Materials

Donepezil HCl and B27 supplement, Quant-it Ribogreen RNA Assay kit, High-Capacity cDNA Reverse Transcription Kit, Taqman™ Gene expression Maxter Mix, Taqman™ assays (GAPDH, Tnf, IL-1β, BACE-1), Amyloid beta 42 ELISA kit, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Memantine HCl, brain-derived neurotrophic factor (BDNF), and retinoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). ON-TARGET plus BACE-1 siRNA and siGLO Red siRNA were purchased in Horizon Discovery (Lafayette, CO, USA). Egg L-α-phosphatidyl choline (Egg-PC) (95%), cholesterol, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased in Avanti Polar Lipids (Alabaster, AL, USA). Human astrocytes, poly-L-lysine (10 mg/mL) were purchased in ScienCell Research Laboratories (Carlsbad, CA, USA). SH-SY5Y, HBEC-5i, Calu-3 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Aβ42 were purchased in Anaspec (Fremont, CA, USA). Caspase-3/7 assay kit was purchased from Promega (Madison, WI, USA). Float-A-Lyzer dialysis device and CE dialysis tubing were purchased in Repligen (Waltham, MA, USA).

Preparation of Drug and siRNA-loaded Liposomes

The liposomes were prepared using the thin-layer hydration method using Egg PC and cholesterol in a molar ratio of 11:9, as previously described (31). Donepezil or memantine was incorporated into the liposome via the ion-gradient method, as previously described (32). Cationic liposomes were prepared using Egg-PC, cholesterol, and DOTAP in a mass ratio of 25:11:12.5, as previously described (33). BACE-1 siRNA was dissolved in nuclease-free citric acid buffer (0.1 M, pH 4), added to the flask containing a dry lipid film, hydrated until the lipid film was fully dissolved and dry sonicated, extruded, and purified by dialysis (Float-A-Lyzer MWCO 100 kD) at 4 °C overnight in nuclease-free NaCl 0.9% solution. The size distribution, zeta potential (ZP) and polydispersity index (PDI) of liposomes were analyzed with Malvern Zetasizer Nano (Malvern Instruments, Malvern, UK). The prepared liposomes were stored at 4 °C for further analysis.

Determination of Encapsulation Efficiency

The encapsulation efficiency (EE) of drugs in the donepezil (DL) and memantine liposomes (ML) was determined by dialysis. Quantification of donepezil was performed using the previously reported HPLC method (34) using 60:40 (v/v) methanol/0.02 M phosphate buffer as the mobile phase (pH 7.5, 1 mL/min flow rate, 10 µL the injection volume) and a reverse phase C18 column (Waters, 150 × 4.6 mm, Milford, MA, USA) at 25 °C. Donepezil was quantified at wavelength 268 nm using a UV detector. Since memantine does not have a chromophore, it cannot be directly detected by a UV detector (35). Memantine was derivatized with 9-fluorenyl methyl-chloroformate (FMOC) to improve detectability using the previously reported method (36). The derivatized memantine was analyzed using the UV-HPLC system. 80:20 (v/v) acetonitrile/0.05 M phosphate buffer (pH 4, adjusted with orthophosphoric acid) was used as a mobile phase (2 mL/min flow rate 20 µL injection volume). The mobile phase was filtered with a 0.45 μm nylon filter and degassed for 10 min. The following equation was used to calculate the drug encapsulation efficiency:

\(\:EE\left(\%\right)=\frac_}_}\times\:100\), where the \(\:_\:\text\text\text\:C}_\) are the concentrations of a drug before and after dialysis, respectively.

The EE of liposomes containing BACE-1 siRNA (BACEL) was determined by the Ribogreen Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The measurement was performed by a previously reported method with some modifications (37). Briefly, BACEL was disrupted with 2% Triton-X 100 for 15 min at 37 °C. The total siRNA concentration in the liposomes was measured based on the calibration curves with known standards. siRNA was measured at wavelengths of 480/520 nm. The following equation was used to calculate the EE:

\(\:EE\left(\%\right)=\frac_-_}_}\times\:100\), where Ct is the concentration of BACE-1 siRNA with Triton X-100, C0 is the concentration of BACE-1 siRNA without Triton X-100, and Ci is the initial concentration of BACE − 1 siRNA.

In Vitro Release of Drugs and SiRNA From Liposomes

The in vitro release of donepezil and memantine was evaluated by a reverse dialysis sac technique modified from the previously reported method (38) using float-A-Lyzer Dialysis tube (100 kD MWCO, Repligen, Waltham, MA, USA) in phosphate-buffered saline (PBS) at 37 °C. The released drugs were analyzed by HPLC. The in vitro release of BACE-1 siRNA and BACEL was evaluated using the conventional dialysis method with a 100 kD cellulose ester membrane. using a Ribogreen Assay (Thermo Fisher Scientific, Waltham, MA, USA).

Cell Culture

SH-SY5Y cells were cultured in T-75 flasks in a 1:1 mixture of EMEM/F12 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) solution. Calu-3 cells were cultured in T-75 flasks with EMEM (10% FBS and 1% P/S). Primary human astrocytes (HA) were cultured in poly-L-lysine (2 µg/cm2) precoated T-75 flasks with complete astrocytes medium (2% FBS, 1% P/S solution and 1% astrocytes growth supplement). HBEC-5i cells (ATCC, Manassas, VA, USA) were cultured in DMEM: F12 with 40 µg/mL endothelial growth supplement (Sigma, St. Louis, MO, USA), FBS 10%, and 1% P/S solution. The flasks for HBEC-5i cells were pre-treated with 0.1% gelatin (ATCC, Manassas, VA, USA) according to the manufacturer’s protocol. All cells were incubated at 37 °C with 5% CO2.

Evaluation of Transport across Calu-3 Cells

Calu-3 cells were used to model the nasal transport of liposomes. Calu-3 cells are lung carcinoma cell lines that share characteristics similar to those of the nasal epithelium. They can be cultured at an air-to-liquid (A-L) interface and form tight junctions with a uniform mucus layer (39,40,41). Calu-3 cells were cultured in T-75 flasks, and once they were confluent, cells were trypsinized using 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) and incubated (3 × 105 cells/cm2) on 0.4 μm pore polyester membrane insert in 24 mm Transwell (Corning #3450, Corning, NY, USA). The transepithelial electrical resistance (TEER) values were recorded daily with epithelial volt/ohm meter EVOM 2 (World Precision Instruments, Sarasota, FL, USA), and once the cells were confluent and the TEER value reached over 500 Ω·cm2, the media from the apical side was removed to create an A-L interface. The medium was changed every other day, and once it reached day 6 of the A-L interface, free and liposomal formulations of donepezil, memantine, and fluorescein isothiocyanate (FITC)-labeled BACEL were tested for transport studies. Each therapeutic agent was incubated at the apical membrane, and the media on the basolateral membrane was collected at 30, 60, 90, 120, 150, 180 min. The concentration of collected samples of donepezil and memantine were analyzed with UV-HPLC. FITC-BACEL was measured at wavelength of 490/520 nm.

Cellular Internalization

To evaluate the cellular internalization of liposomes, 1 mg/mL of FITC- and Cy3-siRNA (siGLO Red Transfection Indicator, Horizon Discovery, Lafayette, CO, USA) were labeled during liposomal preparation. All the liposomes were sonicated, extruded, and dialyzed using the previous method. HBEC-5i or HA cells were seeded in the Nunc Lab-Tek II Chamber Slide system (4 wells) at 10,000 cells/cm2 and incubated for 24 h. Then, the cells were treated for 24 h with FITC-Cy3-siRNA-liposomes filtered with a 0.22 μm PES filter. The cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) three times and fixed with 4% formaldehyde for 5 min. Then, the cells were washed three times with DPBS and stained with 4’,6-diamidino-2-phenylindole (DAPI) (300 nM) for 10 min, protected from light. The stain solution was removed and washed three times with DPBS, and the cells were analyzed with a confocal microscope.

Cell Differentiation

SH-SY5Y cells were differentiated with retinoic acid and brain-derived neurotrophic factor (BDNF) by a previously reported method with several modifications (42, 43). Briefly, the cells were seeded (12,500 cells/cm2) in a Petri dish and incubated for 24 h. Then, the cells were incubated with 10 µM retinoic acid in EMEM/F12 complete media (ATCC, Manassas, VA, USA; Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS (media #1), and the medium was changed every other day with media #1. After 4 days of differentiation, cells were further incubated with 10 µM retinoic acid, 50 ng/mL BDNF, and 1 x of B-27 supplements in EMEM/F12 media without FBS (media #2) for 2 days. After a total of 6 days of differentiation, cells were used for the experiment and further cultured in media #2.

Cytotoxicity Studies

The cytotoxicity of all different treatment groups was evaluated using a water-soluble tetrazolium salt (WST) assay. Briefly, SH-SY5Y cells (5,000 cells/well) were seeded on 96 well plates and incubated for 24 h. SH-SY5Y cells were differentiated according to the previous protocol before the treatment. Various concentrations of free donepezil, DL, free memantine, ML, free BACE-1 siRNA, BACEL, free triple-drug cocktail, and liposomal triple-drug cocktail were incubated for 48 h to investigate the cytotoxicity of the drug to each cell line. Also, differentiated SH-SY5Y cells were incubated with different concentrations of Aβ42 peptides (2.5, 5, 10, 20 µM) to evaluate any cytotoxic effect. The peptides were dissolved in 1% NH4OH and further diluted with PBS to obtain a 1 mg/mL concentration. 10 µL/well of WST-1 reagent was added to 100 µL cell culture medium and incubated at 37 °C for 2 h. The wells were shaken for 60 s, and the absorbance was read at 440 nm, reference wavelength of 600 nm. All samples were analyzed in triplicate.

Caspase Activity

SH-SY5Y (5,000 cells/well) were seeded in 96 well plates and differentiated for 6 days. Undifferentiated and differentiated cells were preliminarily incubated with staurosporine (20 nM) or Aβ42 (5µM) for 24 h. Staurosporine is an agent that induces caspase activity and is used as a positive control for the assay. Then, cells incubated with peptides were treated with free memantine (1, 5, and 10 µM) for 24 h. The activity of caspase-3/7 was measured using a Caspase-Glo assay kit (Promega, Madison, WI, USA). The reagent was added to the culture medium and incubated at room temperature for 1 h. The luminescence intensity was measured using a TECAN Genios Pro reader (Tecan, Morrisville, NC, USA). Differentiated SH-SY5Y cells were incubated with various concentrations of Aβ42 (5, 10, 20 µM) for 24 h to evaluate the caspase activity. Various treatment groups were also used to assess caspase activity, including DL 2µM, ML 10µM, BACEL 50 nM, and triple liposome.

mRNA Expression

The expression of targeted mRNAs was analyzed using the reverse transcription (RT) quantitative polymerase chain reaction (qPCR). Differentiated SH-SY5Y cells whereas incubated with Aβ42 (5 µM) for 24 h. Then, the cells were treated with DL, ML, BACEL, and triple drug cocktail for 48 h. The cells were processed using the RNeasy Plus mini-Kit protocol (Qiagen, Germantown, MD, USA). RNA was quantified using NanoDrop and reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) based on manufacturer protocol. Then, 40 ng of each cDNA was analyzed by qPCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as an endogenous housekeeping gene to normalize BACE-1, TNF-α, and IL-1β expression levels. The data was calculated based on the comparative Ct method (2−ΔΔCt) – a relative quantification strategy that uses threshold cycles (CTs) from qPCR data to calculate relative gene expression levels between samples.

Enzyme-linked Immunosorbent Assay (ELISA)

The quantification of Aβ42 proteins was performed using an Amyloid β 1–42 ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, differentiated SH-SY5Y cells were incubated with Aβ42 5µM for 24 h. Cells were treated for 48 h with various groups: (1) DL 2 µM, (2) ML 10 µM, (3) BACEL 50 nM, and (4) triple-drug cocktail liposome. Then, the culture media was collected and supplemented with protease inhibitors. The suspension was centrifuged at 13,000 rpm for 15 min, and the supernatant was used for the detection of Aβ42. For detection of Aβ42 in the cell membrane, SH-SY5Y cells were washed in PBS twice and lysed in buffer with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP, 0.5% sodium deoxycholate, 0.1% SDS, 5mM EDTA (RIPA buffer), and one complete protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated in lysis buffer on ice, scraped, and centrifuged at 13,000 rpm, 4 °C for 15 min. The supernatant was collected for ELISA assay. Proteins from each sample were normalized with a Pierce ® protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and were analyzed with an ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) based on the manufacturer’s protocol.

Statistical Analysis

All the data are presented as the mean ± SD. All the data were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA for grouped analysis, followed by Dunnett post hoc analysis for multiple comparison tests. The level of statistical significance was set at p < 0.05 by using GraphPad Prism (Graphpad Software, Boston, MA, USA).