Synthesis of polymer lipid nanoparticles loaded with Quercetin: Potential for Activating Autophagy to Promote Apoptosis of Breast Cancer Cells

doi:10.21203/rs.3.rs-2291061/v1

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Synthesis of polymer lipid nanoparticles loaded with Quercetin: Potential for Activating Autophagy to Promote Apoptosis of Breast Cancer Cells

https://doi.org/10.21203/rs.3.rs-2291061/v1

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Studies have shown that autophagy plays an important role in breast cancer progression and treatment. We have emphasized on preparation and optimization of polymer lipid nanoparticles loaded with quercetin (Q-PLNs) and nano-targeted therapeutic strategies on autophagy modulation. Q-PLNs had uniform particle size, good stability, and sustained release effect. Q-PLNs greatly promoted cellular uptake with lower IC₅₀ and increased apoptotic rate. It was discovered that low-dose autophagy inhibitor CQ could partially block behavior of Q-PLNs, suggesting that Q-PLNs could induce autophagy in MCF-7 cells. Apoptosis and Western blot experiment reflected that Q-PLNs activated autophagy and apoptosis of MCF-7 cells by regulating AMPK/mTOR/ULK1 signal activity. In conclusion, polymer lipid nanoparticles were good delivery carriers to improve stability and pharmacological activity of quercetin. The pro-apoptotic effect of Q-PLNs was related to autophagy activation. The initiation mechanism of quercetin induced autophagy in MCF-7 cells presented a basis for further investigation of molecular biological mechanism.

Polymer lipid nanoparticles

Breast cancer

Autophagy

Cell apoptosis

Quercetin

Cell autophagy is a self-digestion process under the action of stimulation factors or in lack of energy, which can degrade their own proteins, organelles, and cytoplasmic lysosome into energy-matter [1], thus achieving the goal of organelle renewal and metabolism. Autophagy plays a vital biological role in the stability of cells and the body’s internal environment [2], which can not only relieve the pressure brought by tumor proliferation but also meet the continued growth of energy requirements of cells during environmental stress [3]. Autophagy has the dual function of tumor inhibition or promotion [4], depending on the survival environment and the tumor stages. In the earliest stage of tumor growth, autophagy acts as a tumor suppressor that restricts inflammation, tissue damage, and genomic instability [5]. Impaired autophagy possibly leads to oxidative stress, genomic instability, chronic tissue damage, and tumorigenesis, and is involved in inflammation and immune activation [6]. Therefore, the regulation of autophagy is conducive to becoming a target for tumor treatment. When tumor cells are exposed to the stress of growth, metastasis, or chemotherapy, autophagy tends to induce the metastatic process by spreading cells and maintaining survival, thereby promoting tumor progression [7, 8], which have an important part in normal cell survival and balance [9]. Independent of genotoxic stress and genomic instability, autophagy also reduces sensitivity to anti-cancer therapies, eliminates organelle damages, and maintains cellular integrity [10].

Quercetin (QUE) is the most widely distributed natural flavonoid [11]. Due to its powerful antioxidant effects, QUE has been used to prevent and treat a variety of diseases [12], such as cancer, or heart and liver diseases [13]. According to previous studies, QUE have an influence on the biological activity of autophagy. It can induce autophagy-related death when acting on specific tumor cells. QUE enhances TNF-related apoptosis-inducing ligand (TRAIL) mediated death of lung cancer A549 cells, which is related to QUE-induced autophagy [14]. Hyun et al. [15] have found that QUE inhibited the expression of HSP70 and induce autophagy in pancreatic cancer PANC-1 and MIA PaCa-2 cells. The production and regulation of autophagy are mediated by multiple signals, among which the adenylate activated protein kinase (AMPK), Mammalian Target of Rapamycin (mTOR), UNC-51-like kinase 1 (ULK1) are the classic signaling molecules regulating autophagy [16, 17]. Since autophagy is a key link in tumor progression, we hypothesized that QUE promotes apoptosis of breast cancer cells by activating autophagy. However, on account of the complexity of autophagy functions, the mechanism of QUE between apoptosis and autophagy in breast cancer cells still needs to be investigated, so an in-depth investigation was conducted in this study.

Due to the presence of phenolic hydroxyl group and carbonyl group in the structure of QUE, it has the ability of strong free radical scavenging and binding with transition metal ions [18]. QUE rapidly binds to glucuronic acid or sulfate when passing through the intestine and liver, and part of the metabolites are also methylated, resulting in low bioavailability and poor medicinal properties [19]. Nano drug delivery system (NDDS) refers to a new drug delivery system that uses special technology to embed drugs in nanocarriers to reduce toxic and side effects, and improve bioavailability and tissue distribution characteristics in vivo [20]. NDDS show excellent performance in increasing the solubility of hydrophobic drugs, and improving drug stability and pharmacological activity [21]. Polymer lipid nanoparticles (PLNs) have good stability and encapsulation efficiency due to their intact structures [22]. Herein, we constructed polymer lipid nanoparticles to effectively transport quercetin, and the physical and chemical properties and quality evaluation of the nanoparticles were investigated. We also compared cell apoptosis, autophagic flux, and expression changes of autophagy-related proteins after administration, aiming to elucidate the role of QUE in apoptosis and autophagy of breast cancer cells.

2.1 material

Quercetin (95%) was provided by Chengdu Phytochemical Pure Biotechnology Co., Ltd. (Sichuan, China). Poly-lactide-co-glycolide (PLGA) was provided by Jinan Daigang Biological Engineering Co., Ltd. Egg yolk lecithin (PC-98T) and Distearoyl phosphoethanolamine-PEG₂₀₀₀ (DSPE-mPEG₂₀₀₀) were provided by Aweituo Pharmaceutical Technology Co., Ltd. (Shanghai, China). Acetone and ethanol (HPLC grade) were provided by Guangdong Guanghua Company. DMEM cell medium and phosphate buffer saline (PBS; pH 7.4) were purchased from Gibco Life Technologies (New York, USA). Fetal Bovine Serum (FBS) was purchased from Ausgenex Inc.

2.2 Preparation and optimization of Q-PLNs

Q-PLNs were prepared by the nano-precipitation method [23]. QUE and polymer PLGA were dissolved in acetone and slowly added into PC-98T solution. The polymer core was formed after heating volatiles of acetone, then the core-shell structure was formed by self-assembly through hydrophobic action. In order to prepare Q-PLNs with good stability, the preparation process parameters were optimized. It was found that Q-PLNs were prepared at the emulsifying temperature of 70°C, emulsifying time of 60 min, and the oil-water ratio of 1:20 had smaller particle size and PDI, and higher encapsulation efficiency. Through single factor experiment and Box-Behnken design, the optimal preparation prescription of Q-PLNs was determined as follows: 13.15 mg PLGA, 2.42 mg QUE, 42.64 mg PC-98T, 5 mg DSPE-mPEG₂₀₀₀, acetone, and 4% ethanol solution dissolved with 1% Tween-80 in a ratio of 1:20 (v/v).

2.3 Encapsulation efficiency (EE) of Q-PLNs

High performance liquid chromatography (HPLC) was used to determine the encapsulation efficiency (EE). HPLC conditions: The maximum detection wavelength of QUE was 370 nm, the mobile phase was methanol: 0.2% phosphoric acid aqueous solution (55:45), the flow rate was 1.0 mL/min, and the column temperature was 25℃. The Q-PLNs solution was placed in a 10 KD ultrafiltration centrifuge tube and centrifuged at 10000 rpm/min for 10 min. The solution in the outer tube of the ultrafiltration tube was analyzed by HPLC to determine the amount of free drug (W_f). Demulsification was performed by ultrasound for 30 minutes, and the supernatant after centrifuged at 1000 rpm/min for 5 min was taken to analyze the total drug amount (W_t). The calculation formula was as follows: EE = (W_t-W_f)/ W_t*100%.

2.4 Characterization of Q-PLNs

Zetasizer Nano ZS90 (Malvern, England) was used to measure the particle size, polymer dispersity index (PDI), and zeta potential of Q-PLNs. The morphology of Q-PLNs was observed by transmission electron microscopy (TEM, Tecnai 10; Royal Philips, Netherlands), which were negatively stained with 2% phosphoric acid and observed under dark field. The thermal properties of Q-PLNs were analyzed by thermal gravimetric analyzer (PerkinElmer, USA). Thermogravimetric analysis (TGA) was performed with temperature scans ranging from 35°C-600°C with nitrogen as the covering gas at a flow rate of 10°C/min.

2.5 Release of Q-PLNs in vitro

Membrane dialysis method was used to study the drug release in vitro. Q-PLNs and QUE Propylene Glycol solution (2.5 mg mL^− 1) were placed in a dialysis bag (MW 12000–14000 kDa) and soaked in 100 mL release medium (consisting of 35% anhydrous ethanol and 65% distilled water) [24], which were kept at 37°C under stirring at 100 rpm/min. Samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 h to analyze the concentration by HPLC at 370 nm. The cumulative release of QUE and Q-PLNs in the release medium was investigated.

2.6 Stability investigation of Q-PLNs

The hydroxyl and pyranose structures of QUE are the reasons for its poor chemical stability [25], and the stability of QUE is greatly affected by temperature and light. Therefore, the effects of different temperatures and light conditions on the particle size and encapsulation efficiency of Q-PLNs were investigated.

2.6.1 Temperature stability

Q-PLNs and QUE ethanol solution (50 µg mL^− 1) was used as the control group. The samples were stored at room temperature and 4°C under dark conditions. The changes of degradation, particle size and encapsulation efficiency at different temperatures for 30 days was investigated.

2.6.2 Illumination stability

Q-PLNs and QUE ethanol solution (50 µg mL^− 1) were placed under (4500 ± 500) LX light. Samples were taken at 0, 5, and 10 days, respectively. The degradation, particle size and encapsulation efficiency were determined.

2.7 Antioxidant activity of Q-PLNs

The antioxidant activity can be evaluated by detecting the DPPH scavenging ability of Q-PLNs. The samples were mixed with DPPH anhydrous ethanol solution (200 µM) at a ratio of 1:2 (v/v), and the reaction was conducted at room temperature for 30 min or 60 min without light. The absorbance was measured at 515 nm by a microplate analyzer (Bio-Tek Winooski, VT, USA). Antioxidant activity percentage (AA) was calculated according to the following formula: AA = (A_DPPH-A_Sample)/A_DPPH×100%. The A means the absorbance and the AA half reduction concentration IC₅₀ means the sample concentration of the mixture when AA is 50%.

2.8 Cell Culture

Human breast cancer MCF-7 cell line was purchased from Nanjing Keygen Biotechnology Co., Ltd. MCF-7 cells were cultured in DMEM medium (containing 1% double antibody and 10% FBS) at 37°C and 5% CO₂.

2.8.1 Cellular uptake

In order to investigate the uptake of Q-PLNs by MCF-7 cells, coumarin-6 (C6) replaces QUE to prepare C-6 PLNs with the final concentration of 200 µg mL^− 1. MCF-7 cells were inoculated in confocal dishes at a cell density of 5×10⁴ cells/well. The MCF-7 cells were treated with medium containing C-6 PLNs for 2 h, washed with PBS and added with 4% paraformaldehyde for 10 min. Finally, MCF-7 cells were stained with DAPI for 5 min. Cellular uptake was observed by laser confocal microscopy.

The uptake of C-6 PLNs by MCF-7 cells was quantitatively analyzed. MCF-7 cells were inoculated in a 6-well plate at a cell density of 2×10⁵ cells/well. The MCF-7 cells were treated with medium containing C-6 PLNs for 2 h. The cells were digested and the fluorescent dye on the cell surface was washed by PBS. Fluorescence intensity was measured by Flow cytometry (FACSCalibur, BD, USA), and data were analyzed by FlowJo V10 software.

2.8.2 Cytotoxicity

MCF-7 cells were inoculated in 96-well plates at a cell density of 4×10³ cells/well. Different concentrations of QUE, PLNs and Q-PLNs were added, respectively. A drug-free control group and a blank group were set up, and each group was set with 6 complex wells. 50 µL MTT culture medium was added for 3 h, then DMSO solution was added to dissolve formazan. The OD value was detected by the microplate analyzer at 490 nm, and the IC₅₀ was calculated by GraphPad 6.0 software.

Cell viability (%) = (OD _drug - OD_blank)/( OD_control - OD_blank) ×100%

2.8.3 Autophagy flux

The Cyto-ID® Autophagy Detection Kit 2.0 can qualitatively and quantitatively detects the autophagy of living cells, because the green dye will excite bright fluorescence in the vesicles produced during autophagy. Chloroquine (CQ), a kind of lysosomal inhibitor, is set as negative control. MCF-7 cells were inoculated in confocal dishes at a cell density of 5×10⁵ cells/well. Q-PLNs (40 µM), CQ (10 µM), and CQ + Q-PLNs (40 µM) were incubated for 24 h, and the wells without drug treatment was the control group. 5% paraformaldehyde solution was added for 20 min. 500 µL of dye solution (PBS: Cytoid: Hoechst 33342 = 996:2:2, v/v) was added and incubated in dark for 30 min. The cells were observed by laser confocal microscopy, and the effects of different concentrations of Q-PLNs on autophagy flux of MCF-7 cells were detected by flow cytometry.

2.8.4 Apoptosis

Cell apoptosis was detected using the AnnexinV-APC/PI Apoptosis Assay Kit. MCF-7 cells were inoculated in 6-well plates at a cell density of 2×10⁵ cells/well. Cells were cultured in QUE (40 µM), Q-PLNs (20, 40, 60 µM), CQ (10 µM), and CQ + Q-PLNs (40 µM) medium for 24 h, respectively. Annexin V-APC and PI were added respectively and incubated in dark for 15 min. Fluorescence intensity was measured by flow cytometry and the data was analyzed by FlowJo V10 software.

2.8.5 Western blot

MCF-7 cells were inoculated in 6-well plates at a cell density of 1×10⁶ cells/well and protein was extracted from blank group, Q-PLNs (40 µM), CQ (10 µM), and CQ + Q-PLNs (40 µM) groups. The protein concentration was determined by the BCA method, and the total protein content was 30 µg/well. Proteins with different molecular weights were separated by electrophoresis. The membrane was transferred and sealed in PBST containing 5% skim milk powder. After rinsing with PBST, the PVDF membrane and primary antibody was incubated overnight at 4℃. Next the PVDF membrane and secondary antibody was incubated for 2 h, and ECL A solution and B solution were mixed in equal amounts and dropped into the PVDF membrane, which was exposed and developed on the gel imaging system (Tanon-5200Multi, Shanghai).

2.9 Statistical analysis

All tests were conducted in three or more repeated trials. All test data were expressed as mean ± SD. GraphPad Prism 6.0 software was used for statistical analysis. One-way ANOVA analysis was used to test the difference between the sample data. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.

3.1 Characterization of Q-PLNs

From Fig. 1A, PLNs presented a clear blue opalescence, while Q-PLNs presented a light-yellow transparent solution. Figure 1B showed that Q-PLNs were spherical without aggregation under the electron microscope. As measured in Fig. 1C-D, the zeta potential was (-16.1 ± 7.14) mV, particle size was (109.3 ± 0.216) nm, and PDI < 0.3, indicating that Q-PLNs were uniform and had good stability. Multiple quadratic regression equations were obtained by Design Expert 12.0 software: Y= -75.51–4.26A − 2.14B + 0.7512C + 0.9725AB + 2.58AC − 0.615BC − 3.35A2 -3.01B2 -5.02C2 (R² = 0.9620), The encapsulation rate of Q-PLNs was predicted to be 77.55%. As shown in Table 1, the average encapsulation rate of three batches of Q-PLNs prepared in parallel was 77.31%, and the deviation from the predicted value was less than 2%, suggesting that the regression model fitted well and the predicted value was accurate and reliable.

The thermal properties of Q-PLNs were analyzed by the relationship between mass and temperature. The TGA curve of QUE declared that due to the volatilization of water, the first weightlessness occurred near 60–118°C. The second weightlessness occurred at about 209–349°C, and the weightlessness rate was 30.3%. From Fig. 1E, Q-PLNs experienced two times of weightlessness. The first time started at about 220°C, and the weightlessness rate was 76.4%. The initial degradation temperature of Q-PLNs was higher than that of PLNs, implying that PLNs loaded with QUE had higher thermal stability. Maybe the strong interaction between QUE and preparation components (such as hydrogen bond formation) accounted for delaying the degradation of Q-PLNs. The second time started at about 350°C, and the weight loss rate was 20.5%. In the second degradation process, the weight loss rate of Q-PLNs was higher than that of PLNs, possibly because the temperature was within the degradation temperature range of QUE, which increased the weight loss rate of Q-PLNs. The total degradation efficiency of Q-PLNs was 96.9%, which was much higher than that of QUE (51.4%). Compared with QUE, Q-PLNs with the same mass had smaller particle size and larger content of lipid soluble preparation excipients. Therefore, the total mass of Q-PLNs degradation was larger under high temperature conditions.

3.2 Cumulative release rate of Q-PLNs in vitro

According to Fig. 1F, QUE was completely released within 8 h. The degradation rate of Q-PLNs was fast in the first 8 h, and the release rate was close to 80%. After that the release rate began to slow down, exhibiting a gentle trend, and the release rate arrived 90% at 36 h. Eventually the cumulative release of Q-PLNs was 92.67% within 72 h, showing the characteristics of sustained release. The research proved that the introduction of PLN carriers could avoid the effect of rapid release of QUE on drug efficacy, accomplishing the purpose of slow and controlled release and improving bioavailability.

The release data were fitted with Origin Pro 9.1 to study the release mechanism of Q-PLNs. From Table 2, among the three release kinetic models, R² of the first-order release kinetic model was the largest, so QUE was released through the first-order model. The behavior of Q-PLNs in the release medium conformed to the first-order release kinetic model (R² = 0.9982). This was due to QUE was wrapped in the hydrophobic core. QUE can only be released into the medium through the interface between the hydrophobic core and water molecules. In the rapid release stage, QUE diffused quickly owing to being loosely bound with PLN carriers. The release curve tended to be flat in the second stage with the degradation of PLN carriers. In general, Q-PLNs achieved a slow-release state and the action time of QUE can be prolonged.

3.3 Stability of Q-PLNs

3.3.1 Temperature stability

As shown in Fig. 2A, the degradation rate of Q-PLNs at the same temperature was much lower than that of QUE. The degradation rate of Q-PLNs at room temperature (3.2%) was slightly higher than that of Q-PLNs at 4°C (3.0%) but much lower than that of QUE (15.5%). Figure 2B showed the variation of Q-PLNs particle size within 30 days at different temperatures. With the extension of storage time, the encapsulation efficiency of Q-PLNs decreased slightly, while the increasing trend of particle size at room temperature is more obvious, demonstrating that Q-PLNs have good stability in a low-temperature environment. Moreover, Q-PLNs effectively improved the stability of QUE, which was because polymer lipid nanoparticles prevented the contact between QUE and the external environment, thus reducing the chance of QUE being oxidized and degraded.

3.3.2 Illumination stability

Figure 2C displayed the changes of QUE degradation, encapsulation efficiency and particle size of Q-PLNs at different illumination time. After 10 days of continuous irradiation, the appearance of Q-PLNs solution remained clear and transparent. The particle size and encapsulation efficiency changed little. The results reflected the degradation rate of Q-PLNs was only 6.6%, which was much lower than that of QUE (18.7%), turning out to be that Q-PLNs had good illumination stability.

3.4 Antioxidant activity of Q-PLNs

The antioxidant activity of Q-PLNs was determined by the DPPH assay. Electrons provided by the phenolic hydroxyl groups in QUE structures can eliminate DPPH free radicals, so the absorbance of the DPPH solution depends on the free radical scavenging activity and concentration of QUE. From Fig. 2D, the antioxidant activities of Q-PLNs and QUE are positively correlated with the concentration. From Fig. 2E, The IC₅₀ of the reaction for 60 min is smaller than that of the reaction for 30min. The smaller the IC₅₀, the more sufficient reaction and the better the DPPH radical scavenging effects. In addition, at the same reaction time, the IC₅₀ of Q-PLNs was almost the same as that of free QUE, which means that polymer lipid nanoparticles would not reduce the antioxidant activity of free QUE.

3.5 Qualitative and quantitative cellular uptake

Polymer lipid nanoparticles can stably encapsulate hydrophobic drugs and protect them from degradation in vitro and in vivo. Moreover, due to the ideal particle size, it can pass through cellular internal and external barriers, which is conducive to increasing the blood circulation time and reaching the tumor site [26]. Figure 3A-C showed the cellular uptake capacity of C-6 and C-6 PLNs. The blue fluorescence is DAPI-stained nuclei and the green fluorescence is C-6 entering cells. The C-6 PLNs group showed significant fluorescence signals (1874) compared to that of the C-6 group (446), indicating that polymer lipid nanoparticles can enable more C-6 to be taken up by breast cancer MCF-7 cells.

3.6 Cytotoxicity

Figure 3D revealed that PLNs were less cytotoxic, suggesting that the preparation materials of polymer lipid nanoparticles were generally safe. Figure 3E showed that Q-PLNs had a stronger inhibitory effect on MCF-7 cells at the same concentration compared with QUE. The IC₅₀ of Q-PLNs (56.59 µM) was lower than that of QUE (211.2 µM), which might be related to the enhanced cellular uptake ability and the continuous release behavior of Q-PLNs.

3.7 Autophagy flux

Different from apoptosis and programmed cell death in general, autophagy death is one of the new programmed cell pathways and is strictly controlled by autophagy signals [27]. Stimulated by autophagy signals, the serine-threonine protein kinase (ULK1) complex can be activated for autophagy initiation [28, 29]. LC3-II is applied as a marker of autophagy to reveal autophagosomes and quantify autophagy in cells [30, 31].

The Cyto-ID® Autophagy Detection Kit 2.0 utilizes novel dyes to detect autophagy vesicles and monitor autophagy flow in living cells. Selective labeling of accumulated autophagy vesicles is one of rapid quantitative methods for monitoring autophagy in living cells without cell transfection. From Fig. 4A-B, compared with the control group (1.0), the green autophagy signal of Q-PLNs (3.25) was increased, suggesting that Q-PLNs can induce autophagy of MCF-7 cells, and the increase of autophagy vesicles leaded to the accumulation of probes in MCF-7 cells. CQ is a dose-dependent autophagy inhibitor, which blocks autophagy by inhibiting lysosomal proteases and preventing autophagosomes from fusing with autophagolysosomes. The low concentration of CQ (10 µM) could preserve part of autophagy activities. While the concentration of CQ exceeded 50 µM, the autophagy activities were completely inhibited. Herein, low concentration CQ (10 µM) combined with Q-PLNs was used to treat MCF-7 cells, and the green fluorescence signal was decreased (2.97), explaining that CQ (10 µM) could partially inhibit the autophagy induced by Q-PLNs.

3.8 Autophagy and apoptosis

As shown in Fig. 4C-D, compared with QUE (15.89%), Q-PLNs greatly stimulated the apoptosis of MCF-7 cells, indicating that nanocarriers can promote drug uptake and improve efficacy. Meanwhile, the apoptotic rates of Q-PLNs improved with the increase of concentration. Compared with Q-PLNs (19.96%), CQ combined with Q-PLNs resulted in reduced cell apoptosis (14.90%). It was preliminarily concluded that Q-PLNs promoted the apoptosis of MCF-7 cells, which was probably relative to the induction of autophagy.

3.9 The expression of autophagy-related proteins

Western blot was exploited to detect the expression of p-AMPK, AMPK, mTOR, p-mTOR, ULK1, p-ULK1, LC3-I, LC3-II, and other autophagy-related proteins of MCF-7 cells. To explore whether Q-PLNs regulate autophagy by affecting the AMPK/mTOR/ULK1 pathway and thereby promote breast cancer apoptosis. AMPK and mTOR are two key targets of autophagy regulation. Activation of AMPK enhances autophagy activity, while mTOR inhibits autophagy. From Fig. 5A-B, compared with the control group, the expressions of AMPK, mTOR, and ULK1 in Q-PLNs and CQ groups were significantly different (p < 0.05). In contrast with CQ group, Q-PLNs group observed that the expression of p-AMPK, AMPK, ULK1, and p-ULK1 were significantly up-regulated, while the expressions of mTOR and p-mTOR were significantly decreased (p < 0.05). Adding CQ in the Q-PLNs group could inhibit the effects of Q-PLNs to a certain extent, and the difference was statistically significant (p < 0.05). These results fully proved that Q-PLNs can promote AMPK and ULK1 protein phosphorylation and inhibit mTOR protein phosphorylation, finally activating AMPK/mTOR/ULK1 signaling activity.

Beclin-1 and LC3 are the markers of autophagy, wherein Beclin-1 is the initiation protein of autophagy, and LC3 is the specific marker protein of autophagy, including LC3-Ⅰ and LC3-Ⅱ protein subtypes. During the formation of autophagosomes, cytoplasmic LC3-I will enzymatically remove a small fragment of a polypeptide, and then bind to PE to transform into membrane LC3-II [32]. The transformation of LC3-I to LC3-II is an important marker of autophagy [32] to symbolize autophagosome maturation. Consequently Beclin-1 expression level and LC3-II/LC3-I ratio can be detected to estimate the level of autophagy. Figure 5C-D, showed that Beclin-1 and LC3-II protein expression in Q-PLNs group was significantly increased compared with that in control group and CQ group, whereas LC3-I expression was significantly decreased. The difference between LC3-II/LC3-I ratio was statistically significant (p < 0.05). Compared with control group and CQ group, the expression of pro-apoptotic protein Bax in Q-PLNs group was significantly increased, the expression of anti-apoptotic protein Bcl-2 was significantly decreased, and the ratio of Bax/Bcl-2 was statistically significant (p < 0.05). It was demonstrated that Q-PLNs induced autophagy of MCF-7 cells could promote apoptosis and inhibit tumor growth.

Due to its strong antioxidant activity, QUE is easy to be degraded under light, heat, or ultraviolet conditions. Meanwhile, QUE is almost insoluble in water, which further limits the development of its biological activity. The development of effective nano preparation has great advantages of improving the solubility, stability, and efficacy of hydrophobic drugs. Polymer lipid nanoparticles are core-shell nanocarriers constructed by combining the advantages of both polymer nanoparticles and lipid nanoparticles [33]. PLNs consist of three parts, namely the drug-carrying polymer core, the internal lipid layer surrounding the polymer core, and the external lipid-PEG layer [33]. The polymer system provides a large specific surface area, which is effectively in favor of the drug solubility. The internal lipid layer can enhance the biocompatibility of the polymer nucleus and prevent water expansion, thus reducing the rate of polymer degradation [34]. The outer PEG layer can not only prolong circulation time, but also achieve targeted drug delivery through PEG terminal functional groups conjugation with targeted ligands such as folic acid, monoclonal antibodies, and peptides [35]. The experimental results clarified that Q-PLNs improved the light stability and temperature stability of QUE, and they have similar antioxidant activity. In vitro release study illustrated that Q-PLNs were released slowly in the first-level model, achieving continuous and effective release. The results of cellular uptake and MTT assay elucidated that Q-PLNs enhanced the drug accumulation in MCF-7 cells and cytotoxicity of QUE. Therefore, the optimized Q-PLNs have good safety and stability as well as they can achieve the purpose of slow controlled release and targeting MCF-7 cells [36].

Alterations in autophagy pathways are present in a variety of diseases, including cancer, metabolic diseases, autoimmune diseases, and aging [9]. The allele deletion of the autophagy regulatory factor Beclin-1 was observed in 40–75% of human breast tumors, stating that autophagy can inhibit tumorigenesis [37]. In addition, metabolic stress induced by autophagy deficiency promotes DNA damage and chromosomal instability [38]. When DNA is damaged, autophagy-deficient cells exhibited reduced homologous recombination repair of damaged DNA, illuminating that autophagy plays an important part in maintaining genomic integrity [39]. Taken together, this evidence identifies that autophagy may be beneficial in inhibiting malignant transformation and tumor development.

According to research findings, the main mechanism of autophagy includes ULK1 and its upstream effectors such as AKT, mTOR, and AMPK, which initiate the processes of vesicular double-membrane formation of autophagy [40]. In the presence of nutrient deficiency or hypoxia, adenosine monophosphate (AMP)/adenosine triphosphate (ATP) and adenosine diphosphate (ADP)/ATP ratios increase, and LKB1 phosphorylate AMPKα to activate AMPK and directly inhibit mTOR complex 1 (mTORC1) [41, 42]. Dissociation of mTORC1 promotes rapid phosphorylation of ULK1 to activate autophagy, which plays a critical role in autophagy initiation. AMPK can also activate autophagy by directly inducing phosphorylation of ULK1, VPS34, and Beclin-1 [43, 44]. Under nutrient-rich conditions, active mTORC1 phosphorylates S757 at ULK1 to disrupt ULK1-AMPK interactions, inhibits phosphorylation of S317 and S777 at two AMPK loci, and keeps ULK1 inactive to inhibit autophagy [45].

LC3, Beclin-1, and P62 were the proteins most closely related to autophagy [46]. The autophagy regulatory protein LC3 is a mammalian homolog of yeast ATG8, widely existing on the autophagosome membrane [47], which can be detected in the whole process of autophagy. In mammals, LC3 protein expression varies with different types of tissue and cells [48, 49]. At the initial stage of autophagy, LC3-I in the cytoplasm binds to phosphatidylethanolamine to form LC3-II, which locates on the autophagosome membrane. After the start of the autophagy, LC3-II can be cleaved by ATG4 at its carboxyl-terminal and degraded to regenerate LC3-I in the cytoplasm [49]. Accordingly, the relative ratio of LC3-I/LC3-II is correlated with the formation amount of autophagosomes, perhaps to some extent determining the degree of autophagy induced by a specific stimulus.

Beclin-1, the first identified mammalian autophagy protein, which is involved in the formation of autophagosomes and has the role of initiating and promoting autophagy [50]. Beclin-1 gene is an important marker to measure autophagy ability, for autophagy functions will be inhibited when heterozygosity loss or abnormal methylation of Beclin-1 gene occurs. The deletion and mutation of Beclin-1 can promote tumor cell proliferation and is also an important factor in the connection between autophagy and tumor cell apoptosis [51].

Apoptosis is gene-regulated programmed cell death and an important way of tumor cell death. The process of apoptosis is regulated by many factors, among which Bcl-2, Bax, and caspase protein families are particularly crucial and perform effectively in the whole process of apoptosis [52, 53]. Caspase 9 regulates upstream apoptotic signals and then activates downstream caspase 3 through cascade reactions [54]. Bcl-2 inhibits apoptosis by inhibiting protein activity of caspase 3, while Bax promotes apoptosis by activating caspase 9. The relationship between autophagy and apoptosis is inseparable, for instance, P62 can effectively activate apoptosis-related protein caspase 8 and promote the co-localization of caspase 8 and LC3 [55]. On the one hand, Beclin-1 inhibits autophagy by binding to apoptosis-related protein Bcl-2 [50]. On the other hand, caspase also can inhibit autophagy by cutting Beclin-1 [56]. In different tumor types and backgrounds, the relationship between the two is also convertible: autophagy can promote apoptosis or inhibit apoptosis, which has an important influence on the survival of tumor cells.

In this experiment, Q-PLNs activated phosphorylation of AMPK, inhibited phosphorylation of mTOR, and activated ULK1 to induce autophagy. Autophagy flux and flow cytometry results demonstrated that Q-PLNs induced autophagy and promoted apoptosis of breast cancer MCF-7 cells. Western blot results showed that Q-PLNs significantly activated activity of AMPK/mTOR/ULK1 signaling pathway, promoted the expression of autophagy-related proteins Beclin-1 and LC3-II, and increased the proportion of pro-apoptotic proteins (p < 0.05) compared with control group and inhibitor CQ group. Studies have clarified that QUE has inhibitory effects on various tumors by affecting the cell cycle, inducing apoptosis, regulating autophagy, and inhibiting angiogenesis [57]. QUE also can inhibit the AKT/mTOR signal to regulate the metabolism, thereby inhibiting breast cancer growth [58]. In addition, QUE inhibits glycolysis to promote autophagy, thus inhibiting the growth and metastasis of breast cancer [59]. Consequently, our study has deepened the understanding of the mechanism between Q-PLNs-induced autophagy and breast cancer apoptosis, and the mechanism of autophagy on the occurrence and development of breast cancer needs to be further investigated, which is conducive to providing a scientific basis for the design of nano-preparations loaded with natural active ingredients for the breast cancer treatment.

The shape of optimized Q-PLNs was spheroid, and the encapsulation rate was 77.31%. The particle size of the Q-PLNs was 109.3 nm and PDI was 0.216 < 0.3, indicating that they have uniform particle size and good dispersion. It has been reported that nano preparations with particle size of about 100 nm can better avoid rapid elimination in vivo [60]. Hence, Q-PLNs can effectively prolong systemic circulation time, and improve the solubility, stability, and pharmacological activities of QUE. Q-PLNs were released in a first-order model, and the cumulative release amount reached 92.67% within 72 h, showing an obvious slow-release characteristic. Cellular uptake and cytotoxicity experiments revealed that Q-PLNs had good biocompatibility and the nanomaterials were safe. At the same time, Q-PLNs can promote cellular uptake and improve the pharmacological activities of QUE. Confocal microscopy and flow cytometry analysis proved that Q-PLNs exhibited significant effects on anti-growth by inducing autophagy and promoting apoptosis of breast cancer. Western blot experiment further demonstrated that Q-PLNs promoted autophagy initiation by activating AMPK/mTOR/ULK1 pathway, however, CQ inhibited the effects of Q-PLNs on pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2, suggesting that the pro-apoptotic effect of Q-PLNs was related to the promotion of autophagy triggered by AMPK/mTOR/ULK1 pathway.

QUE	Quercetin
TRAIL	TNF-related apoptosis-inducing ligand
AMPK	Adenylate activated protein kinase
mTOR	Mammalian Target of Rapamycin
ULK1	UNC-51-like kinase 1
NDDS	Nano drug delivery system
PLGA	Poly-lactide-co-glycolide
PC-98T	Egg yolk lecithin
DSPE-mPEG₂₀₀₀	Distearoyl phosphoethanolamine-PEG₂₀₀₀
FBS	Fetal Bovine Serum
HPLC	High performance liquid chromatography
EE	Encapsulation efficiency
PDI	Polymer dispersity index
TGA	Thermogravimetric analysis
AA	Antioxidant activity percentage
CQ	Chloroquine

Consent for publication

Not applicable.

Competing interests

The authors have no conflicts of interest regarding the publication of this paper.

Ethical Approval

Not applicable.

Authors' contributions

Meng Lan performed the construction and mechanism research of Q-PLNs formulation components, and made substantial contributions to acquisition, analysis, and interpretation of data, and was major contributors in writing the manuscript; Fansu Meng was responsible for the evaluation of Q-PLNs quality; Qi Li undertook the establishment of cell models; Mujuan Pang, Fengjie Liu and Zhaodi Kong screened formulation components of Q-PLNs; Tiange Cai, Zhenjiang Yang and Yu Cai were involved in drafting the manuscript and revising it critically for intellectual content. All authors read and approved the final manuscript.

Funding

This study was financially supported by the projects of the Guangdong Key Laboratory of Traditional Chinese Medicine Information Technology.

Acknowledgements

Not applicable.

Availability of data and materials

The data that supports the findings of this study are available in the supplementary material of this article.

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Table 1 and 2 are available in the Supplementary Files section.

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Synthesis of polymer lipid nanoparticles loaded with Quercetin: Potential for Activating Autophagy to Promote Apoptosis of Breast Cancer Cells

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 material

2.2 Preparation and optimization of Q-PLNs

2.3 Encapsulation efficiency (EE) of Q-PLNs

2.4 Characterization of Q-PLNs

2.5 Release of Q-PLNs in vitro

2.6 Stability investigation of Q-PLNs

2.6.1 Temperature stability

2.6.2 Illumination stability

2.7 Antioxidant activity of Q-PLNs

2.8 Cell Culture

2.8.1 Cellular uptake

2.8.2 Cytotoxicity

2.8.3 Autophagy flux

2.8.4 Apoptosis

2.8.5 Western blot

2.9 Statistical analysis

3. Results And Discussion

3.1 Characterization of Q-PLNs

3.2 Cumulative release rate of Q-PLNs in vitro

3.3 Stability of Q-PLNs

3.3.1 Temperature stability

3.3.2 Illumination stability

3.4 Antioxidant activity of Q-PLNs

3.5 Qualitative and quantitative cellular uptake

3.6 Cytotoxicity

3.7 Autophagy flux

3.8 Autophagy and apoptosis

3.9 The expression of autophagy-related proteins

4. Discussion

5. Conclusion

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