, high repeatability, and easier industrialization, can provide an important direction for the clinical application of nanodrug transformation in the future [20]. 3-Aminophenylboronic acid (PBA), a stable and controllable small molecule with high affinity for sialic acid receptor (SA receptor), possesses the ability to target pancreatic cancer in nanodelivery systems [21–23]. SA receptor is a widely overexpressed in metastatic tumors, and it is positively correlated with malignancy and metastasis of various cancers [24]. In addition, the low clearance rate of nanomicelles in blood circulation can be achieved by the hydration film after polyethylene glycol (PEG) modification [25]. PEG is easy to construct micelles by connecting hydrophobic drugs and ligands, and the micelles formed are uniform and stable in structure. Therefore, we chose PEG linked to PBA and DOX loaded with PI3K inhibitor PF to achieve effective treatment of pancreatic cancer and, meanwhile, explored the synthesis, surface properties, structure, and drug-carrying function of nanomicelles, as well as the efficacy of nanomicelles in the treatment of pancreatic cancer.

2. Materials and Methods

2.1. Materials and Reagents

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) were obtained from Aladdin (Shanghai, China); NH₂-PEG-COOH (2000Da), 4-carboxyphenylboronic acid (PBA), and doxorubicin hydrochloride (DOX) were obtained from Shanghai Macklin Biochemical Co., Ltd.; CCK-8, DAPI, DMEM-H, and fetal bovine serum were obtained from Thermo Fisher (Shanghai, China).

2.2. Cell Culture

Pancreatic cancer cells (BxPC-3 cells) were cultured in DMEM-H medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in a carbon dioxide incubator (HERACELL 150i, Thermo Fisher) at a constant temperature of 37°C. The trypsin containing EDTA was used for passage.

2.3. Combination Therapy of Free DOX and PF In Vitro

Pancreatic cancer cells (BxPC-3 cells) were planted in the 96 well plates (5000 cells per well). After 12 hours, DOX and PF with the maximum concentration of 1 μg/mL were diluted in the complete culture medium. The drug was diluted gradient, and the cells were cultured for two days. Then, the complete culture medium with drugs was sucked, and the medium with 10% CCK-8 reagent was added. Similarly, the 10% CCK-8 medium was added to the well without cells as blank, and the same CCK-8 medium was added to cells without treatment as control. The 96 well plates were incubated at 37°C for 1 hour, and the absorbance was detected at 490 nm by microplate reader (Multiskan GO, Thermo Fisher). The cell viability was calculated as follows:

In order to explore the combined efficacy of two drugs in the treatment of pancreatic cancer, we prepared nanomicelle with appropriate concentration. We used two combinations with different concentrations of drugs and calculated the combined medication index of the two drugs by SynergyFinder 2.0, referring to the previous research methods [26].

2.4. Synthesis of PBA-PEG-COOH

PEG was modified by similar protocol according to the previous study [27]. 10 mL of DMSO was added appropriately to dissolve 0.2 g of DMAP, 0.4 g of EDCI, and 0.4 g of PBA. Then, the solution was stirred at 50°C for 30 minutes on the magnetic stirrer (DF-101S, Shanghai Licheng). After that, 0.5 g of NH₂-PEG-COOH was added into the solution and reacted with other materials in the solution for one day. Finally, the above solution was dialyzed (3000 Da) in deionized water and freeze-dried.

2.5. Synthesis of PBA-PEG-DOX

5 mL of DMSO was added appropriately to dissolve each of the three groups of mixtures, namely, 0.10 g of DOX and 0.20 g of 1,1-carbonyldiimidazole (CDI), 0.12 g of DOX, and 0.24 g of CDI, as well as 0.14 g of DOX and 0.28 g of CDI, respectively. After these solutions were stirred at 50°C for 2 hours, 0.4 g of PBA-PEG-COOH, 0.08 g of DMAP, and 0.16 g of EDCI, which were activated for 30 minutes, were added to the three mixed solutions and stirred on the magnetic stirrer for one day, respectively. Finally, the above solutions were dialyzed (3000 Da) in deionized water and freeze-dried.

2.6. Preparation of PBA-PEG-DOX Nanomicelle (PPD NPs) and PF-Loading PBA-PEG-DOX Nanomicelle ([email protected] NPs)

DMSO was added appropriately to dissolve 10 mg of PBA-PEG-DOX. When the solution was completely dissolved, it was placed in the dialysis bag and dialyzed for one day (7000 Da). Then, the PBA-PEG-DOX nanomicelles (PPD NPs) and PF-loading PBA-PEG-DOX nanomicelles ([email protected] NPs) were collected from the dialysis bag. The nanomicelles with appropriate size were selected for the subsequent experiments. 10 mg of PBA-PEG-DOX with 1 mg of PF04691502 was added for the preparation of drug-loaded nanomicelles by the same protocol.

Both the nanomicelles were filtered using a microporous membrane (450 nm), and the size and zeta potential were measured on a dynamic light scattering instrument (Nano-ZS90, Malvern). The test was conducted using an argon ion laser, with the wavelength of 658 nm, temperature of °C, and DLS angle of 90°. The zeta potential was determined at the same time under 11.4 v/cm, 13.0 mA, and 25°C. The sample solvent was diluted with distilled water. For scanning, the two kinds of nanomicelles were dropped on the copper mesh for air-drying. Then, they were stained with 2% phosphotungstic acid solution and air-dried before being photographed on a transmission electron microscope (TecnaiG2 F20, FEI).

2.7. Detection of Nanomaterials

The Fourier transform infrared spectrometer (FTIR) and hydrogen nuclear magnetic resonance spectroscopy (1H NMR) were applied to detect the synthesis of nanomaterials. Firstly, the PBA-PEG-COOH and PBA-PEG-DOX were mixed with KBr, and then, the mixture was pressed and detected on the FTIR instrument (Nicolet™ iS20 FTIR, Thermo Fisher). After that, the samples were dissolved in DMSO-d6 and detected on the 1H NMR instrument (AV-500, Bruker).

2.8. Drug Loading and Drug Release In Vitro

In order to detect the modification of PBA on PEG, we dissolved PBA with DMSO and diluted it with ultrapure water to prepare 5 PBA aqueous solutions with the concentrations of 2, 4, 6, 8, and 10 μg/mL, respectively. Then, the absorbance (285 nm) was detected to calculate the PBA in PBA-PEG-COOH and PBA-PEG-DOX.

The concentration of 2, 4, 6, 8, and 10 μg/mL of DOX or PF was detected at 508 nm and 346 nm on the ultraviolet visible light absorption instrument (BlueStar A, Labtech Group), respectively. The PBA-PEG-DOX materials and [email protected] NPs were diluted to 20 μg/mL, and the above absorbance was detected for testing the drug loading.

10 mL of prepared [email protected] NPs was placed in the dialysis bag and put in a beaker with 30 mL of PBS medium. Then, the beaker was placed on a shaker (BSD-TX345, Boxun) at a constant temperature of 37°C and shaken at a rate of 75 rpm. The PBS in the beaker outside of the dialysis bag was replaced at different time points, and the volume and absorbance of the previous PBS were measured to obtain the in vitro release rate of nanomicelles. The release rate of the DOX or PF was calculated as follows: where is the drug release rate at hour; and were the volume of PBS and drug concentration in PBS at hour in the beaker, respectively; is the volume of PBS in dialysis bag; and is the initial concentration of the nanocomposites sample ( 48 hours; both V₀ and C₀ are 0).

2.9. Toxicity of Nanomicelle In Vitro

Pancreatic cancer cells (BxPC-3 cells) were planted in the 96 well plates (5000 cells per well). At 12 hours, DOX, PBA-PEG-DOX, PF, and [email protected] NPs with different concentrations were added. Two days later, the culture medium with drug was sucked, and the complete medium with 10% CCK-8 reagent was added. The 96 well plates were incubated at 37°C for 1 hour, and the absorbance was detected at 490 nm by a microplate reader (Multiskan GO, Thermo Fisher).

2.10. Cell Migration Assay

Pancreatic cancer cells were planted in 24 well plates (10⁵ cells per well). When they grew to 90%, they were treated with DOX, DOX + PF, and [email protected] NPs with different concentrations (150 ng/mL for DOX and 96 ng/mL for PF). The treated cells were photographed at 0 hour, 6 hours, and 12 hours by the laser confocal microscope (AE2000, Motic), and then, the mobility was calculated using the Image J software.

2.11. In Vitro Intracellular Uptake

Pancreatic cancer cells were planted in 6 well plates (210⁵ cells per well) and treated with DOX, DOX + PF, and [email protected] NPs with different concentrations (5 μg/mL for DOX and 3.2 μg/mL for PF) when they grew to 50%. The treated cells were dyed with DAPI (1 μg/mL) and photographed at 2 hours, 4 hours, and 6 hours, and then, the cellular uptake was calculated using the Image J software.

2.12. Statistical Analysis

The data were measured in parallel for 3 times and expressed as . The data were -tested by GraphPad 7.0 and drawn by Origin 2017 and GraphPad 7.0. For the difference between two groups, and were considered significant, and was considered highly significant.

3. Results

3.1. Cytotoxicity and Combination Index of Free Drugs

CCK-8 Kit was used to detect the toxicity of doxorubicin (DOX) and PF04691502 (PF) to BXPC-3 cells. We firstly analyzed the cytotoxicity of free DOX and free PF on the pancreatic cancer cells. The results showed that the cell survival rate decreased gradually with the increase of the drug concentration. And the IC₅₀ of DOX was larger than that of PF (0 μg/mL for DOX and μg/mL for PF). To evaluate the synergistic effect of DOX and PF, we treated BXPC-3 cells with the two drugs in different combination. When the concentrations of PF and DOX were 0.1 μg/mL and 0.2 μg/mL, respectively, the cell survival rate reduced to , which was lower than before. Then, the combined synergy scores of the two drugs were analyzed using SynergyFinder 2.0. As shown in Figure 1(c), the DOX and PF have a certain synergistic effect on BXPC-3 cells, with a synergy score of 12.5. Thus, the toxicity experiment of free drugs provided a certain reference significance for the ratio of drug-loaded nanomicelles.

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3.2. FTIR and 1H NMR Analysis

Figure 2 demonstrates the successful synthesis of nanomaterials by FTIR and 1H NMR. It can be seen that 4-carboxyphenylboronic acid has a vC=O peak of the carboxyl group conjugated to the benzene at 1689 cm^-1. Meanwhile, it has merging peaks of carboxyl between 3000 and 3500 cm^-1, while NH₂-PEG-COOH has a carboxyl vC=O peak at 1716 cm^-1. When the two react, the peaks at 1689 cm^-1 of phenylboronic acid (PBA) and 1716 cm^-1 of NH₂-PEG-COOH disappear, and instead, an amide bond peak appears at 1648 cm^-1, indicating the successful grafting. The data of 1H NMR confirm the grafting of PBA on NH₂-PEG-COOH. In the 1H spectrum of PBA, the wide peak at about 13 ppm is the carboxyl peak, which disappears after the reaction. The methylene peak at 3.51 ppm in NH₂-PEG-COOH corresponds to PBA-PEG-COOH after the reaction. Besides, the 1H NMR spectrum multiple peaks between 7.5 ppm and 8.5pmm of the benzene ring in PBA indicate the success of the PBA grafting indirectly. For PBA-PEG-DOX, the FITR spectrum proves little but the 1242 cm^-1 vC-O of the phenol in the DOX. In NMR spectrum, the increased and enhanced peak between 7.5 ppm and 8.5 ppm shows the increase of benzene ring substances. At the same time, the peaks of the hydroxyl hydrogen or hydrogen on hydroxyl o-carbon from DOX appear between 4 ppm and 5.5 ppm. These data above indicate the successful synthesis of our nanomaterials.

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3.3. Size, Zeta Potential, and Electron Microscopy Analysis of Nanomicelle

In Section 2.5 and Section 2.6, we synthesized three kinds of PBA-PEG-DOX with different hydrophobic substitution degrees and prepared them to detect their size distribution. The size of the nanomicelles with the ratio of 10 : 3 (PBA-PEG-COOH: DOX) is smaller than those of the other two (Figure S1A and S1B). Therefore, we prepared blank NPs and PF-loading NPs with the ratio of 10 : 3 (PBA-PEG-COOH: DOX). Referring to the toxicity test of free drugs, we selected a more effective combined concentration of the two drugs (0.3 μg/mL of DOX and 0.2 μg/mL of PF) for the subsequent experiments and verified the two nano micelles by DLS and TEM. As shown in Figure 3, the size of PPD NPs was nm with its PDI of , and the size of [email protected] NPs was nm with its PDI of . The size of the nanomicelle increased with the PF loading. Meanwhile, the zeta potentials of the PPD NPs and [email protected] NPs were mV and mV, respectively. The results of transmission electron microscopy showed that the nano micelle was relatively spherical in the shape, with the size less than 200 nm, meeting the requirements of subsequent experiments. In addition, we also tested the stability of blank nanoparticles over time. As shown in Figure S2, the particle size of nanoparticles does not change significantly within 5 days of placement, and the results demonstrated that aggregation does not occur, indicating that the prepared NPs and nanomaterials are of high stability in aqueous solution.

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3.4. Drug Loading and Drug Release of Nanomicelles

To detect the mass fractions of PBA, DOX, and PF in nano micelles, absorbance method was applied for calculation. We detected the UV absorption of PBA-PEG-COOH at 285 nm. By using the standard curve method, the content of PBA in PBA-PEG-COOH was . The dual absorbance method was used to detect the content of DOX in PBA-PEG-DOX material and the drug loading of DOX and PF in nano micelles. The results showed that there was DOX in the PBA-PEG-DOX materials. We selected the drug-loaded nano micelles for follow-up experiments. Firstly, we measured the drug loading of DOX and PF of the nano micelles, which were and , respectively. After that, we performed drug release experiments. As shown in Figure 4, free DOX and PF release faster within the first 16 hours, with the drug release rates of and , respectively, while the release rates of DOX and PF in [email protected] NPs were and , respectively. After 48 hours, the release rates of DOX and PF from [email protected] NPs were and , respectively, indicating that the nanomicelles prepared in this study could effectively delay drug release.

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3.5. Cytotoxicity of [email protected] NPs

The toxicities of materials and drugs to BxPC-3 cells were detected using CCK-8 Kit. As shown in Figure 5, the survival rate of tumor cells was at the DOX concentration of 300 ng/mL, and the survival rate was for PBA-PEG-DOX. Therefore, PBA-PEG-DOX appears to have stronger cytotoxicity than DOX. At the same time, the cell viability rate for PBA-PEG-DOX combined with PF was at the maximum concentration, while the rate for drug-loaded NPs was smaller than it, with the cell activity of . Hence, it can be concluded that under the same drug concentration, our [email protected] NPs have higher cytotoxicity.

3.6. Cellular Uptake Assay In Vitro

We continued the cellular uptake experiments to investigate the relationship between toxicity and cellular uptake. The free drug showed a certain uptake at 2 hours (Figure 6), and the fluorescence of DOX coincided with that of DAPI, indicating that its intracellular distribution was mainly in the nucleus, as same as the [email protected] NPs. The uptake of the NP group was higher than that of the free drug group through Image J quantification. Compared with the figures at 2 hours, the intracellular drug contents of the free drug group and the NP group were higher at 4 and 6 hours, but there was no significant difference in cellular uptake for the free drug group at 4 and 6 hours, while the DOX fluorescence in cells of the NP group continued to increase. Meanwhile, the DOX accumulated in the NP group around the nucleus (in the cytoplasm) at 4 and 6 hours, and the fluorescence in the cytoplasm was enhanced at 6 hours, indicating the continuous accumulation of DOX in the cells. However, there was no statistically significant difference in the uptake of free DOX between these two time points.

3.7. Cell Migration Assay

In order to explore the effect of nano micelles on tumor migration, we carried out cell scratch experiment (Figure 7). After 6 hours, the metastatic rates of the three experimental groups were significantly lower than those of the control group. After 12 hours, the migration rate of the DOX group was significantly increased, indicating that DOX-treat alone may have a limited inhibition effect on tumor migration after a certain period of time, while the dual-drug combination and drug-loading nano micelle group both showed a better inhibition effect on migration. However, there was no significant difference in the effect of free drug combination and NPs on BXPC-3 metastasis.

4. Discussion

The multi-drug combination therapy has been widely adopted in the treatment of tumors, especially the drug-resistant tumors. The use of Epithelial-Mesenchymal Transition (EMT) inhibitors can significantly improve the efficacy of traditional small molecule drugs, reduce tumor drug resistance, reduce metastasis and recurrence, and improve the prognosis of patients [28]. The complex environment in vivo is prone to inaccurate drug toxicity. Therefore, using nano-drug loading system to accurately locate drugs in tumors can reduce toxic and side effects, thus improving the curative effect. At present, only liposomes have been approved for clinical use. Similar to them, the organic nano micelle, including the hydrophilic group and hydrophobic group, is simple in structure, which makes it stable and reproducible, thus easier to be used in industrial production [29–31]. Therefore, it is one of the promising carriers for tumor treatment. However, nano micelles are also facing various challenges. For example, a specific size (100-200 nm) of nano micelles is usually required to achieve the passive targeting by the EPR effect, or the surface modification is required to target for efficient tumor uptake [32–36]. Besides, it is unstable in plasma. For example, the adsorption of plasma proteins is also one of the factors limiting nano micelles [37–42].

In this study, we successfully synthesized nano materials based on PEG. We used the carboxyl group on phenylboronic acid (PBA) and the amino group on doxorubicin (DOX) to react with the amino group and carboxyl group on NH₂-PEG-COOH, respectively. Generally, amide bonds can be kept in aqueous solution for a long time at low temperature. In the stability experiment, our nanomicelles were kept at low temperature for 5 days without obvious change. Meanwhile, urea was formed by reactant acid as the main by-product of the catalytic reaction of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and it was significantly reduced after the addition of 4-dimethylaminopyridine (DMAP). We removed the by-products of PBA and PBA-PEG-COOH by dialysis as they were of small molecular weight and good water solubility. Compared to PBA-PEG-DOX with poor water solubility and PBA-PEG-COOH with high molecular weight, the by-products and unreacted substances are more likely to pass through the dialysis bag with DMSO during dialysis. Therefore, appropriately prolonging the dialysis time can reduce the impurities in nanomaterials, which also reveals that the tested nanomaterials are connected conjugated compounds rather than a single mixture.

In the preexperiment, we synthesized PBA-PEG-DOX with various proportions of DOX. As DOX is both a drug and a hydrophobic group, the increase or decrease of it can change the drug loading and the size of nanoparticles simultaneously. Therefore, the optional range of DOX substitution degree is low, and PBA preferably reacts completely with the amino groups for the high cellular uptake. It has been found that a small change in the proportion of DOX has a great impact on the size of the material. Therefore, we chose the proportion with the smallest size in the previous experiment for the subsequent experiments. Additionally, with the combination experiment of free drugs, more appropriate combination ratio can be determined. So, in the present study, we did not prepare nano micelles with various PF loadings for cell experiment. We successfully prepared blank and drug-loaded nano micelles and found that the size of drug-loaded nanomicelles was larger than that of blank nanomicelles, and the negative value of membrane potential decreased. The hydrodynamic sizes of dual nanoparticles were less than 250 nm and less than 200 nm, respectively, in TEM. Because of the multiple nitrogen atoms of PF, the surface negative potential of the nano micelle can be reduced by the inclusion.

Due to the strong toxicity of the two drugs, lower drug loading is allowed. The prepared drug-loaded nanomicelles, with a double drug loading rate (~10%, either), can significantly reduce the drug release rate, which showed effect in biological experiments both in vivo and in vitro. We conducted cytotoxicity experiments and found that the cytotoxicity of free PBA-PEG-DOX was slightly stronger than that of free DOX at the same DOX concentration, implying that the material synthesis does not affect DOX toxicity. At the same time, the results of uptake experiment have revealed that the PBA-PEG-DOX vector can absorb DOX into the cytoplasm when the DOX in the nucleus is relatively saturated and help to achieve long-term endocytosis. This may explain why the cytotoxicity of PBA-PEG-DOX was higher than that of the DOX and the cytotoxicity of nanomicelles was higher than that of free drugs.

Finally, we conducted a migration experiment. After 6 hours of treatment, the mobilities of the three groups were low, while after 12 hours, the mobility of the free DOX group got relatively higher. Combined with the fact that the intracellular content of free DOX tended to remain stable or even decrease after 6 hours, it can be speculated that the rate of free drug entering the cell after 4-6 hours was balanced with the working rate of tumor drug resistance related to pumps or other resistance genes. Different from free drugs, the concentration of nanomicelles in cells increased continuously in 4-6 hours, indicating that nanoparticles can be continuously ingested by cells through the mediation of receptors. At the same time, PBA-PEG-modified may increase the toxicity through reducing the intracellular clearance of drugs by its high molecular weight.

5. Conclusion

Novel nanocarriers self-assembled from DOX, PEG, and PBA were synthesized and characterized. Amphiphilic nanomicelle materials were synthesized through amide reaction, and the size of nanoparticles was optimized by adjusting the proportion of DOX in the reactants. With a more appropriate proportion, the size of nanoparticles increased regardless of the proportion of DOX. The synergistic effect of PF and DOX was verified through the combination experiment of free drugs, and the appropriate ratio of the two drugs was identified for dual drug codelivery. The obtained [email protected] micelle could control the release, cause long-lasting cell intake, and exert cytotoxicity. The in vitro cell experiments have shown that nano micelles can inhibit the growth and metastasis of pancreatic cancer cells. Hence, the [email protected] micelle delivery system can be a potential nanoscale drug for the treatment of pancreatic cancer.

Data Availability

The data used to support the findings of this study are included in the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Xuan Zeng, Xiaoxiao Fan, Chunyan Fu, and Jialu Yang contributed to the investigation. Jiahui Tian and Qian Peng contributed to funding resource. WeiGuo Qin and Yi Wu contributed to conceptualization and funding acquisition. Xuan Zeng and Xiaoxiao Fan contributed equally to this work.

Acknowledgments

We thank the support of grants from the Hunan Provincial Health Commission (No. 202202082769).

Supplementary Materials

The supporting information is available free of charge on the Hindawi website. Figures S1 and S2: screening the appropriate feeding ratio of nanoparticles; study on the stability of nanoparticles with time in aqueous solution. (Supplementary Materials)

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Copyright © 2022 Xuan Zeng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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