Development of inhalable quinacrine loaded bovine serum albumin modified cationic nanoparticles: Repurposing quinacrine for lung cancer therapeutics

Journal Pre-proofs Development of Inhalable Quinacrine Loaded Bovine Serum Albumin Modified Cationic Nanoparticles: Repurposing Quinacrine for Lung Cancer Therapeutics Bhuvaneshwar Vaidya, Nishant S Kulkarni, Snehal K Shukla, Vineela Parvathaneni, Gautam Chauhan, Jenna K Damon, Apoorva Sarode, Jerome V Garcia, Nitesh Kunda, Samir Mitragotri, Vivek Gupta PII: DOI: Reference:

S0378-5173(19)31056-7 https://doi.org/10.1016/j.ijpharm.2019.118995 IJP 118995

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

25 July 2019 18 December 2019 22 December 2019

Please cite this article as: B. Vaidya, N.S. Kulkarni, S.K. Shukla, V. Parvathaneni, G. Chauhan, J.K. Damon, A. Sarode, J. V Garcia, N. Kunda, S. Mitragotri, V. Gupta, Development of Inhalable Quinacrine Loaded Bovine Serum Albumin Modified Cationic Nanoparticles: Repurposing Quinacrine for Lung Cancer Therapeutics, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118995

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Development of Inhalable Quinacrine Loaded Bovine Serum Albumin Modified Cationic Nanoparticles: Repurposing Quinacrine for Lung Cancer Therapeutics

Bhuvaneshwar Vaidya1, Nishant S Kulkarni2, Snehal K Shukla2, Vineela Parvathaneni2, Gautam Chauhan2, Jenna K Damon3, Apoorva Sarode4, Jerome V Garcia3, Nitesh Kunda2, Samir Mitragotri4, Vivek Gupta1,2*

1School

of Pharmacy

Keck Graduate Institute, Claremont, CA 91711

2College

of Pharmacy and Health Sciences

St. John’s University, Queens, NY 11439

3Department

of Biology

University of La Verne, La Verne, CA 91750

4John

A Paulson School of Engineering and Applied Sciences Harvard University, Cambridge, MA 02138

*To whom correspondence should be addressed: Dr. Vivek Gupta, College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY – 11439. Phone: 718-990-3929. Email: [email protected]

Abstract: Drug repurposing is on the rise as an atypical strategy for discovery of new molecules, involving use of pre-existing molecules for a different therapeutic application than the approved indication. Using this strategy, the current study aims to leverage effects of quinacrine (QA), a well-known anti-malarial drug, for treatment of non-small cell lung cancer (NSCLC). For respiratory diseases, designing a QA loaded inhalable delivery system has multiple advantages over invasive delivery. QA-loaded nanoparticles (NPs) were thus prepared using polyethyleneimine (PEI) as a cationic stabilizer. While the use of PEI provided cationic charge on the particles, it also mediated a burst release of QA and demonstrated potential particle toxicity. These concerns were circumvented by coating nanoparticles with bovine serum albumin (BSA), which retained the cationic charge, reduced NP toxicity and modulated QA release. Prepared nanoparticles were characterized for physicochemical properties along with their aerosolization potential. Therapeutic efficacy of the formulations was tested in different NSCLC cells. Mechanism of higher anti-proliferation was evaluated by studying cell cycle profile, apoptosis and molecular markers involved in the progression of lung cancer. BSA coated QA nanoparticles demonstrated good aerosolization potential with a mass median aerodynamic diameter of significantly less than 5µm. Nanoparticles also demonstrated improved therapeutic efficacy against NSCLC cells in terms of low IC50 values, cell cycle arrest at G2/M phase and autophagy inhibition leading to increased apoptosis. BSA coated QA NPs also demonstrated enhanced therapeutic efficacy in a 3D cell culture model. The present study thus lays solid groundwork for pre-clinical and eventual clinical studies as a standalone therapy and in combination with existing chemotherapeutics. Key Words: Quinacrine; cationic nanoparticles; aerosolization; non-small cell lung cancer; autophagy; apoptosis

INTRODUCTION: Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 14% of the total cancer diagnoses. The American Cancer Society (ACS) has estimated about 234,030 new cases on lung cancer in the United States and about 154, 050 deaths from lung cancer for 2018 (American Cancer Society, 2018). Non-small cell lung cancer (NSCLC), one of the two forms of lung cancer, constitutes >80% of all lung cancers (Torre et al., 2016). While chemotherapy is one of the most commonly used and successful approaches to treat lung cancer, it still suffers from innumerable drawbacks including poor selectivity toward cancerous and metastatic cells, carcinogenicity, and acquired drug-resistance. Majority of these limitations of formulations and chemotherapeutic agents stem from the fact that these agents have a high biodistribution in tissues other than lungs. These drawbacks can be countered by developing an inhalable formulation of chemotherapeutic agents (Rayamajhi et al., 2011). Inhalation route is deemed to be highly beneficial for delivering payload to the lungs, especially deep lungs. It is a non-invasive process with localized particle accumulation at the site of action leading to reduction in therapeutic dose, thus further reducing systemic side effects and improving patient compliance than invasive procedures. Loading chemotherapeutic agents on a suitable inhalable carrier system can enhance the prognosis of lung cancer (Abdelaziz et al., 2018). New drug discovery and development is associated with numerous complexities due to which emphasis of translational research has shifted toward leveraging already available resources for discovering novel anti-cancer regimens (Sleire et al., 2017). One of the more frequent approaches include drug repositioning, i.e. testing an already FDA approved drug for different disease indication/s, which comes with advantages of reported safety profile, less toxicity to normal cells and tissues, and major insights into formulation and delivery strategies (Wurth et al.,

2016). These investigational drugs could not only reduce the dose-related side effects of chemotherapeutic agents but may also improve the efficacy by either additive or synergistic effects (Sleire et al., 2017; Wurth et al., 2016). Quinacrine (QA), a decades-old anti-protozoal and antimalarial drug, has recently been investigated primarily as synergistic agent to enhance anti-cancer potential of several other chemotherapeutic agents (Bhateja et al., 2018; Dermawan et al., 2014; Gallant et al., 2011; Wang et al., 2010). Few studies also report anti-cancer potential of quinacrine as a standalone therapy against a wide range of cancers including breast (Das et al., 2017; Preet et al., 2012; Preet et al., 2016), colon (Jani et al., 2010; Mohapatra et al., 2012), cervical (Fasanmade et al., 2001), gastric (Wu et al., 2012), ovarian (Khurana et al., 2015), and renal (Gurova et al., 2005). Quinacrine is reported to exhibit its anti-cancer activity through different mechanisms including DNA intercalation (Gurova, 2009; Hossain et al., 2008), topoisomerase inhibition (Preet et al., 2012), tumor suppressor gene p53 activation (Wu et al., 2012), cyclin-dependent kinase inhibitor p21 activation (Mohapatra et al., 2012), and autophagy modulation (Das et al., 2017; Khurana et al., 2015), and therefore is a truly polypharmacological agent (Ehsanian et al., 2011). More importantly, no report has been published citing its carcinogenic potential on long-term use (Sokal et al., 2000; Sokal et al., 2010). In a recent report, it was established that quinacrine was able to sensitize the resistant lung cancer cells for erlotinib-mediated cytotoxicity, thus exhibiting a significant synergism with erlotinib, in both in-vitro and in-vivo studies (Bhateja et al., 2018; Dermawan et al., 2014). It has also been reported that when loaded in cationic liposomes with vinorelbine, quinacrine showed improved anti-cancer activity against NSCLC (Li et al., 2015). Despite these advantages, quinacrine suffers from several drawbacks including low bioavailability/permeability at tumor site, localized skin deposition leading to skin pigmentation following chronic exposure, rashes and other immunological complications (Gurova, 2009). While

some of the side-effects are reported to be transient and temporary, there is still a significant need to attain local therapeutic concentrations at tumor site while reducing the overall physiological exposure. Nanoparticles based delivery of anti-cancer agents has been historically shown to improve localized and selective therapeutic activity with reduced side-effects. Hence, development of quinacrine nanoformulation would be a promising approach to reduce its side effects and also to improve efficacy against lung cancer by enhancing localized drug concentrations at tumor site. There are few published reports of quinacrine-loaded nanoparticles used as single agent or in combination with other chemotherapeutic agents (Li et al., 2014; Li et al., 2015; Nayak et al., 2016; Nayak et al., 2017; Zhang et al., 2012). However, there is no study reporting a detailed description of their formulation development/optimization parameters, characterization, and detailed mechanistic profile. Further, no study has discussed the use of quinacrine for lung cancer as a standalone therapy; either as a solution or a nanoformulation. Our study presents the first report of utility and efficacy of quinacrine-loaded nanoparticles as a repurposed agent for NSCLC therapy. MATERIALS AND METHODS: Materials: Quinacrine hydrochloride, poly vinyl alcohol (PVA), bovine serum albumin (BSA), and polyethyleneimine (PEI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly (lacticco-glycolic) (PLGA 50:50, 25,000-35,000 Da) was procured from PolyScitech (West Lafayette, IN). All other solvents including HPLC grade solvents and chemicals, unless otherwise specified, were purchased from Fisher Scientific. Preparation of quinacrine-loaded PLGA nanoparticles:

Quinacrine-loaded PLGA nanoparticles were prepared using multiple emulsion solvent evaporation method as described in previous publications from our lab (Gupta et al., 2011; Vaidya et al., 2018), with minor modifications. Briefly, polymer (20 mg/mL) was dissolved in dichloromethane (3 mL) to form organic phase. Aqueous quinacrine solution (0.5 ml) was used as an internal aqueous phase whereas 2% w/v aqueous PVA solution or 2% PEI in 2% PVA was used as an external aqueous phase. Internal aqueous phase was added dropwise in the polymer solution and sonicated for 1 min using probe sonicator (10 sec on/off cycle; Branson 450) to form primary emulsion. Primary emulsion was further added to external aqueous phase and sonicated for 2 min to form w/o/w multiple emulsion (10 sec on/off cycle). Developed w/o/w emulsion was left overnight under moderate magnetic stirring for evaporation of organic solvent. Residual organic solvent was removed by vacuum rotary evaporator. Hardened nanoparticles were collected by refrigerated centrifugation at 15,000 ×g for 20 min and washed with water thrice. Entrapment efficiency was calculated by measuring free drug amount in the supernatant by measuring absorbance at 425 nm using UV-visible spectrophotometry and some samples were analyzed using ultra pressure liquid chromatography (UPLC; Waters, Inc.) eluted by using 0.1% o-phosphoric acid:acetonitrile (35:65) as mobile phase. Preparation of BSA-coated PLGA Nanoparticles: BSA solutions with different concentrations were prepared by dissolving BSA in deionized water with stirring. The BSA-coated PLGA nanoparticles were prepared by adding PEI stabilized PLGA nanoparticles dropwise to the BSA solution. After dropwise addition of PLGA nanoparticles to BSA solution, the mixture was stirred at 200 rpm for 30 minutes at room temperature, and nanoparticles were collected by centrifuging at 15,000 ×g for 20 minutes. Pelletized particles were dispersed in deionized water for further characterization.

Physicochemical characterization of PLGA nanoparticles: Prepared nanoparticles were characterized for size and size distribution, zeta potential, shape and morphology, and entrapment efficiency. Particle size, Poly dispersity index (PDI) and Zeta Potential: Size and size distribution of the nanoparticles were determined by dynamic light scattering method using Zetasizer nano ZS (Malvern Instrument, UK). Polydispersity indices (PDI) were measured to check the uniformity of the particles’ size. Surface charge of nanoparticles was determined by measuring zeta potential of the nanoparticles using Zetasizer nano ZS (Malvern Instrument, UK). The morphology and internal structure of plain as well as coated PLGA nanoparticles was characterized using electron microscopy. Sample preparation for scanning electron microscopy (SEM) involved vacuum drying of 10µL dilute particle suspension on an aluminum stub, followed by coating with platinum-palladium (EMS 150T S Metal Sputter Coater, Quorom Technologies, UK). The sputter-coated samples were imaged using the Supra55VP Field Emission Scanning Electron Microscope (ZEISS, Germany) at an acceleration voltage of 5 kV and a working distance of 2.5 mm. Particle diameter measurements were made using SmartSEM image acquisition and analysis software. For transmission electron microscopy (TEM), 300-mesh Formvar® carbon-coated copper grids (Electron Microscopy Sciences, PA) were made hydrophilic using glow discharge plasma treatment for 30s. Approximately 5μL of dilute nanoparticle suspension was placed on the treated grids and allowed to dry at room temperature. TEM micrographs were obtained using JEOL 1230 TEM (JEOL USA, Inc., MA) operated at 120kV. Entrapment efficiency and loading efficiency:

Entrapment efficiency of the nanoparticles was determined by measuring the amount of free drug in supernatant collected after centrifugation of the dispersion at 15,000 ×g for 20 min. The following equation was used to calculate the entrapment efficiency (EE) and loading efficiency (LE) of the particles:

𝐸𝐸% =

[

]

𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑡𝑎𝑘𝑒𝑛 𝑖𝑛𝑖𝑡𝑖𝑎𝑙𝑙𝑦 ― 𝑢𝑛𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒𝑑 𝑑𝑟𝑢𝑔 × 100 𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑡𝑎𝑘𝑒𝑛 𝑖𝑛𝑖𝑡𝑖𝑎𝑙𝑙𝑦

𝐿𝐸% =

[

]

𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑒𝑛𝑡𝑟𝑎𝑝𝑝𝑒𝑑 × 100 𝑇𝑜𝑡𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

In-vitro drug release studies: In-vitro drug release profile from quinacrine-loaded nanoparticles was determined in the phosphate buffered saline (PBS, pH 7.4) using an earlier reported method (Kaur and Tikoo, 2013). Briefly, specific amount of lyophilized nanoparticles were dispersed in 1.5 mL of PBS (pH 7.4) in microcentrifuge tubes. Tubes were placed in the incubator at 37°C and 50 rpm for different time intervals. At each specific time point, a tube was withdrawn and centrifuged to collect the supernatant. Amount of drug released at specific time point was determined by measuring drug in the supernatant. Differential Scanning Calorimetry (DSC) Studies The calorimetric studies were carried out to evaluate the thermal state of drug and polymer during encapsulation. The thermograms were generated using DSC 6000 (PerkinElmer, Inc; CT, USA) equipped with an intracooler accessory. Accurately weighed sample was sealed in aluminum pan and analyzed over a temperature range of 30°C to 300°C against an empty pan maintained as

reference. The heating rate was maintained at 10°C/min under the nitrogen purge having flow rate of 50 mL/min. Powder X-ray Diffraction (PXRD) Studies Diffractometry was performed to investigate the impact of encapsulation process on physical state of the drug. Powder X-ray diffraction studies were carried out using XRD-6000 (Shimadzu, Kyoto, Japan). The diffractometry was performed using graphite monochromator and the source consisting of copper. The Kα radiation ( of 1.5418 Å) was obtained at a voltage of 40 kV and current of 30 mA. The samples were spread uniformly on a glass micro sample holder and were analyzed over 2θ range of 10° to 80° at scanning rate of 2° min-1. Aerosolization of QA loaded Nanoparticles The 170 Next Generation Impactor (NGI) was employed to assess the aerosol performance of QA uncoated and coated particles. Briefly, 2 mL of QA NPs were loaded into the nebulizer cup attached to a PARI® LC plus nebulizer. The samples were drawn through the induction port into the NGI using a vacuum pump (MSP Corp, Shoreview, MN, USA) operated at a flow rate of 15 L/min for 4 minutes. Prior to running the NGI, the plates were refrigerated at 4ºC for 90 minutes to cool the NGI plates. The samples were collected using a binary mixture of acetonitrile and water (50:50) and centrifuged at 21,000 xg for 45 mins. Thereafter, the supernatant was analyzed using UPLC method, as mentioned above. The Fine Particle Fraction (FPF, %) was determined as the fine particle dose (dae < 5.39 μm) of the total emitted dose deposited in the NGI. The calculation for FPF (%) was done by dividing the fine particle dose (amount of drug deposited from stage 3 to stage 8) by the total emitted dose (amount of drug emitted from mouthpiece to stage 8). The mass median aerodynamic diameter (MMAD, D50%) was obtained by determining the aerodynamic

diameter that corresponds to 50% of the % cumulative deposition. MMAD was calculated using a log plot of aerodynamic diameter (ECD) vs cumulative deposition (%) of the drug. MMAD is D50%, the aerodynamic diameter (ECD) on y axis corresponding to cumulative % of 50% on x axis in micron. The geometric standard deviation (GSD) was calculated using the following equation from the log-probability analysis (n=3).

𝐺𝑆𝐷 =

𝐷84.1% 𝐷15.9%

In-vitro cellular uptake studies: To determine the impact of PEI and BSA on intracellular uptake of nanoparticles, coumarin-6 loaded nanoparticles were prepared by following the previously described method, while replacing quinacrine with coumarin-6. For uptake studies, A549 NSCLC cells (25,000 cells/well) were seeded in 8-well chambered glass slides (NuncTM Lab-TekTM II Chamber Slide, Thermo Fisher Scientific), and were allowed to adhere overnight. After adherence, cells were incubated with coumarin-6 loaded nanoparticles for different time intervals. At each time point, cells were washed twice with ice-cold PBS (pH 7.4) and were fixed with 4% paraformaldehyde (PFA) for 10 min. Fixed cells were washed two times with ice-cold PBS, permeabilized with 0.1% Triton X-100 followed by washing with ice-cold PBS. Rhodamine phalloidin (Thermo Fisher Scientific) was used to stain the cells and coverslips were mounted with Vectashield® hardset mountant (Vector Labs, Burlingame, CA). Following appropriate drying, slides were imaged using confocal fluorescence microscope (Leica Microsystems, Inc., Buffalo Grove, IL). To quantify the difference in intracellular uptake of quinacrine with or without encapsulation in cationic nanoparticles, flow cytometry analysis was done after incubating quinacrine-loaded

nanoparticles (1 µM quinacrine equivalent) with the A549 cells (1*106 cells/well in 6-well plate) for 3 hours, followed by two times washing with ice-cold PBS. The cells were trypsinized and washed again with ice-cold PBS and pelletized by centrifugation. Pellets were dispersed in PBS and the cells (10,000 counts/sample) were analyzed for quinacrine signals using flow cytometer (BD Accuri™ C6, BD Biosciences) and data were processed using FlowJo software. Mean fluorescence intensities (MFI) of samples after incubation with different nanoparticles were compared with MFI from quinacrine solution. In-vitro Cytotoxicity Assay: A well-reported MTT assay was adopted to determine anti-proliferative activity of the formulations. Briefly, cells (2,000 cells/well) were seeded in 96-well tissue culture plates and left overnight for adherence in CO2 incubator (5% CO2, 370C). After adherence, cells were treated with different concentrations (0.31-9.0 µM) of plain quinacrine and quinacrine loaded nanoparticles for 72 hours. Simultaneously, in a separate set of experiments, A549 NSCLC and normal Human Embryonic Kidney (HEK293) cells were incubated with blank nanoparticles (PEI stabilized and BSA coated) to measure the toxicity profiles of blank nanoparticles. Blank nanoparticles concentrations were chosen equivalent to QA loaded nanoparticles. In addition, to determine the safety profile of PEI-stabilized and BSA-coated nanoparticles, acute cytotoxicity of blank nanoparticles were measured by incubating A549 cells with nanoparticles for short period of time, i.e. at 6 hours. At the end of treatment period, media was removed from the wells followed by addition of MTT solution (1 mg/mL) and incubation for further 2 hours in incubator to develop formazan crystals in the cells. Crystalized formazan was dissolved by adding dimethyl sulfoxide (DMSO) in the wells after removing MTT solution. Plates were shaken for 10 min on plate shaker and absorbance

was measured at 570 nm using spectrophotometer. Untreated cells were used as control and cell viability was determined against control cells. Clonogenic assay: To determine the long-term effects of quinacrine loaded nanoformulations in resistant cancer cells, a well-established clonogenic assay was used according to the earlier reported method (Preet et al., 2012). Briefly, 500 cells/well were seeded in 6-well plate and left overnight for attachment. After attachment, cells were treated with plain quinacrine or quinacrine loaded nanoparticles for 24 hours, after which the drug/nanoparticle containing media was replaced with fresh media; and cells were further incubated for 5 days. Media was changed on each alternate day. After incubation period, media was removed, cells were washed with ice-cold PBS (pH 7.4) two times and were fixed with 4% PFA solution for 10 min. Fixed cells were washed with ice-cold PBS twice and incubated with crystal violet dye (0.2% w/v) for 1 hour. After staining, cells were washed with distilled water and images were captured using a digital camera. Cell colonies were counted by colony counter and compared with control cells. Mechanism of Improved Anti-proliferative Activity: Cell cycle and apoptosis analysis: Following treatment for specific time period, cell cycle and apoptosis were studied with propidium iodide (PI) assay using flow cytometry. Briefly, A549 cells (1*106 cells/well in a 6-well plate) were treated with various concentrations of plain drug or nanoformulations for 72 hours. After treatment, the cells were collected by trypsinization followed by washing two times with PBS. After washing, cells were fixed in cold 70% ethanol for 1 hour followed by two times washing with PBS. Thereafter, cells were incubated with RNA-ase (100 µg/mL) and PI (10 µg/mL) for 30

min in dark at room temperature. Cells (10,000 counts/sample) were analyzed for PI signals using flow cytometer (BD Accuri™ C6, BD Biosciences) and data were processed using FlowJo software. Effect of nanoformulations on molecular markers: To evaluate the mechanism of action of quinacrine-loaded NPs, different cellular markers involved in the progression and migration of lung cancers were quantified. Cells were seeded in 100 mm culture dishes and incubated at 5% CO2/37°C to attain 70-80% confluency. After reaching desired confluency, cells were treated with formulations (plain drug and nanoformulations) for 72 hours. After treatment period, cells were harvested by scraping and were washed with PBS. Cell pellets were collected by centrifuging cells at 500g for 3 minutes. Cell lysates were collected by disrupting cells using RIPA cell lysis (Thermo Scientific) buffer with added protease inhibitors for 30 minutes with occasional vortexing. Supernatant was collected after centrifugation at 21,000 ×g for 10 minutes in microcentrifuge tubes. The collected cellular lysate was used for different protein marker analyses by western blotting. SDS-PAGE and western blotting: Total protein content of cell lysates was determined by using BCA protein assay method, according to supplier’s protocol (Thermo Scientific, Waltham, MA). Equal amount of protein was loaded in each well of the precast gels (10% or any Kd gels, Bio-Rad) and separated using standard protocol. Polyvinylidene fluoride (PVDF) membrane with 0.2 μm pore size was used for the transfer of proteins. Membranes were incubated overnight at 4°C with primary antibodies for various protein markers including LC3B (1:3000, Abcam, Cambridge, MA), p62 (1:1000, Sigma), cleaved poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling Technology, Danvers, MA), p21

(1:1000, Abcam, Cambridge, MA), p53 (1:1000, Thermo Scientific, Waltham, MA), ph-p53 (1:1000, Thermo Scientific, Waltham, MA), and cleaved Caspase 3 (1:1000, Cell Signaling Technology, Danvers, MA); whereas goat anti-mouse/anti-rabbit HRP-conjugated secondary antibody (1:3000, Abcam, Cambridge, MA) was incubated for 2 hours at room temperature. ECL substrate was used for visualization of protein band and membranes were imaged using western blot image analyzer (Azure C500, Azure Biosystems, Dublin, CA). For demonstration of equal protein loading in each well, membranes were re-probed with a β-actin antibody (1:2000, Abcam, Cambridge, MA). 3D-Spheroid Cell Culture Study: A 3D cell-based model was developed as previously reported by Kulkarni et al (Kulkarni et al., 2019). Briefly, A549 cells were trypsinized on reaching 80% confluency and plated in ultra-low

attachment U-shaped plates (2,000 cells/well). Cells were allowed to form spheroids overnight and the following day, were treated with different concentrations of quinacrine and quinacrine-loaded nanoparticles. Spheroids generally require a few days to form an intact solid mass, post-seeding. In this study, however, the effect of drug and formulations was tested on spheroids which were in their early stages of formation, mimicking a prophylactic model rather than a therapeutic model. Media replenishment was done every 72 hours by replacing half of the media in wells with fresh media. Images were captured every 72 hours using an inverted color microscope (Laxco, Mill Creek, WA) for 15 days. At the end of treatment period, cell viability was measured using cell titer-glo® 3D (Promega Inc., Madison, WI) as per manufacturer’s protocol. Live/Dead Cellular Assay: At the end of treatment period for previously mentioned A549 spheroids, live/dead assay was performed using a viability/cytotoxicity assay kit for animal live or dead cells (Biotium, Fremont,

CA) as per manufacturer’s protocol. Live cells were stained green by calcein AM and dead cells were stained red by ethidium homodimer III (EthD-III) and spheroids were imaged using an Evos FL fluorescence microscope (ThermoFisher Scientific, Waltham, MA) using GFP (green fluorescence protein) and RFP (red fluorescence protein) filters respectively. Statistical Analysis: All data presented here are mean ± SD or SEM (n=3 to 6). Cytotoxicity studies represent average of 3 independent trials (n=6 for each trial). Unpaired student’s t-test was used to compare two groups whereas to compare more than two groups one-way ANOVA followed by Tukey’s post hoc multiple comparison test was used. P value <0.05 was considered statistically significant. RESULTS AND DISCUSSION: In the present study, we sought to repurpose quinacrine for improved therapy of NSCLC by encapsulating it in biodegradable PLGA nanoparticles so as to exploit nanoparticles’ enhanced intracellular uptake potential. To achieve the objective, positive charge (cationic) nanoparticles were prepared using PEI as a stabilizer during preparation of quinacrine-loaded PLGA nanoparticles. Cationic nanoparticles have been extensively reported for increased intracellular delivery of therapeutics and nucleic acid therapeutics (Chou et al., 2018; Jin and Kim, 2012; Kong et al., 2012; Lee et al., 2008; Sheng et al., 2016). Their high intracellular accumulation is attributed to favorable interactions with cellular lipid bilayer membrane, by absorptive mediated endocytosis process, a reportedly energy dependent process (Pang et al., 2012). Polyethyleneimine (PEI) is a very well-known cationic polymer which increases intracellular delivery by escaping lysosome through proton-sponge effect (Boussif et al., 1995; Pack et al., 2005; Vermeulen et al., 2018). In the first step, quinacrine-loaded PLGA nanoparticles were prepared using well reported method utilizing 2% PVA as stabilizer. Prepared nanoparticles were of sub-200 nm in size (196±8 nm)

with negative charge (-18.1±0.3 mV). However, we observed low entrapment efficiency, i.e., 26.3±1.9% in non-modified anionic nanoparticles. To increase the entrapment efficiency and to impart positive charge, we utilized 2% PEI in 2% PVA (combination of PVA and PEI) as a stabilizer for double emulsion evaporation method. It has been reported earlier (Romero et al., 2010) that the concentration of PEI as stabilizer did not affect the zeta potential significantly however particle size decreased with increasing concentration of PEI. Based on the findings, we selected 2% of PEI as a stabilizer in our studies. Interestingly, we observed significantly higher entrapment efficiency (56.5±1.8%) along with high cationic charge particles (+30.8±0.4 mV). However, size of the particles increased slightly to 224±14 nm (Table 1). The prepared NPs were evaluated for in-vitro drug release profile in PBS (pH 7.4 @370C). Results of the study demonstrated that although use of PEI increased the entrapment efficiency of the particles, drug release in first 0.5 hours increased significantly, i.e., 97.3±2.31% from PEI stabilized particles vs 30.6±3.2% from plain nanoparticles (Table 1 & Fig. 1A). This may be ascribed to partial adsorption/interaction of quinacrine with PEI at nanoparticle surface. QA is hydrophilic and PEI

being a hydrophilic polymer interacts more with the release medium. This high interaction of PEI and release medium attracts more medium towards the particles and QA having more affinity for the release medium, undergoes a burst release in the first few hours. In case of plain nanoparticles, drug was entirely entrapped in matrix of PLGA particles thus retarding release.

Preparation of BSA-coated quinacrine-loaded PLGA nanoparticles: While effective intracellular delivery adjuvants, highly positive charged carrier nanoparticles have a rather narrow efficacy window and are shown to be toxic to the cells by causing disruption of plasma-membrane integrity, mitochondrial damage, and a higher number of autophagosomes than their anionic counterparts. Thus, it is imperative to control the surface charge to maintain the balance between localized tumor targeting and inherent side-effects (Frohlich, 2012). To control the positive charge of the particles and also to increase receptor-mediated uptake of the particles by cancer cells, we have used layered coatings of bovine serum albumin (BSA) as a surface modifier. Albumin-based nanoparticles have shown improved therapeutic efficacy of anti-cancer agents against different types of cancers (Choi et al., 2015; Kim et al., 2017; Shen and Li, 2018; Wan et al., 2016; Yu and Jin, 2016) and paclitaxel-albumin particle formulation (Abraxane®) is approved for clinical use. Also, BSA coating could reduce the burst release of quinacrine from PEI-stabilized nanoparticles. Albumin is the most abundant protein in the plasma and plays an important role in clearance of extraneous particles from circulation (Azizi et al., 2017). Albumin carriers are also reported to help in local drug accumulation at tumor site via two mechanisms, i.e. by passive targeting and by receptor (albondin/glycoprotein 60 (Gp60)) mediated enhanced transcytosis in endothelial cells (Tiruppathi et al., 1997; Wartlick et al., 2004). Further, higher metabolic rate in cancer cells also results in higher albumin cellular uptake, which may contribute to higher accumulation of albumin modified nanoparticles in the cancer cells compared to normal cells (Azizi et al., 2017; Frei, 2011). Different concentrations of BSA (w/v in deionized water) were tested for the coating purpose. Results of the study showed that when concentration of BSA coating solution increased from 0.25% to 2% w/v, zeta potential of particles reduced from +30.8±0.4 mV (no BSA) to +10.4±0.2 mV (1% BSA). However, further increase in BSA

concentration resulted in negative potential (-1.26±0.41 mV) (Fig. 1B). Hence, we optimized 1% BSA coating to prepare BSA coated cationic polymeric nanoparticles. % EE and % drug loading efficiencies were found to be 48.6±2.1 and 7.9% respectively for BSA coated NPs. In-vitro drug release study confirmed the slow release of quinacrine from BSA coated particles compared to PEI stabilized nanoparticles (67.1±6.0% vs 97.3±2.3% from PEI stabilized nanoparticles) (Table 1; Fig. 1C). In line with our hypothesis about controlling the NPs’ positive surface charge for reduced toxicity, we performed acute cytotoxicity studies by incubating A549 cells with blank nanoparticles (non-BSA vs BSA modified) for 6 hours at different concentrations (1-20 μg/mL). Results of the study showed that PEI-stabilized high (+30 mV) positive charge nanoparticles were toxic to the cells at higher concentration (20 μg/mL; 80.2±6.6% cell viability),

Fig. 1: (A) Comparison of 24 hour in-vitro release of quinacrine from anionic, PEI-stabilized, and BSA-coated nanoparticles. (B) The effect of BSA coating on zeta potential of NPs, (C) Invitro release profile of PEI stabilized PLGA NPs over a period of 5 days. (D) The effect of BSA coating on acute toxicity of PEI stabilized PLGA NP against A549 cells, Data represent mean±SD (n=3 for in-vitro, and n=6 for cell culture experiments).

whereas BSA-coated nanoparticles did not show any toxicity at the same concentration (97.8±6.8%), suggesting significant reduction in positive charge induced acute cytotoxicity by masking (partial) positive charge of PEI with BSA coating (Fig. 1D). Hence, based on the results of charge and acute toxicity, 1% BSA coated quinacrine-loaded PLGA nanoparticles were selected for further studies. Shape and Morphology of quinacrine loaded PLGA nanoparticles: Fig. 2A represents SEM images of plain PLGA NPs (2A-i), PEI stabilized (2A-ii), and BSA coated PLGA nanoparticles (2A-iii). It is evident from the images that the particles were spherical in shape. No significant differences were seen in shape, morphology, and size after coating of particles with BSA. In addition to SEM imaging, a more detailed morphological analysis was also

Fig. 2: (A) SEM and (B) TEM images of plain (i), PEI stabilized (ii), and BSAcoated (iii) quinacrine-loaded nanoparticles. (C) Representative dynamic light scattering (DLS) histograms of plain (i), PEI stabilized (ii), and BSA coated (iii) quinacrine-loaded PLGA NPs.

conducted with transmission electron microscopy studies. Representative TEM images are presented in Fig. 2B; plain NP (2B-i), PEI stabilized (2B-ii), and BSA coated (2B-iii). Sizes of different nanoparticles were observed in accordance to dynamic light scattering (DLS) method (Table 1). The representative DLS histograms are presented for all NP formulations; plain (2C-i), PEI stabilized (2C-ii), and BSA coated (2C-iii). Differential Scanning Calorimetry (DSC): The calorimetric studies were carried out to evaluate the thermal state of drug and polymer during encapsulation. Fig. 3A represents the DSC thermograms of pure quinacrine, blank nanoparticles, and quinacrine loaded particles. As can be seen, two endothermic peaks

were

observed

in

the

DSC

thermogram of quinacrine. Major peak at 264.33°C represents melting point of quinacrine (248-251°C) whereas other peak at 144.0°C might be representing dehydration of the compound. DSC thermogram of the physical mixture of quinacrine exhibited

and a

characteristic

blank

shift

nanoparticles

in

endothermic

quinacrine’s peak

as

observed at 281.66°C which may indicate interaction between quinacrine and blank

Fig. 3: DSC thermograms (A) and XRD spectra (B) of (i) Plain quinacrine, (ii) blank PEI stabilized NPs, (iii) physical mixture of quinacrine and blank PEI NPs, (iv) quinacrine loaded PEI NPs, and (v) quinacrine loaded BSA-coated NPs.

nanoparticles. While present in physical mixture, peaks for quinacrine disappeared in quinacrine loaded nanoparticles (Fig. 3A-iv and –v). Thus, DSC results clearly depicted quinacrine encapsulation in the PLGA nanoparticles. X-ray Powder Diffraction (XRD): To study the effect of encapsulation on crystalline nature of quinacrine, XRD studies were performed. Fig. 3B represents the XRD graphs for pure quinacrine (i), blank nanoparticles (ii and iii), and quinacrine loaded particles (iv and v). As can be seen multiple peaks representing crystalline nature of quinacrine were observed in the XRD graph of plain quinacrine. However, the characteristic crystalline peaks of quinacrine were absent when encapsulated in the PLGA nanoparticles. It can be depicted from the results that loading of quinacrine in the core of PLGA nanoparticles may reduce the crystallinity of quinacrine or change its nature from crystalline to amorphous form (Lee et al., 2015). Aerosolization of QA loaded Nanoparticles: The total dose recovered from the nebulizer cup and the NGI including FPF (%) for both the formulations, PEI stabilized NPs and BSA coated NPs are given in Fig. 4C. Also present are the estimated values for MMAD and GSD for the same. The graphical representation of the deposition pattern of the two formulations is given in the Fig. 4B, whereas the cumulative mass recovered as a function of Effective cut-off diameter is given in Fig. 4A. FPF data suggests good aerosolization performance and the MMAD values suggest that majority of the emitted dose will be delivered to respirable region of the lungs. Further, these data suggest that both BSA-coated and uncoated NPs are capable of efficiently delivering QA to the lungs. However, since uncoated and coated NPs are suspended in water, the long-term stability of the formulation may be compromised resulting in

pre mature drug release from the NPs and aggregation of NPs over time resulting in poor aerosolization performance. Future studies will include the development of a dry powder formulation of uncoated and coated QA NPs that will address these above-mentioned problems. (A)

(B)

(C)

Fig. 4: In-vitro aerosol performance of PEI stabilized NPs and BSA coated NPs (A) Cumulative mass recovered as a function of the effective cutoff diameter (B) Deposition pattern of quinacrine loaded nanoparticles in various stages of Next Generation Impactor (NGI). (C) Summary of critical data sets and particle attributes obtained from aerosolization study using NGI. Data represent mean ± SD (n = 3).

In-vitro Cellular Uptake in NSCLC Cell Lines: To detect nanoparticles in the cellular environment, fluorescence nanoparticles were prepared using a hydrophobic dye, coumarin-6 (C-6). Hydrophobic dyes typically release very slowly (negligible amount in specified time of cellular uptake studies) in aqueous external environment, hence clearly depicting the uptake of intact nanoparticle by the cells (Vaidya et al., 2018). Fig. 5A represents qualitative fluorescence images of A549 NSCLC cells incubated with C-6 loaded nanoparticles for 3 and 24 hours respectively. Qualitatively, PEI stabilized nanoparticles demonstrated higher uptake compared to plain NPs. Further, BSA coated NPs also demonstrated

higher uptake compared to plain NPs. It was also observed that nanoparticles retained in the cells for longer period of time as higher fluorescence intensity was observed after 24 hours of incubation. This confirms the higher uptake of cationic and BSA coated cationic particles, thus underlining sustained release characteristics of polymeric nanoparticles in cellular environment. To further determine effect of controlling surface positive charge on NPs’ cellular uptake, a flow

Fig. 5: (A) In-vitro cellular uptake of coumarin-6 (c-6) loaded PLGA NPs in A549 cells following 3 hours and 24 hours exposure. (B) Raw flow cytometric data representing higher cellular uptake of surface modified (PEI stabilized and BSA-coated) nanoparticles. (C) Quantification of cellular uptake of PEI stabilized and BSA coated PLGA NPs by flow cytometry analysis following 3-hour incubation expressed as relative mean fluorescence intensities (MFI) vs QA solution. Surface modified nanoparticles reported 2-fold increase in cellular uptake of courmarin-6. Data represent representative images (4A and 4B), and mean±SD (4C) (n=3; *p<0.05 vs plain PLGA NPs). Green: coumarin-6; Red: Cells stained with rhodamine phalloidin.

cytometry quantification experiment was performed after incubating cells with quinacrine loaded nanoparticles for 3 hours. As can be seen in Figs. 5B & 5C, mean florescence intensity after incubation with PEI stabilized, and BSA coated NPs showed 1.5-fold higher uptake compared to plain NPs (Figs. 5B & C). Higher uptake with cationic nanoparticles may be ascribed to chargebased interaction between positive charge of NPs and negative charge of cell membrane. Additionally, BSA also helps in cellular uptake of NPs. Hence, BSA coated particles with lesser positive charge (+10 mV vs +30 mV of PEI stabilized NPs) also showed similar uptake profile. In-vitro Anti-proliferative and Cytotoxic Studies: This set of experiments was aimed at improved therapeutic efficacy of quinacrine against nonsmall cell lung cancer cells by encapsulation in cationic nanoparticles. Cellular cytotoxicity of prepared NPs was determined in four different lung cancer cells (Fig. 6). It was observed that nanoformulation improved anti-proliferative activity of quinacrine in three cell lines, i.e., A549, H4006, and H157, while no significant difference was observed in H460 cells. It was also observed that IC50 value of quinacrine against A549 cells was reduced ~2.5-folds (3 μM of plain quinacrine vs 1.3 μM of BSA coated particles) when encapsulated in the BSA coated nanoparticles (Fig. 6A, Table 2). Similarly, in H4006 and H157 cells, IC50 values was reduced to ~2.5 and ~2-folds, respectively (Table 2). However, in H460 cells where IC50 for plain quinacrine was in submicron concentration, nanoparticles did not show any further improvement in activity. Improved anticancer activity of QA nanoformulation is consistent with higher uptake of NPs in the cellular uptake studies (Fig. 5).

As discussed earlier, while PEI is a phenomenal surface coating agent for imparting

positive charge, its toxicity is of major concern when used as an integral formulation component.

(A)

(B) 100

100

% Cell Viability

% Cell Viability

H4006

A549

120

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Fig. 6: Cytotoxicity profile of quinacrine (QA) loaded PEI stabilized and BSA coated PLGA NPs against different non-small cell lung cancer (NSCLC) cell lines (A) A549, (B) H4006, (C) H157, and (D) H460, after 72 hours of treatment. (E) Cellular toxicity of blank PEI stabilized PLGA nanoparticles in A549 NSCLC cells following 72 hours incubation. (F) Cytotoxicity of blank particles on Human Embryonic Kidney Cells (HEK293) following a 72-hour incubation. Blank NP concentrations were chosen based on corresponding quinacrine concentrations (in case of drug-loaded NPs). Data represent mean±SD of at least 3 individual experiments with n=6.

Hence, to confirm the toxicity of cationic nanoparticle against cancer cells, A549 cells were

incubated with equivalent concentrations

of

blank

nanoparticles

for

same

exposure time (72 hours). It was observed that blank nanoparticles did not show any significant toxicity against cancer cells at concentrations used for quinacrine loaded formulations (Fig. 6E). The toxicity profile of blank NPs (both PEI-Stabilized and BSA-Coated) was also analyzed on normal Human Embryonic Kidney cells (HEK293) and both type of blank

particles

had

negligible

toxicity on these normal cells, as indicated confirming

in

Fig.

negligible

6F,

thus

untoward

effect on normal human cells upon administration. Long-term

Efficacy

Studies:

Clonogenic Colony Formation Assay:

Fig. 7: Effect of treatments on clonogenic properties of A549 cells. (A) Representative images of the colonies after staining with crystal violet. (B) Quantitative representation of clonogenic assay as % reduction in number of colonies with plain quinacrine (QA) solution and QA loaded nanoparticles treatment as compared to control cells. Data represent mean±SD (n=3). *p<0.05, **p<0.01, ***p<0.001 vs control.

To see the effect of nanoformulation’s enhanced cellular uptake and sustained release properties, long term effect studies were conducted using clonogenic study. Fig. 7 represents the colony growth after incubating the A549 cells for 5 days in drug/formulation free media, following treatment with quinacrine or quinacrine-loaded NPs for 2 days. Representative images (Fig. 7A) show a significantly smaller number of colonies after treatment with drug-loaded NPs. Quantitatively, nanoformulations treatment resulted in significant (p<0.001 vs quinacrine solution treatment) reduction of colony formation (70% of the control) (Fig. 7B). The clonogenic assay measures the ability of drug treatment on the colony forming propensity of individual cells in heterogeneous population of cancerous cells (Anichini et al., 2018; Franken et al., 2006; Heydt et al., 2018). This assay also determines the ability of treatment on growth inhibition and reproduction on cellular level. Results of the study showed that nanoformulation treatment resulted in reduced number of colonies, which could be implicated to higher cellular uptake and prolonged release of quinacrine from nanoparticles. Cell Cycle and Apoptosis Analysis: To study the mechanism of higher anti-proliferative activity of quinacrine nanoformulations against NSCLC cells, cell cycle and apoptosis analyses were performed utilizing propidiun iodide (PI) based flow cytometric assay. PI staining method measures DNA content of the cells and is very rapid and reliable method for simultaneous estimation of cell cycle and apoptosis (Riccardi and Nicoletti, 2006). Results (Figs. 8A & 8B) of the study indicated that treatment of A549 cells with quinacrine-loaded nanoformulations (5 µM) resulted in cell cycle arrest at the G2-M phase. It was observed that plain quinacrine treatment at 5 µM did not have any effect on cell cycle progression whereas quinacrine loaded cationic nanoparticles (PEI-stabilized as well as BSAcoated) significantly increased the number of cells in G2-M phase. It was also observed that both

formulations decreased the number of cells in the G1-G0 phase after treatment (Fig. 8B). No differences were observed in the S-phase among all treatments. Furthermore, it was also observed that quinacrine when loaded in cationic PLGA nanoparticles (PEI-stabilized as well as BSAcoated) increased the apoptosis process in the NSCLC cells. Results of the study (Figs. 8C and 8D) showed that plain quinacrine demonstrated slightly higher apoptosis (p>0.05), as compared to control cells. However, nanoformulation increased the apoptosis activity by approximately 6folds compared to control and 3-fold compared to plain quinacrine (Fig. 8D). By analyzing the

Fig. 8: Effect of QA and QA-loaded nanoparticles on phases of cell cycle and apoptosis. Briefly, A549 cells were growth arrested for 24 hours, and were treated with quinacrine (5 µM; plain or NP encapsulated) for 72 hours. After 72 hours, cells were subjected to propidium iodide mediated cell cycle analysis. (A & B) Cell cycle analysis of A549 cells following 72 hour treatment with QA or QA-loaded NPs. QA treatment inhibited cellular proliferation by arresting G2/M phase, with significantly more G2/M arrest with QAloaded NPs. (C & D) Pseudo-apoptosis measurements for A549 cells showed that QA treatment induced apoptosis A549 lung cancer cell lines, which is in line with western blotting data. NPs showed better efficacy in apoptosis induction than drug alone. Data represent mean±SD (n=3-5). *p<0.05 as compared to plain quinacrine treatment.

data, it could be implicated that quinacrine induced a G2-M arrest eventually resulting in enhanced apoptosis, and thus cell death. It has earlier been reported by Preet et al 2011 that quinacrine increases sub-G1/apoptotic population at higher concentration (20 μM) after 24 hours of incubation. Hence, it could be concluded that higher apoptosis with nanoformulation treatment may be attributed to higher availability of quinacrine followed by increased nanoparticle-mediated cellular accumulation of the drug. Mechanism of Action: To further study the detailed mechanism of improved anti-cancer efficacy of quinacrine loaded nanoparticles against lung cancer cells, different molecular markers involved in cellular growth and proliferation were measured using western blot analyses. We analyzed the expression level of ph-p53, p53, p21, LC3B, p62, cleaved caspase-3, and cleaved PARP after exposure of A549 cells with quinacrine or quinacrine-loaded NPs for 72 hours (Figs. 9A & 9B). It was observed that cleaved caspase-3 level increased when the cells were treated with plain quinacrine which further increased after treatment with nanoformulations. Cleaved PARP level also increased after treatment with quinacrine loaded nanoparticles. PARP is involved in DNA repair process and hence breakdown of PARP could block DNA repair process, thus in turn inducing the apoptotic cascade (Boulares et al., 1999). This effect was also corroborated by DNA fragmentation assay using flow cytometry (Fig 8). P53 and p21 play an important role and are major contributors to cell cycle regulation and apoptosis. P53 acts independently or via p21 to arrest cell cycle and to induce apoptosis. We found that quinacrine nanoparticle treatment increased the activity of p53 (increased ph-p53/p53 ratio) and also increased the expression level of p21 (Fig. 9B). Thus, it could be implicated that cell cycle arrest and apoptosis induction by quinacrine treatment is via p53 dependent p21 signaling pathway (Mohapatra et al., 2012). Quinacrine is a well-known

autophagy inhibitor and acts via inhibiting autophagosome lysosome fusion similarly to chloroquine (Gupta et al., 2010). We studied the effect of quinacrine nanoformulations on autophagy and measured autophagy markers, LC3B-II and p62. It was observed that quinacrine increased the expression of LC3B-II after 72 hours treatment, which was further increased when A549 cells were treated with quinacrine-loaded cationic nanoformulations. LC3-II protein

Fig. 9: Effect on cellular markers of apoptosis and autophagy in A549 cells after treatment with quinacrine (QA) formulations at equivalent dose (5 µM). (A) Representative western blot images (B) Quantitative estimation. Data represent mean±SD (n=3). C: Control; QA: quinacrine; QNP: QA loaded PEI-stabilized PLGA NPs; QCT: QA loaded BSA-coated PLGA NPs.

accumulates in the double-membrane vesicle, autophagosome, during the autophagy process and degrades after fusion of autophagosome to lysosome. However, any dysregulation in autophagy leads to accumulation of autophagosomes in cytosol which results in accumulation of LC3-II (Liu

et al., 2017). However, increase in LC3-II is not inevitably related to reduction/inhibition of autophagy, as it may also be increased with increase in autophagy as increased autophagy also results in the formation of more autophagosomes with higher LC3-II (Wang et al., 2017). A quinacrine acts as a potent autophagy inhibitor by blocking the autophagosome lysosome fusion at the late phase of autophagy, we measured the expression of a cargo protein p62 that degrades during autophagy process (Wang et al., 2017). A cargo protein p62 plays a key role in the transport of several proteins and cell organelles to autophagosomes for further processing in autophagy cascade. During the final steps of autophagy cargo protein p62 degrades hence levels of p62 increase when autophagy process blocks at the late stage (Wang et al., 2017). Knowing the mechanism of quinacrine as autophagy inhibitor, delivery of higher amount of quinacrine in the cytosol may technically result in improved efficacy. In our study, we observed that quinacrineloaded nanoparticles resulted in the higher accumulation of p62. Thus, intracellular delivery of quinacrine using cationic nanoparticles exhibits better autophagy inhibition and improved apoptosis. All these mechanisms result in higher anti-proliferative activity of quinacrine nanoparticles. 3D-Spheroid Cell Culture Study: Repurposed drugs and formulations are usually screened across a variety of cell lines pertaining to a particular disease for evaluating their cytotoxic potential. Treatments are done on a monolayer of adherent cells, which result in a uniform distribution of drugs and formulations across all cells present in that monolayer. Physiologically, this is not the case. A physiological tumor is a solid mass of cells, present in multiple layers across all three dimensions. Drugs’ or formulations’ cytotoxic potential is directly proportional to its efficacy in penetrating the solid mass of cells and so a monolayer study may not be accurate enough to depict the true fate of treatments in in-vivo

scenarios. Animal models thus have been established for studying physiological fate of drugs and formulations. However, growing social concerns with animal models along with high experimental

(A)

(B)

(D)

Control

(C)

Quinacrine

PEI NPs

BSA NPs

1.5 µM

Fig. 10: Cytotoxic potential of quinacrine (QA) and quinacrine loaded PEI stabilized and BSA coated PLGA NPs on 3D-Spheroid Cell Culture model of A549 cells. Figure represents normalized (with respect to volume) growth of spheroids over a period of 15 days with (A) 1.5 µM QA and QA loaded NPs and (B) 0.78 µM of QA and QA loaded NPs. (C) Comparison of cell viability of 3D-spheroids between QA and BSA coated PLGA NPs. (D) Live/Dead cell staining assay for quinacrine (QA) and quinacrine loaded PEI stabilized and BSA coated PLGA NPs treated spheroids on day 15. Green stain indicates live cells and red stain indicates dead cells. Data represent mean±SD (n=3-6). **p<0.01, ***p<0.001, ****p<0.0001 vs control. Scale bar represents 400µM.

costs and inability to have high throughput screening (HTS) require us to explore alternatives. All these limitations can be overcome by utilizing in-vitro 3D cell culture models, which have been demonstrated to have comparable results to in-vivo models, in terms of their performance (Yang et al., 2017). Keeping this in mind, a 3D-spheroid cell culture study (3D-SCC) was designed to better predict the physiological interaction of QA loaded NPs with tumors. Two concentrations (1.5 µM and 0.78 µM) of QA and QA-loaded NPs were tested on A549 cell spheroids, as described in Methods. Fig. 10A represents higher concentration of treatments. It can be seen that the control

group of spheroids keep growing in size over a period of 15 days, whereas the growth of treated spheroids is significantly inhibited. Un-treated spheroids were 1.72±0.36 times larger on day 15 as compared to day 1 whereas BSA coated NPs were 0.94±0.04 times the size as on day 1, indicating inhibition in their growth. Fig. 10B represents lower concentration of treatments and similar results were obtained as for the higher concentrations with respect to spheroid growth inhibition. After 15 days of incubation, BSA coated NPs treated spheroids were found to be 0.99±0.10 times their size on day 1. This inhibition was significantly different than un-treated spheroids. Representative images of spheroids grown is shown in Figs. S1 and S2. While providing significant results, visual representation of spheroids may not accurately indicate the potency of treatments as simple imaging is not indicative of necrotic cells within the spheroid mass. To accurately evaluate the cytotoxic potential of treatments, a cell-titer GLO 3D assay was performed, which is represented in Fig. 10C. It can be clearly seen that there is no significant difference between viability of un-treated spheroids and QA treated spheroids (85±10.6%). However, a significant difference was observed in viability between un-treated spheroids and BSA coated NPs treated spheroids (71.1±7.0%), indicating effective tumor penetration and higher cellular necrosis. Live/Dead Cellular Assay: To further evaluate the potency of QA loaded NPs accurately, a live/dead cellular assay was performed using two fluorescent dyes followed by microscopic imaging. Calcein AM stains live cells green, owing to their cellular permeability and esterase binding capacity in live cells. On the other hand, ethidium homodimer III (EthD-III) is a DNA dye which is impermeable in live cells due to intact cellular membrane. This assay overcomes the limitation of visual representation of spheroids, by specifically labelling necrotic and live cells. As seen in Fig. 10D, higher number of

necrotic cells are present in BSA coated NPs treated spheroid groups as compared to PEI stabilized NPs treated spheroids, QA treated spheroids and un-treated spheroids. Treatment with lower concentration of QA and QA loaded NPs is showed in Fig. S3 This assay, coupled with cellular viability of 3D spheroids, clearly highlights the potency of QA-loaded BSA coated NPs in inhibiting cells in the form of a solid mass. CONCLUSION: With the aim of repurposing quinacrine as a nanoformulation for improved therapy of lung cancer, we have developed positively charged inhalable PLGA nanoparticles using PEI as stabilizer and BSA as a charge-neutralizing coating material. We were able to increase the entrapment efficiency of quinacrine in PLGA nanoparticles by using PEI as a component during the synthesis of PLGA nanoparticles. Further, use of BSA as a coating agent improved release profile and cellular uptake. Also, BSA-coated nanoparticles were found safer to use, due to controllable cationic charge. Antiproliferative activity studies demonstrated improved therapeutic efficacy of quinacrine-loaded nanoparticles compared to plain quinacrine. Cytotoxic potential on 3D cellular models revealed spheroid growth inhibition over a period of 15 days. Based on results from in-situ spheroid studies, we expect better efficacy of BSA-coated nanoparticles when tested in animal lung cancer models because BSA coating may increase accumulation of particles in the target region. In future studies, it could also be tested in combination with other chemotherapeutic agents to improve the therapeutic efficacy and also to reduce the acquired drug-resistance against established chemotherapeutic agents. ACKNOWLEDGEMENTS AND OTHER DISCLOSURES: This study was supported by start-up funds to Vivek Gupta by Keck Graduate Institute (KGI) and St. John’s University. N.S.K., S.K.S., V.P., and G.C. and were supported by graduate

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Graphical abstract

AUTHOR CONTRIBUTION STATEMENT Development of Inhalable Quinacrine Loaded Bovine Serum Albumin Modified Cationic Nanoparticles: Repurposing Quinacrine for Lung Cancer Therapeutics

Bhuvaneshwar Vaidya1, Nishant S Kulkarni2, Snehal K Shukla2, Vineela Parvathaneni2, Gautam Chauhan2, Jenna K Damon3, Apoorva Sarode4, Jerome V Garcia3, Nitesh Kunda2, Samir Mitragotri4, Vivek Gupta1,2*

1School

of Pharmacy

Keck Graduate Institute, Claremont, CA 91711

2College

of Pharmacy and Health Sciences

St. John’s University, Queens, NY 11439

3Department

of Biology

University of La Verne, La Verne, CA 91750

4John

A Paulson School of Engineering and Applied Sciences Harvard University, Cambridge, MA 02138

Author Name

Contribution and Credits

Bhuvaneshwar Vaidya, PhD

Conceptualization, Methodology, Validation, Investigation, Execution, Formal analysis, Writing – original draft, Software

Nishant S Kulkarni, MS

Methodology, Investigation

Snehal K Shukla, MS

Methodology, Investigation

Vineela Parvathaneni, MS

Methodology, Investigation

Gautam Chauhan, MS

Methodology, Investigation

Jenna K Damon, MS

Methodology, Investigation

Apoorva Sarode, BS

Methodology, Investigation

Jerome V Garcia, PhD

Methodology, Investigation

Nitesh K Kunda, PhD

Methodology, Writing – review and editing

Samir Mitragotri, PhD

Methodology, Investigation

Vivek Gupta, PhD

Conceptualization, methodology, Writing – review and editing, Supervision, Project administration, Funding acquisition, Resources