MicroRNA-21 inhibitor (miR-21i) and Simvastatin-loaded poly (D,L-lactide-co-glycolide)/polyethylenimine (PLGA/PEI) nanoparticles for synergistic anticancer effect in Gastric Cancers

In this study, we have successfully developed a simvastatin (SMV) and miR-21i-loaded poly (D,L-lactide-co-glycolide)/polyethylenimine (PLGA/PEI) nanoparticles (NP) to enhance the therapeutic ecacy in gastric cancers. The nanoparticles were characterized for in vitro physicochemical and biological assays and pharmacokinetic study was performed in rats. CLSM/FACS results clearly showed the ability of SMV/miR-21i-loaded PLGA/PEI NP (PPN-S21i) to deliver the combinational therapeutics of SMV and miR-21i to the cancer cells. Combination of SMV+miR-21i showed signicantly lower cell viability compared to that of free SMV. Our results clearly highlight the importance of simultaneous interaction of SMV+miR-21i and that it could signicantly decrease the cell proliferation in BGC-823, SGC-7901 and HGC-27 gastric cancer cells while it was signicantly less cytotoxic to normal gastric mucosa cells (GES-1). Cell apoptosis of SMV+miR-21i was signicantly higher compared to that of individual drug or miRNA. Finally, pharmacokinetic analysis revealed that PPN-S21i signicantly prolonged the blood circulation time of SMV compared to that of free SMV indicating the potential of carrier system. Overall, results clearly indicate that the combination of SMV+miR-21i (gene + drug therapy) might provide a valuable strategy for the clinical management of gastric cancers.


Introduction
Gastric cancer is one of the most lethal cancers with high rate of mortality across the world. The severity of gastric cancer could be estimated from that fact that completes surgical resection of this tumor remains the primary curative option. However, 5-year overall survival is limited in patients undergoing surgery alone (Haq et al., 2012;Siegel et al., 2013). According to an estimation, ~ 40,000 Americans are diagnosed every year with this cancer and equal amount of people are dying each year with gastric cancers making it fourth largest cancer-related death overall (Pennathur et al., 2013). The delay in diagnosis, early reoccurrence, local relapse and distant metastasis are the common cause of the high death rate. Number of treatment strategies have been tried to improve the chemotherapy e cacy including multiple genetic, transcriptional and microenvironmental changes, however, no improved in survival rate of gastric patient was observed (Orditura et al., 2014;Sener et al., 1999). Besides, most patients have no chance of surgical resection in advance stage or metastasis cancers. This kind of grim scenario has highlighted the importance of new therapeutic approaches (Bailey et al., 2008). Therefore, development of effective therapeutic approaches for the effective management of gastric cancer is required.
In this regard, microRNA (miRNA) has garnered increasing attention in the cancer research and treatment of gastric cancers (Passetti et al., 2009). The miRNAs are group of small gene regulating RNAs consisting of 18-24 nucleotides and controls the overexpression in multiple malignancies including gastric cancers (Ramasamy et al., 2019). The miRNA function by base-pairing with the complementary sequences with the messenger mRNAs and inhibit the protein synthesis. The miRNAs regulate the proliferation, progression, metastasis of cancer cells and thereby controls its apoptosis and cell survival (Blower et al., 2008). The inhibition of overexpressed oncogenic miRNAs or restitution of under-expressed miRNAs could result in the improvement of therapeutic e cacy. Four of the overexpressed miRNAs include miR-155, miR-21, miR-221 and miR-222 and among all, miR-21 is reported to have the highest overexpression in gastric cancers and plays a central role in the cancer cell proliferation and metastasis (Feng et al., 2017). The molecular pathway of miR-21 includes the change in the apoptosis signaling, phosphorylation of AKT pathway and increase in the gene involved in the cell invasion. For example, overexpression of miR-21 downregulates the tumor suppressors phosphatase and tensin homologue (PTEN) and phosphorylates the AKT kinase making it less susceptible to drugs like Gemcitabine. We expect that when miR-21 is inhibited by miRNA-21 inhibitor (miR-21i), cancer cell proliferation will be inhibited (Li et al., 2015). The inhibition of miR-21 will also increase the sensitivity of chemotherapeutic drug and thereby increased apoptosis and cancer cell death could be observed (Hong et al., 2013). However, single agent therapy based on miRNA alone would be ineffective for the higher therapeutic effect.
Simvastatin (SMV) -coenzyme A reductase inhibitors has been indicated in the modulation of in ammation (Cho et al., 2008). Recently several statins have been reported to have cytotoxic effects in cancer cells including colon, ovarian, squamous cell carcinoma, and pancreatic cells like BxPc-3 and MiaPaCa-2 cells (Stine et al., 2016). However, it has also been reported that statins can elevate the risk of cancer incidence. The controversial ndings might be attributed to the type of statin, lipophilic and hydrophilic statins used. The lipophilic statin (SMV) has been known to produce the anticancer effect by inhibiting the cell proliferation and promoting the cell apoptosis (Chang et al., 2011). In one instance, it has been reported that SMV induces the anticancer effect by the inhibition of nuclear factor-kB signal pathway. It must be noted that SMV is a highly hydrophobic drug with high rst pass metabolism and low systemic availability and low aqueous solubility (Rosenson, 2004;Muzykantov, 2013). Similarly, naked miRNA are subjected to enzymatic degradation in the systemic circulation and leads to instability following the systemic administration, necessitating the need for a stable nanocarrier system. The implementation of nanomedicine has done wonders in improving the therapeutic e cacy of encapsulated components . In this study, we have utilized poly (D,L-lactide-coglycolide) (PLGA)-based polymeric nanoparticle system to enhance the delivery and therapeutic e cacy of SMV and miR-21i. PLGA is a US-FDA approved biodegradable and biocompatible polymers that are wide accepted in the clinical applications (Danhier et al., 2012). Speci cally, we have introduced Polyethylenimine (PEI) in order to encapsulate the miRNA on the surface of nanoparticles (Langer, Tirrell, 2004).
Overall, main aim of study was to enhance the delivery and therapeutic e cacy of SMV and miR-21i (combination agents) in gastric cancers using PLGA/PEI nanoparticles. The in vitro anticancer effect of single and combinational drugs was tested in BGC-823 cells (cell viability assay, apoptosis assay, and live/dead assay). The pharmacokinetic performance of nanoparticle was studied in SD rats.

Materials And Methods
Preparation of SMV and miR-21i-loaded PLGA/PEI nanoparticles The poly (D,L-lactide-co-glycolide) (PLGA, 50:50) and Polyethylenimine (PEI) were purchased from Sigma-Aldrich, China. The PLGA (30 mg) and PEI (0.1 mg) was dissolved in 1ml of chloroform. The simvastatin (SMV, 10% w/w) was dissolved in the organic phase and stirred for 15 min. Followed by a mixture of bovine serum albumin (BSA, 5 mg) and miRNA-21i/miRNA21i-FAM' (Shanghai GenePharma Co., Ltd., Shanghai, China) (300 µg) in 200 µl of EDTA buffer was added to the organic phase slowly and immediately vortexed for 2 min. The mixture was probe-sonicated for 6 min (60% Amplitude) in an icebath resulting in the formation of water-in-oil (W/O) emulsions. The so-formed W/O emulsion was added to 5 ml of 2.5% polyvinyl alcohol solution and again sonicated for 4 min in the ice-bath. The emulsion was stirred for 15h at room temperature to allow evaporating all of the organic solvents. The SMV/miRNA-loaded nanoparticles were collected by ultracentrifugation at 12000 rpm for 10 min using a sophisticated centrifuge. The nanoparticles were washed twice and stored at 4°C until further use.

Gel retardation analysis
Gel retardation assay was performed in 2% Agarose gel pre-stained with 0.5 mg/ml of ethidium bromide. Brie y, 2% Agarose gel was prepared in TRIS-acetate EDTA buffer and condensation ability of different formulations in N/P ratio was evaluated. The experiment was carried out at 80 V for 30 min and the retardation at different N/P ratio was analyzed by gel image analysis system.

Nanoparticle characterization
The hydrodynamic particle size and zeta potential of nanoparticles were evaluated by laser particle analyzer (Fritsch ANALYSETTE 22, Germany). The experiments were performed at 25°C in triplicate numbers. The morphology of particles was evaluated by transmission electron microscope (TEM) using PHILIPS TECHNAL-10 (Holland). The TEM was carried out at 100 kV. The diluted samples were placed in a copper grid containing 300-mesh and excess water was removed and in turn stained with 2% uranyl acetate solution.
In vitro SMV/miRNA release study The release study was performed by dialysis method. To evaluate the release of miRNA, miR-21i-FAM was used. The nanoparticle dispersion was mixed with 1 ml of release medium (pH 7.4, phosphate buffered saline) and packed in a dialysis membrane (MWCO 3000 Da) which is in turn placed in a rotary shaker (100 rpm) at 37°C. At xed time interval, 1 ml of release medium was collected and replaced with equal amount of fresh buffer. The release of SMV was determined by HPLC method and miR-21i-FAM was evaluated by measuring the uorescence intensity of FAM (λex 488 nm and λem 520 nm) using a microplate reader (Tecan, Durham, USA). WATERS (E2695) HPLC was used with a C18 column. The mobile phase consisted of acetonitrile/water (20:80) at a constant ow rate of 1.2 ml/min at 242 nm.

Cellular uptake analysis of PPN-S21i
The cellular uptake analysis was performed by confocal laser scanning microscopy (CLSM) and ow cytometer. BGC-823 cells (ATCC, USA) were cultured in RPMI-1640 media supplemented with 10% FBS.
2×10 5 cells were seeded in each well of 12-well plate and kept aside for 24h. The cells were treated with fresh medium containing the PPN-S21i nanoparticle containing rhodamine-B as a uorescent tracker. The nanoparticles were incubated for 3h and then washed twice with PBS and then stained with Lysotracker Green for 10 min. The cells were again washed and xed with 4% paraformaldehyde. The cells were observed under Leica Microsystems, Mannheim, Germany. The cellular uptake was further studied by ow cytometer (BD Biosciences, San Diego, CA). The cells were treated in the same manner as mentioned above and then collected by scrapping and 10,000 events were recorded.

Cell viability assay
The cell viability assay was performed by cell counter kit-8 (CCK8) assay. To begin this assay, 8×10 3 BGC-823 cells (ATCC, USA) were seeded in each well of 96 well plates and incubated for 24h in 100 µl volume. The cells were then treated with a SMV, SMV+miR-21i and PPN-S20i nanoparticles in a concentration range from 1-100 µg/ml. For all concentration of SMV, miR-21i was xed at 50 µg/ml. The cells were incubated for 24h. The cells were washed carefully and 10 µl of CCK-8 solution was added to each well of 96 well-plate and incubated for 1h at 37°C. The respective absorbance was measured using a microplate reader at 460 nm using BioTek, USA.
Flow cytometer-based apoptosis assay The apoptosis assay was performed by Annexin-V/PI staining using ow cytometer. To begin this assay, 2×10 5 BGC-823 cells were seeded in each well of 12 well plates and incubated for 24h. The cells were then treated with a free SMV, free miR-21i, SMV+miR-21i and PPN-S20i nanoparticles in a xed concentration. The cells were incubated for 24h and then cells were collected by trypsinization process and pellet was collected by centrifugation process. The cells were resuspended in 200 µl of binding buffer and stained with 3 µl of Annexin-V and PI solution and incubated for 15 min in dark conditions. The cell apoptosis was evaluated by recording 10,000 events in ow cytometer.
Live/Dead assay The anticancer effect was further studied by Live/Dead assay. To begin this assay, 2×10 5 cells were seeded in each well of 12 well plates and incubated for 24h. The cells were then treated with a free SMV, free miR-21i, SMV+miR-21i and PPN-S20i nanoparticles in a xed concentration. The cells were incubated for 24h. The cells were washed and stained with Acridine Orange (AO, 5 µg/ml) and PI (2.5 µg/ml) and incubated for 15 min and then images were captured using a uorescence microscope (Olympus, USA).

Pharmacokinetic analysis in animal
The Sprague-Dawley (SD) rats were obtained from Animal Facility Center of China-Japan Union Hospital of Jilin University, China. The animal study was approved by Institutional Animal Ethics Committee of China-Japan Union Hospital of Jilin University, Changchun. The experimental protocol for animals was approved by China-Japan Union Hospital of Jilin University Ethical guidelines for Small Animals. The SD rats were given free access to food and water and maintained under ambient conditions with 12h dark/light cycle. The rats were administered with a xed SMV dose of 7.5 mg/kg of SD rats. The SD rats were divided into two groups with six rats in each group and formulations were administered by tail vein injection. Blood samples were collected in a periodical manner from 0.25-24h. The plasma was separated from whole blood and stored in -80°C until further analysis. At the end of study period, mice were sacri ced with the exposure to CO 2 .

Statistical analysis
Results are expressed as mean ± standard deviation (SD). Comparison between two groups was performed using Student's t-test. P<0.05 was considered statistically signi cant.

Formulation of PPN-S21i nanoparticles
The preparation of SMV and miR-21i-loaded PLGA/PEI nanoparticles (PPN-S21i) is depicted in the schematic presentation (Figure 1). The optimized PPN-S21i particles have an average size of 131.5±1.68 nm with an effective surface charge of 19.5±1.77 mV. The miRNA binding ability with nanoparticle was assessed by gel retardation assay in 2% Agarose gel at different N/P ratio of miRNA and PEI of PPN-S21i nanoparticles ( Figure 2a). As shown, naked miRNA was found at the opposite electrode while retardation of miRNA was gradually increased with the increase in the N/P ratio with no bright miRNA bands were observed at the bottom. A complete retardation of miRNA movement or 100% binding e ciency was observed at N/P ratio of 4.
Characterization of PPN-S21i nanoparticles PPN-S21i nanoparticles exhibited an entrapment e ciency of 94.5±1.12 % with an active loading e ciency of 8.79% w/w. TEM images revealed a perfect spherical shaped particle spread uniformly on the 300-mesh copper grid (Figure 2b). A dark core with faint surface reveals the surface characteristics of the prepared nanoparticles. The drug release study was performed by dialysis method. Both SMV and miR-21i showed a sustained and controlled release pro le on pH 7.4 buffer system. However, small molecule and nucleic acid released in a different pattern, for example, approximately ~45% of SMV released after 24h compared to ~25% of miRNA release during the same time period. After 60h incubation, ~90% of SMV released and ~45% of miRNA released from PPN-S21i nanoparticles ( Figure  2c). The release pro le de nitely points to the fact that nanoparticles allow a controlled released of encapsulated therapeutics. The results clearly advocate the relative stability of nanoparticle system in the systemic circulation and avoid the unnecessary release of therapeutic load and release the encapsulated drug in the tumor tissues.

Cellular internalization in BGC-823 cells
The cellular uptake of PPN-S21i nanoparticles in BGC-823 cell was rst evaluated by confocal laser scanning microscopy (CLSM) (Figure 3a). As shown, a dense red uorescence was observed in lysosome region of the cancer cells indicating a typical endocytosis-mediated cellular uptake. Merged image clearly showed a perfect merging of lysosome-stained green uorescence and red uorescence originated from the PPN-S21i nanoparticles. The uptake was further ascertained by ow cytometer analysis (Figure 3b). FACS analysis showed a typical shift in the histogram towards the right side indicating a de nitive cellular uptake. As shown, FACS analysis depicted a typical time-based cellular uptake with increase in the internalization of nanoparticles with the increase in the incubation time.

Effect of PPN-S21i nanoparticles on cell proliferation
We have employed three groups to study the effect of single and combinational regiment on the cell viability of three different cancer cells, BGC-823, SGC-7901 and HGC-27 cells (Figure 4a; Figure S1). The three groups include free SMV, SMV+miR-21i, and PPN-S21i. Separately, miR-21i exhibited a concentration-dependent cell killing effect (data not shown). A xed concentration of 50 ng/ml of miR-21i was used for all the combination dose. As shown, cells treated with combination of SMV+miR-21i showed signi cantly lower cell viability compared to that of free SMV. In general, all the formulations exhibited a concentration-dependent cytotoxic effect in the BGC-823 cancer cells. Similarly, PPN-S21i showed remarkable antitumor e cacy in SGC-7901 and HGC-27 cells. We have evaluated the effect of individual formulations on normal gastric mucosal cell line, GES-1 cells. Although higher concentrations of free SMV, SMV+miR-21i, and PPN-S21i decreased the cell viability of GES-1 cells, however, it was signi cantly (p<0.05) less compared to that in cancer cells such as BGC-823 cells.

Effect of PPN-S21i nanoparticles on cell apoptosis -ow cytometer
The combination effect of SMV+miR-21i on BGC-823, SGC-7901 and HGC-27 cells was studied by ow cytometer after staining with Annexin V/PI markers. Annexin V estimates the early apoptosis cells while PI shows positive sign for dead cells or late apoptosis cells. No obvious apoptosis was observed in nontreated cells (Figure 5a; Figure S2). The transfection of miR-21i resulted in ~18% of cell apoptosis while SMV resulted around ~23% apoptosis of cancer cells. As expected, cell apoptosis of SMV+miR-21i was signi cantly higher compared to that of individual drug or miRNA. Most importantly, 3-fold increase in the cell apoptosis was observed for PPN-S21i treated cancer cells compared to individual formulations.

Live/Dead assay
The anticancer effect of SMV+miR-21i, and PPN-S21i was further con rmed by Live/Dead assay. The cells were exposed to respective formulation and incubated for 24h (Figure 5b). The cells were then stained with acridine orange (AO) and propidium iodide (PI) as a respective live cell (green color) and dead cell (red color). As shown, compared to individual SMV or miR-21i, combination of SMV+miR-21i yielded a remarkably higher red uorescence indicating a predominant apoptosis and cell death, simultaneously, green uorescence decreased. The effect was more pronounced in PPN-S21i treated cells which showed brightest red uorescence with lowest green uorescence. The order to red uorescence was control<SMV<miR-21i<SMV+miR-21i<PPN-S21i, respectively. The results were concordant with the cell viability and apoptosis assay.

In vivo pharmacokinetic study
The plasma concentration of SMV against the time pro le has been presented in Figure 6. The pharmacokinetic parameters were obtained after the intravenous administration (tail vein) of free SMV and PPN-S21i in SD rats. The free SMV concentration in plasma immediate started decreasing after intravenous administration and quanti ed until 4h. The pharmacokinetic behavior of SMV was consistent with the pro le of any intravenously administered free drug. On the contrary, PPN-S21i signi cantly prolonged the blood circulation time of SMV indicating the potential of carrier system. The AUC 0-inf of PPN-S21i (3564±369 ng.h/ml) was signi cantly higher compared to that of free SMV (498±114 ng.h/ml). Moreover, the rate of elimination (K el ) was markedly lower (P<0.01) for PPN-S21i (0.05h) followed by a high signi cant increase in T 1/2 (9.15±1.25h, P<0.001) and MRT (P<0.01) values compared to that of free SMV.

Discussion
PLGA/PEI nanoparticles were prepared by double emulsion method. The inclusion of PEI will introduce the positive surface charge on the nanoparticles that will allow the loading of miRNA based on electrostatic interactions. The SMV is loaded in the core of the nanoparticles which is stabilized by polyvinyl alcohol. The presence of PEG will stabilize the particles in the dispersion and systemic circulations. The optimized PPN-S21i particles have an average size of 131.5±1.68 nm with an effective surface charge of 19.5±1.77 mV. A slight decrease in the surface charge was observed after the loading of miRNA owing to the charge compensation. Overall, a small particle size of nanoparticles will allow the preferential accumulation on the malignant tumors using the well-known enhanced permeation and retention (EPR) effect. A moderate positive charge on the particle surface will allow the favorable interaction with the negatively surface cell membrane and higher cellular internalization.
The cellular uptake of PPN-S21i nanoparticles in BGC-823 cell was rst evaluated by confocal laser scanning microscopy (CLSM). The results clearly suggest the ability of PPN-S21i nanoparticles to deliver the combinational therapeutics of SMV and miR-21i in the cancer cells. It could be hypothesized that a positively charged nanoparticles will interact with the negatively charged cell membrane and internalize in the cancer cells. Moreover, a small particle size would be easier to internalize in the cancer cells.
Cells treated with combination of SMV+miR-21i showed signi cantly lower cell viability compared to that of free SMV. Our results clearly highlight the importance of simultaneous interaction of SMV+miR-21i and that it could signi cantly decrease the cell proliferation. Signi cantly higher cell killing effect was observed with PPN-S21i suggesting the role of delivery carrier that could easily internalize the cancer cells and release the encapsulated components in a controlled manner in speci c ratios. A higher cell killing effect of SMV+miR-21i might be attributed to the different mechanism of action of both the therapeutics.
It is possible that miR-21i could effectively inhibit the expression of oncogenic miR-21 receptors and thereby sensitizing the action of SMV. Consistently, Wang et al. have reported that a combination of SMV and miR-21i was effective in inhibiting the cell migration/invasion, cell proliferation and induced cell apoptosis in salivary adenoid cystic carcinoma (SACC) (Wang et al., 2018). Similarly, Li et al. reported that miR-21 was the key factor involved in the migration and invasion of pancreatic cancer cells and authors have showed that the knockdown of miR-2 by transfection miR-21i antisense oligonucleotide (ASO) signi cantly suppressed the migration of cancer cells and signi cantly inhibited the cell proliferation (Li et al., 2017). Overall, PPN-S21i was effective in inducing a signi cantly higher anticancer effect than the free drug itself. Similarly, signi cant difference was observed in cell apoptosis of PPN-S21i treated cells (~70% apoptosis). It has been reported that miR-21i might inhibit the miR-21-based apoptosis pathway by the downregulation of Bcl-2 pathway and upregulation of PTEN, Bax and P53 pathways (Lin et al., 2018). The combination of miR-21i and small molecule drug might act in a synergistic manner for higher apoptosis of gastric cancer cells.
The pharmacokinetic behavior of SMV was consistent with the pro le of any intravenously administered free drug. On the contrary, PPN-S21i signi cantly prolonged the blood circulation time of SMV indicating the potential of carrier system. The enhanced pharmacokinetic pro le of PPN-S21i was attributed to the prolonged blood circulation property of nanoparticulate system and controlled release of encapsulated drug. The presence of PEG (steric hindrance) on the surface further increased the blood circulation properties of the PPN-S21i by reducing its uptake by the reticuloendothelial system (RES). The prolonged circulation of PPN-S21i allows the greater accumulation potential in the malignant tumors (EPR effect) and enhances the therapeutic e cacy in gastric cancers Ruttala et al., 2019).
In summary, we have successfully developed a simvastatin (SMV) and miR-21i-loaded PLGA/PEI nanoparticles to enhance the therapeutic e cacy in gastric cancers. The SMV was loaded with high loading e ciency and miRNA was maximally loaded at an N/P ratio of 4. CLSM/FACS results clearly showed the ability of PPN-S21i nanoparticles to deliver the combinational therapeutics of SMV and miR-21i to the cancer cells. Cells treated with combination of SMV+miR-21i showed signi cantly lower cell viability compared to that of free SMV. Our results clearly highlight the importance of simultaneous interaction of SMV+miR-21i and that it could signi cantly decrease the cell proliferation. As expected, cell apoptosis of SMV+miR-21i was signi cantly higher compared to that of individual drug or miRNA. The anticancer effect was more pronounced in PPN-S21i treated cells which showed brightest red uorescence with lowest green uorescence in Live/Dead assay. Finally, pharmacokinetic analysis revealed that PPN-S21i signi cantly prolonged the blood circulation time of SMV compared to that of free SMV indicating the potential of carrier system. Overall, results clearly indicate that the combination of SMV+miR-21i (gene + drug therapy) might provide a valuable strategy for the clinical management of gastric cancers. Graphical presentation depicting the preparation of simvastatin and miRNA-21i-loaded PLGA/PEI nanoparticles. The PEI-grafted polymeric nanoparticles were prepared by double emulsion method and miRNA was surface loaded onto the carrier system making a SMV/miR-21i-loaded PEI/PLGA NP.  kV; (c) In vitro release kinetics of SMV and miR-21i from PPN-S21i in pH 7.4 phosphate buffer saline (PBS) for 60h (n=3). The drug release was performed through dialysis method and release rate of SMV was determined by HPLC method and miR-21i-FAM was evaluated by microplate reader.