Targeted Nanostructured Lipid Carrier Containing Galangin as a Promising Adjuvant for Improving Anticancer Effects of Chemotherapeutic Agents

Purpose Resistance to chemotherapeutic drugs is the main limitation of cancer therapy. The combination use of anticancer agents and Galangin (a naturally active avonoid) amplies the effectiveness of cancer treatment. This study aimed to prepare Arginyl-glycyl-aspartic acid (RGD) containing nanostructured lipid carrier (NLC-RGD) to improve the bioavailability of Galangin and explore its ability in improving the anticancer effects of doxorubicin (DOX). Galangin with appropriate characteristics of the delivery system (size: 120 PDI: morphology, and were prepared. Uptake experiments revealed that NLC-RGD promotes the accumulation of Galangin into cancerous cells by integrin-mediated endocytosis. Results also showed higher cytotoxicity and apoptotic effects of DOX + Galangin loaded-NLC-RGD in comparison to DOX + Galangin. Gene expression analysis demonstrated that Galangin loaded-NLC-RGD downregulates ABCB1, ABCC1, and ABCC2 more eciently than Galangin. ndings that of NLC-RGD makes it an effective adjuvant to increase the ecacy of chemotherapeutic agents in cancer treatment.


Introduction
Despite the persistent advancement of anti-cancer therapy, as well as growing understanding of the Therefore, downregulation or inhibition of ABC transporters may enhance the effects of chemotherapeutic agents and made them more e cient in low doses, results in a reduction of chemotherapy side effects. Accumulating evidence shows the positive role of avonoids in enhancing the e cacy and toxicity of chemotherapy drugs by interaction with the ABC transporters (Li and Paxton, 2013). Galangin (3, 5, 7-trihydroxy avone) is an Alpinia galangal root extracted avonoid whose apoptotic effects have been con rmed in different types of cancer (Wen et al., 2020). It is reported that combination use of anticancer drugs with Galangin (GA) ampli es the success of disease management.
For example, combination of GA with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizes lung cancer cells to TRAIL treatment (Han et al., 2016). Furthermore, it has shown that the presence of GA potentiated the apoptotic effect of Cisplatin against the lung cancer cells . Despite GA's bene ts in cancer therapy, its e cacy has been restricted by low water solubility, lack of oxidative stability, insu cient bioavailability, poor intestinal absorption, and rst-pass metabolism (Zhu et al., 2018). Therefore, employing an e cient delivery system may overcome GA's limitations and can broaden its clinical application. Nanoparticle-based drug delivery systems protect the encapsulated drugs from demolition and deliver their cargos to the cancerous cells either via ligands-mediated active targeting or via passive targeting through enhanced permeability and retention (EPR) mechanism (Raj et al., 2019). Recently, Arginyl-glycyl-aspartic acid (RGD) containing nanostructured lipid carrier (NLC-RGD) has been introduced as a suitable drug carrier due to their high loading capacity, improved stability, easy preparation, low toxicity, and targeted delivery (Hajipour et  RGD sequence is recognized by different types of integrins (especially avb3 integrin) and facilitates the uptake of RGD-containing nanoparticles by integrin-mediated endocytosis (Cao et al., 2015, Liu et al., 2017. This investigation aimed to provide GA loaded-NLC-RGD to overcome the therapeutic limitation of GA and evaluate its potential to improve the effects of doxorubicin on the human lung cancer cell line.
Moreover, the effects of GA on gene expression of some ABC transporter were studied to discover the probable mechanisms by which GA overcome the drug resistance.

Preparing GA loaded-NLC-RGD
For production of GA loaded-NLC-RGD, hot homogenizer method along with ultra-sonication was used. In this regard, primarily, DSPE-PEG (2000) Amine and RGD were dissolved in DMSO and stirred for 24 h. Next, the mixture was dialyzed against Milli-Q water (membrane tubing, molecular weight cut-off 1000 Da) for 2 days. Then, the nal solution was lyophilized to obtain RGD-PEG-DSPE product. In the next step, GA was dissolved in 1 ml ethanol, added into the liquid phase (Miglyol and precirol® ATO 5) at 70°C. The aqueous phase was prepared by dissolving RGD-PEG-DSPE and poloxamer 407 (as surfactant) in water and was added dropwise to the lipid phase under high speed homogenizing at 20,000 rpm speed (Silent Crusher M, Heidolph, Germany) for 18 min. then, the nanoemulsion was sonicated with the frequency of 2 kHz (10 % power, 1 min sonication, and 1 min rest). At last, the oil/water nanoemulsion cooled down at 25°C to allow recrystallization and NLC-RGD production. The products were coded as the A1-A7 with the different compositions of lipids. Samples were diluted with distilled water (1:10) and were assessed using a photon correlation spectroscopy (PCS) (Nano ZS, Malvern Instruments, UK) to determine the particle size and zeta potential by measuring the intensity variations overtime of a laser beam (633 nm).
The morphology of the GA loaded-NLC-RGD particles was revealed via scanning electron microscopy (SEM) (SEM-TESCAN MIRA3-FEG). Samples were dropped on a copper grid and then sputtered with a gold coating before imaging.

Loading capacity and physical stability
To evaluate the encapsulation e ciency (EE) and loading capacity (LC), the UV-visible spectrophotometer (UV160-shimadzo -Japan) was utilized. For this purpose, the calibration curve of GA was plotted according to the concentrations of 5-50 µg/ml. In the next step, the unloaded GA was separated from GA loaded-NLC-RGD by using the Amicon lter (molecular weight cutoff: 30 kDa, Millipore, UK), and optical density of unloaded-GA was measured by UV-visible spectrophotometer at λ max 267 nm. Finally, the concentration of unloaded-GA was calculated based on the calibration curve formula. Following equations were used to calculate the EE and LC.
To evaluate the stability of prepared nanoparticles, GA loaded-NLC-RGD was stored for 2 months at 4-8°C. After this period the particle size and percentage of released GA were assessed.

Cell viability assay
Human A549 cells were seeded in 96-well plates (10 4 cells/well) and cultivated in RPMI 1640 medium containing 10 % FBS at 37°C in 5 % CO 2 overnight. After the attachment of cells to the plate, they were treated with GA, DOX, and combination of DOX and GA in the free form and loaded in either NLC or NLC-RGD. After 48 h, the cell media were exchanged with 150-µL fresh media including 50 µL of MTT solution (3 mg/mL), and incubated for 4 h at cell culture incubator. Finally, to solve the blue formazan crystals, 175 µL of DMSO and 25 µL Sorensen's glycine buffer were added to each well and optical density (OD) values were recorded at 570 nm using a microplate ELISA reader (ELX 800, Biotek, USA).

Cellular uptake
To study the cellular uptake, uorescein was used for labeling the developed nanoparticles. For this purpose, 0.01% w/w uorescein regarding the weight of the lipids was added to the formulation.
Afterward, the unloaded uorescein was removed by the Amicon® lter (molecular weight cutoff 30 kDa, Millipore, UK) using centrifuging at 4000 rpm for 20 min and washed several times with PBS. A549 cells (3×10 5 cells/mL) were seeded and incubated for 24 h in six-well plate to reach 90% con uence. In the next step, the cells were treated with uorescein containing NLC with and without RGD for 4 h. Finally, the cells were washed twice with PBS and trypsinized to explore the uorescein uptake using a FACS Calibur ow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) and uorescence microscope (Olympus microscope Bh2-RFCA, Japan). To avoid uorescence dequenching of uorescein, these experiments were performed in dark.

Assessment of apoptosis percentage by ow cytometry
Fluorescein isothiocyanate (FITC) labeled annexin V assay was used to determine apoptosis percentage of cells when treated with GA and GA loaded-nanoparticles. For this purpose, A5459 cells were treated with 1 µM DOX, 1 µM DOX + 61 µM GA or equivalent doses of GA loaded nanoparticles. After 24 h, cells were trypsinized, washed, and were incubated with 200 µl of binding buffer containing 5 µl FITC-labeled Annexin V in a dark room for 15 min. Then, 500 µl binding buffer was used to wash the cells. Next, cells were incubated with 200 µl binding buffer containing 5µl propidium iodide (PI) for 5 min at room temperature. Finally, a FACS Calibur ow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) was used to assess the apoptosis and necrosis rate.

Analysis of apoptotic nuclei by DAPI staining
To analyze, the effects of GA and GA loaded-NLC-RGD on DNA fragmentation and nuclear condensation, the nucleus of A549 cells was stained with DAPI uorescence dye. In the rst step, cells were seeded on coverslip at the density of 4 × 10 5 per well, then treated with 1 µM DOX + 61 µM GA or equivalent doses of GA loaded nanoparticles at 37°C. After 24 h, cells were washed with PBS and xed using paraformaldehyde (4%) for 20 min at room temperature. Next, the cells permeabilized using 0.1% (w/v) Triton X-100 for 15 min and stained with DAPI solution (Sigma, USA) for 40 min in a dark room. Finally, the stained cells were washed twice with cold PBS and the apoptotic morphological alteration of cells was captured using a uorescence microscope (Bh2-RFCA, Olympus, Japan).  Table 1. The Pfa method was used to normalize the expression of target genes regarding housekeeping gene (GAPDH) expression.

Statistical analysis
All of the data were described as the mean ± standard deviation. Statistical analyses were completed using student t-test and ANOVA analyses of variance. Less than 0.05 p-value was considered as signi cant.

Preparation and characterization of GA loaded-NLC-RGD
The several formulations (Table 2) of GA loaded-NLC-RGD were assessed to reach the optimum characteristic. Based on the size, polydispersity index (PDI), and loading capacity, optimum formulation contained 120 mg Precirol as a central substance of nanoparticles, 15 mg Miglyol as a stabilizer, and 75 mg Poloxamer as a surfactant. As shown in Fig. 1a, mean size of the optimum formulation was 119.4 nm with 0.23 PDI. The result of zeta potential analysis showed that the surface charge of prepared nanoparticles at the original pH 7.4 is -15 mV (Fig. 1b). Scanning electron microscope (SEM) imaging con rmed that prepared nanoparticles have a spherical morphology and nano-sized scale (Fig. 1c). The original data are presented as supplementary le 1, 2 and 3.

Drug loading and physical stability
Encapsulation e ciency and loading capacity for optimum formulation were calculated 83.1 ± 4.3 % and 59.3 ± 3.0 mg/g respectively. During the 2 months storage at 4-8°C, we observed any signi cant change in clarity and phase separation of the optimum formulation. Furthermore, the size (127.1 nm) and PDI (0.25) of stored nanoparticles were not signi cantly different from freshly prepared nanoparticles (Fig. 1d). During this period, less than 10 % of GA were released from NLC-RGD (supplementary le 4).

Cellular uptake of nanoparticles
Uptake of prepared nanoparticles into cancerous cells was investigated based on the uorescence intensity of Fluorescein dye using ow cytometry (Fig. 3a) and uorescent microscope imaging (Fig. 3b).
As shown in Fig. 3a, cellular uptake of GA loaded-NLC-RGD is signi cantly higher than those of GA loaded-NLC, suggesting that RGD facilitates nanoparticle absorption into cells. Fluorescence microscopy imaging con rmed the obtained results from ow cytometry and showed that the uorescence intensity of cells treated with GA loaded-NLC-RGD was more than those of cells treated with GA loaded-NLC (Fig. 3b). Original data are presented as supplementary le 6.

Cell apoptosis
Annexin V/PI staining was performed to assess the effects of Galnagin loaded-NLC-RGD on DOX-induced apoptosis in A549 cells. Flow cytometry graphs (Fig. 4a) showed that co-treatment of A549 cells with DOX and GA enhances the apoptosis compared to DOX alone (37.77 % vs 30.0%). Results also con rmed that adjuvant effects of GA loaded-NL-RGD (49.58 %) were higher than GA loaded-NLC (37.36%) and free form of GA (37.77%). The obtained results showed that delivery with NLC-RGD is more capable compared to NLC (supplementary le 7).

DAPI Staining
Nucleus condensation and chromatin fragmentation of cells as apoptosis signs were compared between cells treated with DOX, DOX + GA, DOX + GA loaded-NLC, and GA loaded-NLC-RGD. DAPI staining con rmed the results of apoptosis experiments and showed that at an equal time and concentration, DOX + GA loaded-NLC-RGD induce DNA degradation more remarkably than DOX + GA and DOX + GA loaded-NLC (Fig. 4b).

Expression of ABC transporter
To investigate the GA effects on the expression of the ABC transporter family, the mRNA level of important members of the ABC transporter family were assessed in GA treated lung cancer cells. Within each sample, the mRNA level of each gene was normalized to GAPDH mRNA level, as a housekeeping gene. As shown in Fig. 5 (a, b, c), treatment of A549 lung cancer cells with GA or GA loaded-NLC-RGD, downregulate the expression of ABCB1, ABCC1, and ABCC2 in comparison with the untreated group (control). However, we didn't nd any alteration in the expression of ABCG2, when cells were treated either with the GA or GA loaded-NLC-RGD (Fig. 5d). These results also demonstrated that the effect of GA loaded-NLC-RGD on downregulation of ABCB1, ABCC1, and ABCC2 is more than GA (supplementary le 8).

Discussion
The present study con rmed that the GA loaded-NLC-RGD could signi cantly improve the anticancer potential of DOX. Previously synergistic effects of Gallatin and the chemotherapeutic agents have been . It is also reported that neutral or faintly negative ZP is more compatible to interact with the cell membrane (Lane et al., 2015). The Zeta potential of prepared nanoparticles is high enough to provide acceptable stability, and also is not too negative to prevent nanoparticles and cell membrane interaction.
To develop targeted nanoparticles, RGD was added to the formulation that can deliver the drug directly to a cancer cell by binding to integrins. Given to overexpression of αvβ3 and αvβ6 integrin on the surface of the A549 lung cancer cells ( (Gwak et al., 2011), and inhibition of multidrug resistance (MDR) (Lorendeau et al., 2014). In this regard, Yu et al.  showed that GA decreases the Cisplatin resistance of human lung cancer cells and potentiates apoptosis via Bcl-2 suppression. Inactivating Akt and promoting the Caspase-3 pathway are other mechanisms by which GA inhibits retinoblastoma cell proliferation (Zou and Xu, 2018 There is no logical interpret for this controversial report. Our experiments also revealed that the effects of GA on the expression of ABC transporter were strengthened when used in the form of GA loaded-NLC-RGD, con rming the appropriateness of prepared nanoparticles. Results showed that improved targeting of GA makes the DOX more effective for apoptosis induction. Therefore, low doses of DOX can be more effective for cancer inhibition, which in turn decreases the side effects of DOX.

Conclusion
NLC-RGD is a suitable drug delivery system to convey GA into cancerous cells due to appropriate delivery characteristics and specialized RGD mediated uptake mechanism. The potential of GA in downregulation of ABC transporter leads to an increase of anticancer effects of chemotherapeutic drugs such as DOX. Therefore, overcoming the therapeutic limitation of GA by NLC-RGD as a nanoparticle delivery system makes it an effective adjuvant to enhance the e cacy of chemotherapeutic agents in cancer therapy.