Cationically modified inhalable nintedanib niosomes: enhancing therapeutic activity against non-small-cell lung cancer

Lung cancer is the second-most frequently diagnosed cancer in the USA and is the leading cause of cancer deaths in both men and women [1]. It represents a huge global burden, with more than 2 million cases, thus topping the list as the most common cancer worldwide [2]. Of the two subtypes of lung cancer, non-small-cell lung cancer (NSCLC) accounts for approximately 85% of cases in the USA [3]. Nearly half of NSCLC patients die within a year of diagnosis largely because close to 70% of patients present with localized advanced or metastatic disease at the time of diagnosis [4,5]. Lung tumors have a high degree of molecular heterogeneity, and screening for specific genetic mutations is crucial for guiding clinical treatment [6]. Tumor genotyping has discovered gene alterations targetable by tyrosine kinase inhibitors, such as EGFR mutations, anaplastic lymphoma kinase rearrangements and ROS1 rearrangements [7]. In patients whose tumors have high expression of PD-L1, treatment with immune checkpoint inhibitors is indicated, although most patients eventually develop resistance [8]. Despite advances in targeted therapy and immunotherapy, the overall 5-year survival rate for NSCLC remains low at 24% and even worse at 7% for metastasized cancers [9]. The poor prognosis of lung cancer highlights the dire need to continually search for more effective treatments.

Nintedanib (nint) is a triple tyrosine kinase inhibitor, which binds to VEGFRs, PDGFRs and FGFRs [10,11]. Nint has been approved by the US FDA for treatment of idiopathic pulmonary fibrosis, chronic fibrosing interstitial lung diseases and systemic sclerosis-associated interstitial lung disease [12]. Nint has also been approved by the European Committee for the Advancement of Drugs for treatment of NSCLC in combination with docetaxel [10,11]. Nint is also reported to be explored for treatment of other solid tumors such as prostate cancer, renal cancer, colorectal cancer, ovarian carcinoma and melanoma [13,14]. Nint exerts its anticancer effects by binding to multiple pro-angiogenic receptor tyrosine kinases targeting tumor growth, cellular proliferation, homeostasis, invasion and angiogenesis [10,15]. Additionally, several in vitro studies have confirmed that nint is also reported to inhibit metastasis by repressing epithelial mesenchymal transition and thereby limiting the growth of the tumor [10,15]. Nint has been explored in several clinical trials for its efficacy against NSCLC in combination with other chemotherapeutic agents, wherein nint has demonstrated improvement in overall patient survival response in several mutant and resistant NSCLC patients and in patients otherwise displaying poor tolerance to conventional chemotherapy [16–18].

Nint is a class II drug of the biopharmaceutical classification system, exhibiting low dissolution and poor intestinal absorption resulting into extremely limited oral bioavailability of approximately 5% [13,19]. In addition, nint is reported to undergo first-pass metabolism in liver and efflux by P-gp transporters in intestines, thus requiring a higher dose or repeated administration to achieve therapeutic effects, which hinders the patient compliance [20]. In addition to its low bioavailability and required high dosing, the use of nint as monotherapy for NSCLC is also hindered due to dose-related side effects [21], limited route of administration (oral) [14] and severe interindividual pharmacokinetic variability due to high protein binding (∼98%) [20]. Therefore, it is essential to address the limitations of nint for establishing a successful treatment for NSCLC. We hypothesize that encapsulating nint in a robust and efficient drug delivery system coupled with inhalation as route of administration may be an effective method to overcome poor bioavailability and reduce dose requirements to improve the overall safety and efficacy of nint.

Nanocarriers have been extensively researched as a strategy for delivering therapeutic agents, as demonstrated by several FDA-approved products including Doxil^® as the first FDA-approved nanocarrier delivered drug, and Abraxane^®, an albumin nanoparticle-based breast cancer therapy [22]. Nanocarriers are known to have great potential for delivery of chemotherapeutic agents owing to improved deposition at tumor site, overcoming the biological barriers and reduced off-target adverse effects [23,24]. Large surface area, controlled release rate, surface tunability, improved stability and the biodegradable nature of nanocarriers enhance the therapeutic effectiveness of chemotherapeutic agents [25,26]. Moreover, nanocarriers offer flexibility to investigate potential possible routes of administration to overcome the existing limitations of drug molecules [27,28]. Inhalable nanocarriers have been explored for the treatment of lung cancer due to advantages such as local accumulation at site of action, large alveolar surface, bypassing first-pass metabolism resulting in reduced frequency of administration, decreased side effects and increased bioavailability [29–31]. Among the various nanocarriers for drug delivery, liposomes and niosomes have been widely used for inhalable drug delivery due to their potential to encapsulate both hydrophobic as well as hydrophilic drugs [25,32]. Niosomes are bilayered spherical drug delivery carriers composed of nonionic surfactants and cholesterol [27]. Their composition can be easily manipulated to optimize entrapment efficiency, drug release and cellular uptake [33]. Niosomes' unilamellar or multilamellar structure allows them to encapsulate both hydrophilic and lipophilic drugs, as well as to accommodate drug molecules with varying solubility [34]. Both in vitro and in vivo studies have reported that niosomes have the ability to alter pharmacokinetic properties, including improved bioavailability, delayed clearance and increased selectivity at site of action [35–37]. Niosomes have been researched as a vehicle for proteins and peptides, antineoplastic agents, antibiotics and anti-inflammatory agents through various delivery routes, as they offer good stability and biocompatibility with low cost and versatility in formulation [38,39]. In studies involving anticancer agents, inhalable niosomes have effectively increased tumoricidal activity and addressed dose-limiting toxicity thereby enhancing the therapeutic efficacy of the drug molecules [32,36,38,40,41]. Therefore, niosomes may present a potential strategy to overcome the limitations of nint and enhance its efficacy against NSCLC.

In this exploration, we aim to develop an effective niosomal formulation of nint as inhalable carriers for treatment against NSCLC. To our knowledge, this is the first study of its kind exploring nint-loaded niosomes as a strategy for treating lung cancer. We propose that incorporating nint in niosomes for pulmonary administration by inhalation will improve its chemotherapeutic activity at reduced dose and improve its overall safety.

Materials & methods

Materials

Span 60 was purchased from Millipore Sigma (MA, USA). 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was obtained from Avanti Polar Lipids (AL, USA) and cholesterol was purchased from Echelon Biosciences (UT, USA). Nintedanib ethanosulfate (nint) was procured from LC laboratories (MA, USA). HPLC-grade solvents were obtained from Fisher Bioreagents (Thermo Fisher Scientific, NH, USA). All other reagents and assay kits were purchased from third party vendors, details of which are provided in respective methods below.

Cell lines & culture

Human NSCLC cell lines A549, H2122, H1299, H358 and H460, and human embryonic kidney (HEK) (HEK-293) cell lines were obtained from American Type Culture Collection (VA, USA). NSCLC cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Corning Inc., NY, USA), and HEK-293 were grown in Dulbecco's Modified Eagle's Medium (Corning Inc.). Sodium pyruvate (1%), penicillin-streptomycin (1%) and 10% fetal bovine serum was added to both the media before using (Corning Inc.). For all the other reagents used, details are provided in their respective methods.

Preparation of niosomes

Nint-loaded niosomes were prepared using thin-film hydration technique as reported previously [42,43]. Briefly, a specific molar ratio of span 60 and cholesterol for a total of 10 mM (film-forming constituents) were dissolved in chloroform: a methanol (4:1) mixture containing 1 mg of nint in a round-bottom flask. For DOTAP containing niosomes, ∼1 mg of DOTAP (representing 5% of the total weight of span 60) was added in the organic solvent mixture. Thin film was prepared by using rotary evaporator (WG-EV311, Wilmad Lab-glass, NJ, USA). The solvent was evaporated at 60°C for 45 min under vacuum maintained at 300 Pa pressure. The obtained thin film was hydrated using phosphate-buffered saline (PBS) (pH 7.4) at 60°C for 45 min. The niosomal solution was placed in an ice bath and subjected to probe sonication for 3 min at an amplitude of 40% to obtain uniform nano-sized niosomes. To separate the unentrapped/free drug from obtained niosomal dispersion, the dispersion was washed by centrifuging at 20,000×g for 15 min. The washed niosomes was finally resuspended in 1 ml of PBS.

Blank niosomes were prepared as described above but without addition of nint. Similarly, coumarin-loaded niosomes were made by addition of coumarin-6 instead of nint. All the niosomal formulations were stored at 4°C until further use.

Characterization of niosomes

Vesicle size, size distribution & zeta potential

Vesicle size and size distribution (polydispersity index) of all formulations were evaluated using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Surface charge of the vesicles, also known as zeta potential, was measured by determining the electrophoretic mobility of vesicles under an electric field using the Zetasizer Nano ZS (Malvern Instruments).

Encapsulation efficiency

The amount of drug encapsulated in the formulations was measured by lysing the formulations. Briefly, 20 μl of formulation was subjected to lysis using a mixture of 20 μl of chloroform, 20 μl methanol and 1960 μl solvent mixture of 1:1 acetonitrile:water. The mixture was centrifuged at 22,000×g for 45 min to separate the drug from the lysed formulation. The supernatant was then analyzed using ultra-performance liquid chromatography (UPLC) (Waters Inc., MA, USA) to determine the amount of drug present.

Percentage encapsulation efficiency and percentage drug loading was calculated using the following Equations 1 & 2:

Analysis of nint using UPLC

Nint was quantified by using the Waters ACQUITY UPLC™ (Waters) method as reported previously [44]. The samples were analyzed using ACQUITY UPLC™ HSS T3 C18 column. The separation phase used was orthophosphoric acid at 0.1% concentration and comprised 35% v/v of the mobile phase, while acetonitrile was used at 65% v/v to complete the mobile phase. The mobile phase was eluted in isocratic mode at a flow of 0.55 ml/min. The detection wavelength used was 266 nm. Data were collected and analyzed using Empower 3.0 software (Waters).

Cryo-transmission electron microscopy

Morphological analysis of niosomes was performed using cryo-transmission electron microscopy (cryo-TEM) (FEI Titan Halo 80-300, FEI, OR, USA). Briefly, niosomes sample was placed on lacey carbon-coated copper grids (300 mesh, Ted Pella Inc., CA, USA) to form a very thin film. The film on the sample was placed in liquid nitrogen and then placed into a 626 cryospecimen holder (Gatan, Inc., CA, USA) and imaging was performed at 100 kV. The images were captured using 4k × 4k Ceta 16M complementary metal oxide semiconductor sensors camera (Gatan, Inc.).

Differential scanning calorimetry studies

Calorimetric analysis of the niosomal samples were conducted using differential scanning calorimetry (DSC 6000, PerkinElmer, MA, USA) connected with intracooler accessory. Nint was used as such for analysis while blank nio and nint nio were lyophilized before analysis using LABCONCO FreeZone freeze dryer (MO, USA). The physical mixture of blank nio and nint was prepared by blending nint and lyophilized blank nio in mortar and pestle. The samples were loaded in an aluminum pan which was sealed using a lid and the samples were analyzed. An empty sealed aluminum pan was used as reference. The samples were analyzed over 50–300°C temperature range.

Powder x-ray diffraction studies

Powder x-ray diffraction studies were conducted with a XRD-6000 (Shimadzu, Kyoto, Japan) using copper source to generate Kα radiation (λ of 1.5418 Å). Briefly, the sample was spread on a microglass slide such that it resulted in a thin film. The samples were analyzed at a scanning rate of 2° 2θ/min over the 10–80° 2θ range.

In vitro drug release

In vitro release studies of niosomes were performed using Slide-A-Lyzer dialysis cassettes (3000 molecular weight cut-off), (Fisher Thermo Scientific). Briefly, the dialysis cassettes were prehydrated using PBS pH 7.4, and ∼0.3 ml of formulations were then loaded in the hydrated cassettes. The dialysis cassettes were then immersed in 100 ml of release media comprising PBS (pH 7.4) along with 1% tween 80 added to maintain the sink conditions [45]. The release samples were maintained at 37°C with constant stirring at 100 r.p.m. Samples were withdrawn at specific time intervals and an equivalent volume of fresh media was replenished. The amount of nint released in withdrawn samples was analyzed using UPLC as described previously in the section analysis of nint using UPLC.

Cellular uptake studies

Qualitative cellular uptake using fluorescence microscopy

Cellular uptake studies were performed using coumarin-6-loaded niosomes following a previously reported method [46]. Briefly, A549 cells were seeded in eight-chambered imaging cover glass (Eppendorf, NY, USA) at density of 1.0 × 10⁴ cells/chamber and incubated overnight at 37°C with 5% CO₂. For treatment, coumarin-6 was dissolved in dimethyl sulfoxide (DMSO) at a very high concentration and was then diluted to the required concentration using RPMI 1640 media. The concentration was adjusted such that the sample would contain no more than 1% DMSO. Coumarin-6-loaded niosomes were diluted to the required concentration using RPMI 1640 media. After incubation, cells were treated with either coumarin-6 and coumarin-6-loaded niosomes (reg nio and DO nio) at 1 μg/ml of coumarin concentration for 1 h and 3 h period. Following the treatment period, cells were washed thrice with ice-cold PBS (1×) and were fixed using 4% paraformaldehyde for 10 min and again washed using PBS. After washing, staining of the cells were performed using VECTASHEILD hardset mountant containing 4′,6-diamidino-2-phenylindole (Vector laboratories, CA, USA). The stained cells were then imaged using fluorescent microscopy (Evos FL, Thermo Fisher Scientific) at 20× magnification.

Quantitative cellular uptake using direct measurement

Additionally, the amount of coumarin-6 internalized by A549 cells was quantified by determining the fluorescence intensity of coumarin-6 after lysing the cells. Briefly, A549 cells were seeded in a six-well plate at a density of 1 × 10⁶ cells/well and incubated at 37°C with 5% CO₂. After overnight incubation, cells were treated for 1 h and 3 h with different treatment groups; one group comprised reg nio, and the other comprised DO nio at 1 μg/ml concentration of coumarin. Media was added to the cells considered as control. Following the respective treatment period, cells were rinsed twice using ice-cold PBS, and cells were then scraped and centrifuged. The cell pellet obtained after centrifugation was lysed by adding 100 μl of DMSO, centrifuged, and the supernatant was used for analysis. Florescence intensity of coumarin-6 present in the cell lysates of different treatment groups was measured at excitation wavelength (λ_ex) of 420 nm and emission wavelength (λ_em) of 465 nm using Tecan plate reader (Tecan Group Ltd, Männedorf, Switzerland).

Quantification of intracellular uptake using fluorescent cell counting

Cellular uptake using Nexcelom^® Cellometer Vision (Nexcelom, MA, USA) was performed in A549 cells. Briefly, A549 cells were seeded at a density of 100,000 cells/ml in a tissue-culture-treated petri dish having a growth area of 60 cm² and incubated overnight at 37°C/5% CO₂. Next day, cells were treated with different treatment groups: plain coumarin, coumarin-reg nio and coumarin-DO nio at a concentration of 1 μg/ml and incubated for 3 h. After 3 h, cells were trypsinized, centrifuged at 5000 r.p.m. for 5 min and pellet was redispersed in 50 μl of 1× sterile PBS and imaged on Nexcelom^® cellometer. For quantification, geometric mean of the particles that were internalized by the cells were measured by the Nexcelom^® software and this data was plotted using GraphPad Prism Version 9.4.0.

In vitro aerosolization efficiency

In vitro aerosolization efficiency of nint-loaded niosomes was evaluated using Copley-170 Next Generation Impactor™ (NGI) (MSP Corporation, MN, USA) following a previously reported method [47]. Briefly, NGI was refrigerated at 4°C for 2 h prior to performing the experiment to minimize the solvent evaporation and avoid the shrinkage of nebulized droplets which may impact particle deposition [48]. Then, 2 ml of nint DO nio was loaded in the nebulizer cup of PARI LC PLUS (PARI Respiratory Equipment, Inc., VA, USA) nebulizer kit. Aerosolization study was performed under vacuum maintained at 15 l/min for 4 min and the formulation was deposited in different stages of NGI. Samples from various stages were collected using acetonitrile:water (50:50) and centrifuged at 22,000×g for 45 min. The supernatant was analyzed using UPLC as described previously in the section analysis of nint using UPLC.

The emitted dose was assessed as the amount of the drug deposited in region from mouth to stage 8 of the NGI. Fine particle fraction (%) was calculated as the fraction of emitted dose deposited in NGI stages with d_ae <5.39 μm. Mass median aerodynamic diameter (MMAD) representing d_ae <5.00 μm and geometric standard deviation was determined from log-probability analysis.

Cytotoxicity studies

In vitro cytotoxicity of reg nio and DO nio along with nint was evaluated in six NSCLC cell lines: A549, H2122, H1299, H358 and H460 as reported earlier with slight modifications [48,49]. The chosen NSCLC cell lines are different in their cell types as well as their KRAS mutation status, as can be seen in Table 1 [50]. The goal of the study was to evaluate the therapeutic effect of the drug on the cells influenced by their cell type or mutation status.

Briefly, cells were seeded at 2.5 × 10³ cells/well in a 96-well plate and incubated overnight for adherence at 37°C and 5% CO₂. Nint was dissolved directly in the respective cell culture media used for the treatment and diluted further to obtain the desired concentration. Cells were treated at different concentrations of nint ranging from 0.39–50 μM and incubated for 72 h. Corresponding volumes of nint-loaded niosomes were calculated based on the percentage drug entrapment efficiency. Post treatment period, the treatment was aspirated and 1-methyltetrazole-5-thiol (MTT) (Fisher Bioreagents, NH, USA) was added in each well. Furthermore, DMSO was added to MTT solution to dissolve the formazan crystals. Cytotoxicity of the treatment groups was determined by using Tecan Spark 10M plate reader (Tecan Group Ltd, Männedorf, Switzerland). The absorbance of dissolved formazan crystals was determined at a wavelength of 570 nm. Cell viability percentage was calculated by comparing the absorbance of treatment groups against control (no treatment media). IC₅₀ values were determined using GraphPad Prism Software Version 6.0 (CA, USA).

The cytotoxicity of blank niosomes was investigated against A549 NSCLC cancer cells, while the safety of blank niosomes was determined on HEK-293, normal human embryonic kidney cell line using the MTT assay method as described above.

Scratch assay: assessing impact on metastasis

Scratch assay, also known as cell migration assay or wound healing assay, is used to simulate metastatic properties of tumorigenic cells and to evaluate the efficacy of treatments [51]. For this experiment, A549 cells were seeded at a density of 1.0 × 10⁵ cells per well in 24-well tissue-culture-treated plates and incubated overnight for adherence at 37°C/5% CO₂. The following day, a scratch was made in each well using 100 μl sterile pipette tip and the cells were washed twice with PBS to remove all the nonadhered cells. Treatment was added to the respective well: nint and nint DO nio at two different concentrations; 1.25 μM and 7.5 μM. media (no treatment) was added to the cells used as control. Scratch images were captured at 0 h (after scratch formation but prior to treatments), 24 h and 48 h at the same area at each time point using inverted microscope (LAXCO, WA, USA) at 10× magnification. The percentage scratch closure caused due to migration of cells over the scratch was evaluated by measuring and comparing the width of the scratch using ImageJ software (version 1.44).

Clonogenic assay

Clonogenic assay was performed to assess the in vitro survival ability of single cells which can further reproduce to form colonies [52]. This assay was performed using the protocol as published previously [51]. Briefly, A549 cells were plated in a six-well plate at a seeding density of 2.5 × 10² cells per well and incubated overnight at 37°C/5% CO₂. The following day, the cells were treated with nint and nint DO nio at 1.25 μM and 7.25 μM concentration and incubated for 48 h at 37°C and 5% CO₂. After incubation, the treatment was aspirated and replaced with fresh media. Furthermore, the cells were cultured for 7 days by replacing the media every 2 days. After the culturing period, the media was aspirated, and cells were washed with ice-cold PBS (pH 7.4) twice. The cells were then fixed using 4% paraformaldehyde and stained using 0.2% crystal violet (Fisher Bioreagents), followed by rinsing the cells thrice with water to remove excess dye and air drying the plate. Images of the colonies were captured using a digital camera and the colonies were counted using OpenCFU colony counter software [53].

In vitro tumor simulation studies

In vitro tumor simulation studies, also known as 3D spheroids study or 3D cell culture studies, have been widely used to evaluate the effectiveness and penetration ability of nanoformulations in a 3D tumor [54]. This study was performed in accordance to previously published protocol with slight modifications [55,56]. Briefly, A549 cells were seeded at a density of 5.0 × 10² cells/well in Corning™ 96-Well Ultra-Low Attachment Treated Spheroid Microplates and incubated for 72 h at 37°C and 5% CO₂. After incubation, cells were treated with nint and nint DO nio in two different treatment regimens: 1) single dose, where treatment was provided only once at the start of experiment and 2) multiple dose, with treatment at every 72 h until day 12. While providing treatment, only half volume of media was replaced with treatment (2× concentration) to minimize the chance of tumor aspiration. Spheroid images were captured at predetermined time intervals using LMI-6000 inverted microscope (LAXCO, WA, USA) at 10× magnification. The therapeutic effectiveness of treatment was evaluated by following methods.

Spheroid volume

Spheroid volume was calculated using spheroid diameters obtained from images of spheroids using ImageJ software (NIH, MD, USA).

3D cell viability

3D cell viability in each spheroid mass was measured using CellTiter-Glo^® (Promega Corporation, WI, USA). On day 12, after imaging the spheroids, 100 μl media was aspirated out of 200 μl media in the well. Following this, 100 μl of reagent was added to each well. The plate was mixed very gently for 5 min and incubated in the dark for 30 min. Luminescence was measured using Tecan Spark 10M plate reader (Tecan Group Ltd). Percentage cell viability was calculated by equating the luminescence reading of treatment group to control group.

Live–dead cell assay

Live–dead florescence cell assay was performed using the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Biotium, CA, USA). The assay comprises of two dyes: calcein AM, which stains live cells, and ethidium homodimer-III, which stains dead cells. Briefly, at the end of 3D spheroid experiments (day 12), all the media was carefully removed from the wells and 100 μl reagents were added as per the manufacturer's protocol. Furthermore, the plate was kept at room temperature in the dark for 30 min. Fluorescent cells in spheroids were imaged using florescence microscope EVOS FL cell imaging system (Thermo Fisher Scientific) with 10× magnification. Fluorescent filters used for the study were green fluorescent protein (GFP) for live cells and red fluorescent protein (RFP) for dead cells. Florescence intensity was quantified using ImageJ software (NIH, MD, USA).

Statistical analysis

All the results are addressed as mean ± standard deviation with at least n = 3 unless otherwise mentioned. Statistical significance of data was evaluated using student's t-test, one-way analysis of variance and Tukey's post hoc multiple comparison using GraphPad Prism 6.01 (CA, USA). p < 0.05 was considered statistically significant.

Results

Physicochemical characterization of niosomes

Vesicle size, size distribution & zeta potential

Vesicle size of niosomal formulations assessed using dynamic light scattering are shown in Table 2. As can be seen, formulation (F)1 with 9:1 span 60: cholesterol ratio displayed 195.5 ± 5.0 nm vesicle size. Upon increasing the concentration of cholesterol, the vesicle size was found to be reduced with 182.4 ± 3.7 nm (F2, 7:3, nint reg nio) and 176.1 ± 4.5 nm (F3, 5:5), as illustrated in Table 2. These results are suggestive of cholesterol concentration influencing the niosomes' vesicle size in the formulation, wherein increase in cholesterol concentration resulted in decreased vesicle size. This can be attributed to the fact that increase in the cholesterol concentration causes increased hydrophobicity of the niosomes' bilayer which in turn reduces the surface free energy resulting into decreased vesicle size [57]. However, addition of cationic lipid DOTAP (F4, 7:3, nint DO nio) resulted into moderate increase in vesicle size with 246.2 ± 2.3 nm value. The increased vesicle size is suggestive of the interaction between the drug molecules, DOTAP molecules and the polar heads of the cholesterol molecules. This interaction causes increase in the volume of the aqueous niosomal core due to charge repulsion separating the bilayers thereby increasing the vesicle size [58,59].

Size distribution of the particles were evaluated in terms of polydispersity index (PDI) (Table 2). PDI values of all the formulations were found to be in the range of 0.02–0.2. Low PDI values of niosomal vesicles indicate homogeneity, narrow dispersity and absence of agglomeration in all the developed formulations.

Surface charge of the vesicles, also known as zeta potential, governs their biological properties including cellular uptake and internalization of the vesicles. Additionally, surface charge also determines the stability of the colloidal system, as the charged particles tend to repel one another and therefore prevent aggregation due to adequate electrostatic stability. Table 2 shows the zeta potential values of all the formulations. As can be seen, F1 possess -60.5 ± 3.6 mV zeta potential. Decreased span-60 concentration in the formulation tends to reduce the zeta potential value with -48.5 ± 5.1 mV (F2, 7:3, nint reg nio) and -40.2 ± 2.5 mV (F3, 5:5), as demonstrated in Table 2. This can be due to reduction in the unsaturated alkyl chain heads of span 60 in the formulation, leading to decrease in the surface charge. Inclusion of cationic-charge-inducing agent, DOTAP, in the formulation positively increases the zeta potential value from -48.5 ± 5.1 mV (F2, 7:3, nint reg nio) to -20.5 ± 1.9 mV (F4, 7:3, nint DO nio), as in Table 2.

Encapsulation efficiency

Drug encapsulation studies were performed by lysing the formulation to assess the amount of nint encapsulated in niosomes, as shown in Table 2. F1 comprising of span 60:cholesterol (9:1) demonstrated 33.4 ± 9.1% encapsulation efficiency with 1.5 ± 0.4% drug loading capacity. Increase in the cholesterol amount in F2 (nint reg nio) resulted in significant (p < 0.0001) 2.2-fold increase in drug encapsulation with 73.1 ± 2.7% and 3.1 ± 0.4% drug loading (Table 2). However, further addition of cholesterol caused significant (p < 0.05) 1.3-fold decrease in drug entrapment resulting in 52.4 ± 4.1% encapsulation efficiency along with 2.4 ± 0.2% drug loading capacity (F3, Table 2).

Incorporation of positively charged lipid, DOTAP (F4, nint DO nio, Table 2), in niosomes displayed increase in the percentage encapsulation efficiency with 73.1 ± 2.7% against 65.1 ± 8.0% (F2, nint reg nio) (Table 2). The percentage drug loading capacity of F4 was observed at 3.4 ± 0.1%. Based on the obtained results, F2 and F4 with the highest encapsulation efficiency were selected as the optimized formulations for further studies.

Morphological analysis using cryo-TEM

The morphology of nint reg nio and nint DO nio (F2 and F4, Table 2) was evaluated using cryo-TEM. As seen in Figure 1A, both the niosomal formulations displayed spherical vesicle morphology and homogenous dispersity with no aggregation. The vesicle size of nint reg nio was found to be <200 nm while nint DO nio displayed ∼300 nm (scale bar on the image), which is in accordance with the dynamic light-scattering data (Table 2).

Differential scanning calorimetry studies

Results of calorimetric studies performed on several samples are illustrated in Figure 1B. Nint demonstrated a sharp endothermic peak at 305.0°C (Figure 1B[i]), representing the melting point of the drug as reported in the literature [60]. Thermal analysis of blank niosomes as shown in Figure 1B(ii) displayed small endothermic peak at 50.1°C, indicating glass transition temperature of span 60 [61]. The physical mixture of nint and blank niosomes demonstrated two endothermic peaks at 50.3°C and 305.8°C representing span 60 and nint, respectively (Figure 1B[iii]). The peak at 50.3°C representing the moisture was found to be absent in blank niosomes and drug-loaded niosomes. This can be explained by the absorption of moisture from the environment while physically blending blank nio (lyophilized) with nint to prepare the physical mixture for analysis. During this blending, the lyophilized blank nio might have absorbed moisture from the environment, which may have resulted in the endothermic peak at 50°C. In addition, plain nio and drug-loaded nio were lyophilized and directly subjected to analysis, thereby resulting in minimal contact with environmental moisture. A thermogram of nint-loaded niosomes (Figure 1B[iv]) displayed a glass transition peak of span 60 at 49.8°C, while the endothermic peak of nint was found to be absent. These results indicate the successful loading of nint in the niosomal core which may be dispersed homogenously in the noncrystalline form.

Powder x-ray diffraction studies

Diffractogram of nint demonstrated several characteristic crystalline peaks at 9.4°, 11.5°, 14.1°, 17.3°, 18.7° and 19.9° 2θ values as shown in Figure 1C(i), indicating the crystalline nature of nint [60,62]. Blank niosomes displayed crystalline peak at 31.8° 2θ value which can be attributed to the presence of span 60 in the formulation (Figure 1C[ii]). Diffraction studies of physical mixture of nint and blank niosomes (Figure 1C[iii]) displayed the respective crystalline peaks of both the components with diminished intensity. The diffractogram of nint-loaded niosomes demonstrated the span 60 crystalline peak at 31.6° 2θ value, while the characteristic peaks of nint was found to be absent. Data of diffraction studies further support the results of calorimetric study indicating encapsulation and homogenous distribution of nint in the niosome vesicles.

In vitro nint release

Figure 1D represents the in vitro release profile of the formulations evaluated via membrane diffusion technique. As seen, all the formulations showed biphasic drug release pattern, wherein an initial burst release was observed within 30 min of release followed by a slower or controlled release of drug. F1 showed the highest burst release with 60.5 ± 6.9% drug released in 30 min followed by slower drug release (Figure 1D). This can be attributed to the permeation and/or desorption of nint molecules present in close vicinity to the niosomes' surface [63]. F2 resulted into decreased initial burst release of 50.2 ± 6.6% drug released, while F3 resulted into further reduction with 44.4 ± 5.3% initial burst release as seen in Figure 1D. These results suggest that increase in the cholesterol amount in developed niosomal F1–F3 resulted in decreased initial burst release and provided controlled drug release from the vesicles. This can be accredited to the stringent packing of the lipid bilayer due to cholesterol as reported previously which helps to prevent the drug leakage from the niosomal vesicles [56,64].

Inclusion of positively charged lipid, DOTAP as in F4, demonstrated a further decrease in the initial burst release. F4 displayed 40.3 ± 3.9% initial burst release as compared with 50.2 ± 6.6% for F2 having the same molar ratio composition for span 60 and cholesterol (Figure 1D). Additionally, F4 displayed the highest controlled release of nint among all the other formulations with 59.7 ± 2.6% drug release at the end of 72 h.

Cellular uptake studies

Qualitative cellular uptake using fluorescence microscopy

Cellular uptake studies were performed to evaluate the impact of niosomes on the internalization of entrapped drug. In this study, we have used coumarin-6-loaded niosomes (reg nio and DO nio) instead of nint due to their inherent florescence, as nint does not possess any florescence. The florescence images in Figure 2A show that niosomes demonstrated increased internalization of coumarin-6, as seen in the cytoplasm surrounding the nuclei. Among both the niosomes, DO nio demonstrates enhanced internalization of coumarin-6 compared with reg nio. Moreover, the internalization of niosomes was also found to be time dependent, wherein increase in incubation time results in increase in fluorescence intensity of coumarin-6, as seen in Figure 2A.

Quantitative cellular uptake using direct measurement

Additionally, the amount of coumarin-6 internalized in A549 cells was also quantified to further validate the results obtained from imaging. As seen in Figure 2B, coumarin-6 was able to internalize the A549 cells with no significant change observed over increased time interval. Incubation of the niosomes in A549 cells displayed significant (p < 0.05 for reg nio and p < 0.001 for DO nio) increase in fluorescence intensity as compared with coumarin-6, as seen in Figure 2B. DO nio demonstrated ∼1.6 fold significant (p < 0.05) increase in fluorescence intensity as against reg nio after incubation for 1 h. After 3 h, DO nio exhibited ∼1.5 fold significant (p < 0.001) increase in fluorescence intensity as compared with reg nio (Figure 2B). These results suggest that encapsulation of nint in niosomes significantly enhances the internalization of drug in the cells.

Quantification of intracellular uptake using fluorescent cell counting

Fluorescent images of cells stained with coumarin is widely used for qualitative assessment. However, it fails to provide the quantitative measurement of actual particle accumulation inside the cells. For this reason, cellular uptake using Nexcelom^® Cellometer Vision (Nexcelom) was performed and to eliminate the bias; cells supplied with media were taken as control because cells themselves emit some sort of fluorescence. As shown in Supplementary Figure 1, following 3 h incubation with 1 μg/ml coumarin-6 concentration, a significantly higher fluorescence shift was observed with coumarin-DO-nio, as compared with both plain coumarin control and coumarin-nio. When the results were quantified, GFP intensity was found to be the highest for DO nio as compared with any other treatment group (Supplementary Figure 2). The control group showed minimal florescence. DO nio treatment resulted in tenfold higher florescence intensity as compared with reg nio and coumarin. Upon quantification of geometric mean, it was found that DO nio displayed ∼threefold increase in the GFP intensity compared with plain coumarin, while reg nio displayed no significant difference in the fluorescent intensity as compared with coumarin. These results are in accordance with our imaging study and the quantification study performed, suggesting the enhanced cellular uptake of DO nio in the NSCLC cells.

Cytotoxicity studies

The aim of developing nint-loaded niosomes was to reduce the dose of nint for the chemotherapeutic action, and hence enhance its therapeutic activity. In vitro cytotoxicity of optimized formulations were assessed on five different NSCLC cell lines via MTT assay. Figure 3A–E represents the cytotoxic potential of the treatments in five NSCLC cell lines – A549, H2122, H1299, H358 and H460, respectively. As can be seen, results demonstrate a significant reduction in the IC₅₀ values upon treatment with nint DO nio as against nint reg nio and nint (Table 3). As can be seen, nint DO nio resulted into ∼2.3-fold reduction (p < 0.01) in IC₅₀ value in A549 cells with 1.5 ± 0.8 μM as compared with 3.5 ± 0.5 μM (nint) and 3.4 ± 0.4 μM for nint reg nio (Figure 3A & Table 3). In H2122 cells (Figure 3B & Table 3), the cytotoxicity of nint DO nio resulted in ∼1.8 fold significant (p < 0.05) reduction in in IC₅₀ value with 1.8 ± 0.3 μM as compared with nint (3.3 ± 0.1 μM). Cytotoxicity of nint DO nio displayed significant (p < 0.0001) reduction in IC₅₀ values in H1299 and H358 cells as well, as seen in Figure 3C, D & Table 3. The IC₅₀ value of nint ∼5 μM was reduced by nint DO nio ∼2.5 fold in H1299 cells (2.1 ± 0.8 μM) and ∼4.5 fold in H358 cells (1.1 ± 0.4 μM). Assessment of cytotoxicity in H460 cells displayed significant reduction in IC₅₀ values with 4.8 ± 0.6 μM for nint compared with 1.3 ± 0.5 μM for nint DO nio (Figure 3E & Table 3). These findings indicate superior cytotoxic potential of niosomal formulations against the NSCLC cells as compared with plain nint. Among the niosomal formulations, nint DO nio displayed significantly higher cytotoxicity compared with nint reg nio: this can be accredited to the enhanced intracellular internalization and accumulation in the cells which is in agreement with our cellular uptake studies (Figure 2). Based on these results, we performed all further experiments using nint DO nio as our optimized formulation.

We performed cytotoxicity studies using blank reg nio and blank DO nio in A549 cells and HEK cells. As seen Figure 3F, blank particles of reg nio and DO nio demonstrated >95% cell viability in A549 cells at 3.125 μM, and twice the IC₅₀ of nint DO nio, after incubation for 72 h. In addition, the cell viability was >88% at 6.25 μM, four-times the IC₅₀ for nint DO nio (Figure 3F). The IC₅₀ of blank DO nio in A549 cells after 72 h incubation was found to be 20.2 μM. These results establish optimized niosomes as nontoxic delivery carriers and confirm that the cytotoxic effects are produced by nint, released intracellularly. We have also evaluated the safety profile of the developed niosomes in normal cells, HEK, as shown in Figure 3G. The data revealed that both the blank niosomes had >85–90% cell viability after incubation for 72 h at concentration ranging from 0.32–6.25 μM with an IC₅₀ value of 47.3 μM for blank DO Nio. These findings indicate the safety and tolerability of DOTAP in the niosomal formulation when used at much higher concentrations than the IC₅₀ values.

In vitro aerosolization efficiency: assessing pulmonary deposition behavior

Local delivery to the lungs has been widely explored for delivery of therapeutics against various respiratory diseases due to increased local accumulation and reduced exposure to other organs [65]. Aerodynamic properties govern the deposition behavior of inhalable delivery carriers and reflect their ability to reach their target site [66]. Our primary objective was to develop inhalable delivery carrier for delivering nint, so as to achieve localized accumulation in the lungs, that would require reduced dose to demonstrate the antitumor activity. The aerodynamic assessment of nint DO nio was performed using NGI, and the results are demonstrated in Figure 4. Pulmonary deposition pattern of the formulation is represented graphically in Figure 4A wherein niosomes were found to be largely deposited in stage 3 and below of the NGI representing the broncho-alveolar deposition. Cumulative deposition of mass of particles in each stage of NGI as a function of effective cut-off diameter is represented in Figure 4B. As can be seen, 63.1 ± 3.3% of niosome vesicles are deposited below the effective cut-off diameter of 3.30 μm which represents the stage 4 of NGI. From the aerosolization experiment, it was found that the niosomes possessed fine particle fraction of 78.7 ± 2.5% suggesting that niosomes possessed good aerosolization properties. MMAD of 4.3 ± 0.2 μm and geometric standard deviation of 2.0 ± 0.1 as shown in Figure 4C further confirm that the majority of the emitted dose will reach the respirable region of the lungs. Several studies have reported that MMAD value of 2–5 μm is essential for deposition in the peripheral region of the lungs [46].

Assessing impact on metastasis: scratch assay

The developed nint DO nio formulation was assessed against the metastatic property of NSCLC cells using scratch assay. Scratch assay evaluates the influence of treatment on the migration of cells, an important property which governs the metastasis process [67,68]. Figure 5 represents the influence of treatment of nint and nint DO nio on A549 cells at 1.25 μM and 7.5 μM concentrations. As can be seen in Figure 5A, scratch was fabricated at 0 h (pretreatment), and migration of cells was observed in control (media only) group at 24 h, leading to complete closure of the scratch at 48 h. Treatment with nint and nint DO nio resulted into inhibition of scratch closure as compared with the control. This inhibition was found to be dose dependent with prominent effect being observed at higher dose i.e., 7.5 μM (Figure 5A). Scratch closure percentage was quantified using Image-J software (NIH, MD, USA) with respect to the no-treatment control cells (0 h). After 24 h, treatment with 1.25 μM concentration of nint displayed 74.6 ± 4.8% scratch closure while migration of cells was found to be significantly inhibited upon treatment with nint DO nio demonstrating 62.6 ± 4.1% scratch closure (p < 0.05); this closure increased further with dose, wherein nint displayed 31.6 ± 9.2% scratch closure and nint DO nio exhibited 15.9 ± 3.9% scratch closure. Upon treatment for 48 h, the inhibitory effects against cell migration were found to be significantly (p < 0.001) intensified, with nint DO nio (1.25 μM) showing 75.2 ± 5.5% scratch closure against 100% scratch closure by nint. Increase in nint DO nio concentration to 7.5 μM resulted into 39.0 ± 5.8% scratch closure while nint exhibited 59.1 ± 8.6% scratch closure. These results suggest that nint DO nio has a significant inhibitory impact on metastatic properties of NSCLC cells.

Clonogenic assay

Clonogenic assay, also known as colony formation assay, is an in vitro assay which assesses the ability of a single cell to form a colony or result in clonal expansion [69]. This assay reflects the influence of treatment on the inhibition of cell growth and colony formation, and thereby its impact on the tumor growth. Figure 6 illustrates the impact of nint and nint DO nio treatment on the colony formation ability of A549 cells. As can be seen in Figure 6A, nint DO nio demonstrates increased inhibition in the colony formation as compared with nint. At 1.25 μM concentration, nint DO nio demonstrated significant (p < 0.01) reduction in colony growth with 25.3 ± 5.2% as against nint displaying 44.6 ± 2.1% reduction in colony growth (compared with control, considered 100% growth) (Figure 6B). This effect was found to be dose dependent with pronounced inhibition on colony formation being observed at 7.5 μM concentration. At increased concentration, nint displayed 14.1 ± 6.2% reduction in colony growth while nint DO nio exhibited 8.3 ± 3.1% reduction in colony growth, as seen in Figure 6B. Therefore, it could be inferred that nint DO nio significantly reduced colony growth, can potentially inhibit any probability of NSCLC metastasis and would serve as a promising strategy.

In vitro tumor simulation studies: investigating the antitumor activity

In this study, we have used 3D spheroid model to validate the results of in vitro cellular studies performed on nint and nint DO nio, and evaluate the therapeutic activity against NSCLC cells.

Spheroid volume

Figure 7A represents the spheroid volume at different time intervals for 12 days after single-dose treatment. As can be seen, control group spheroids kept on growing and displayed 7.6 ± 1.2 mm³ spheroid volume on day 12 (Figure 7A). Treatment with nint and nint DO nio demonstrated inhibition in tumor growth as compared with control. Treatment with nint (5.0 μM) displayed suppression in tumor growth with 5.8 ± 1.5 mm³ spheroid volume at the end of the treatment regimen. Treatment with nint DO nio resulted into inhibition in tumor growth displaying 5.6 ± 1.3 mm³ spheroid volume as seen in Figure 7A. The results did not show any statistical significance for single-dose study at both 1.0 μM (data not shown) and 5.0 μM dose.

For multiple-dose treatment regimen, the untreated group (control group) showed increase in tumor growth resulting into 8.3 ± 1.6 mm³ spheroid volume on day 12. Treatment with nint and nint DO nio displayed inhibition of tumor growth as seen in Figure 7B & C. At lower concentration of 1.0 μM concentration, nint displayed spheroid volume of 7.2 ± 1.0 mm³, while nint DO nio treatment inhibited tumor growth resulting in 5.8 ± 1.3 mm³ spheroid volume. Tumor growth was found to be inhibited superiorly at a higher concentration of 5.0 μM as compared with the control groups. Treatment with nint DO nio demonstrated spheroid volume of 2.3 ± 0.4 mm³ as against 6.2 ± 1.2 mm³ spheroid volume for nint, displaying significant (p < 0.05) difference of ∼2.7 folds in controlling the spheroid volume, that is, reduction in actual tumor size. Evidently, this can be supported by the shrinkage of the tumor upon treatment with nint DO nio at 5.0 μM concentration. These results establish the superior antitumor activity of nint DO nio against NSCLC cells.

3D cell viability assay

We performed 3D cell viability assay on the spheroids at the end of experiment, day 12, to assess the influence of treatment in the tumor core. As seen in Figure 8A, nint DO nio at 5.0 μM concentration demonstrated a significant reduction in percentage cell viability with 52.3 ± 7.3% as compared with 72.5 ± 10.8% cell viability for nint (p < 0.05). However, at 1.0 μM concentration, no statistically significant difference was observed between nint and nint DO nio. This establishes the improved efficacy of nint DO nio against nint, which was not well reflected by spheroid volume data quantified by optical imaging for single-dose treatment regimen.

Treatment with multiple-dose regimen as in Figure 8A resulted in significant reduction in percentage cell viability with nint DO nio showing 70.1 ± 11.4% cell viability as against 94.1 ± 10.2% cell viability for nint (1.0 μM) (p < 0.01). Upon increasing the dose to 5 μM, nint DO nio demonstrated ∼2.2 fold significant reduction in percentage cell viability, with 25.9 ± 11.4% cell viability when compared with nint exhibiting 56.9 ± 2.2% cell viability (p < 0.0001) (Figure 8A). These results of cell viability assay of 3D spheroids support the spheroid volume data and further establish the improved antitumor activity of nint DO nio against NSCLC cells at both single-dose and multiple-dose treatment regimens.

Live–dead assay

Live–dead assay was performed to further corroborate the improved antitumor activity claim of nint DO nio against 3D spheroids. In this assay, the cell membrane integrity and esterase activity of cells are measured using calcein AM dye, which stains live cells with green fluorescence. On the other hand, ethidium homodimer-III permeates cells with compromised membrane integrity and stain the dead cells red [48,56]. As seen in Figure 8B, for single-dose treatment, nint DO nio demonstrated intensified red fluorescence in the tumor core as compared with nint. At concentration of 1.0 μM, nint DO nio demonstrated 6.3 ± 0.9 unit RFP intensity/mm² spheroid area which was significantly (p < 0.05) higher when compared with nint displaying 2.2 ± 1.4 unit RFP intensity/mm² spheroid area (representative images shown in Figure 8C). Upon increasing the concentration to 5.0 μM, nint DO nio displayed significant (p < 0.05) increase in RFP intensity, with 8.0 ± 0.3 unit RFP intensity/mm² spheroid area against nint demonstrating 4.7 ± 1.6 unit RFP intensity/mm² spheroid area (Figure 8B & C).

For multiple dose treatment, nint DO nio at 1.0 μM concentration exhibited a significant (p < 0.05) increase in RFP intensity, with 5.3 ± 0.7 unit RFP intensity/mm² spheroid area against nint demonstrating 2.8 ± 0.2 unit RFP intensity/mm² spheroid area as seen in Figure 8B & C. This effect was pronounced with increase in the dose of treatment. Nint DO nio displayed 10.4 ± 1.4 unit RFP intensity/mm² spheroid area, which was significantly (p < 0.001) higher than nint displaying 3.8 ± 0.1 unit RFP intensity/mm² spheroid area. In both the dose treatments, red cells were found to be present in the core of the spheroids. This can be explained by the presence of necrotic region in the core of the spheroids. 3D spheroids comprise of three different microregions within them: the proliferation zone (peripheral region), quiescent zone (middle region) and necrotic zone (core region). In the necrotic zone, which is in the core region of the spheroids, there is lack of oxygen and nutrients, which triggers the necrotic cell death. Additionally, there is metabolic waste buildup in the core region, which further triggers the necrotic cell death. Hence, cell death in the core of the third spheroids is very commonly observed due to the existing necrotic region [3,4]. These results establish the superior antitumorigenic activity of nint DO nio against nint in NSCLC cells, which can be attributed to the improved penetrability of nint after encapsulation in niosomes.

Discussion

Nint, a multityrosine kinase inhibitor, is effective against several solid tumors and is an FDA-approved first-line treatment against idiopathic pulmonary fibrosis, interstitial lung diseases and systemic sclerosis-associated interstitial lung disease [12]. Nint is also know to show efficacy for treatment against NSCLC, as demonstrated by results of clinical trials [16–18]. However, application of nint for NSCLC treatment is challenging due to several limitations such as poor solubility, low bioavailability, dose-related adverse effects, frequent dosing and limited route of administration [14,20,60,62]. Therefore, designing an appropriate delivery carrier for nint can serve as a promising strategy to address the challenges for delivering and enhancing its therapeutic effectiveness.

In this study, we investigated the development of nint-loaded niosomes as inhalable delivery carriers for treatment against NSCLC. Niosomes were formulated using span 60 and cholesterol via thin-film hydration method. DOTAP was added as a positively charged lipid to the formulation. The formulation optimization highlighted that cholesterol plays a vital role in the entrapment of drug in niosomal vesicles. The influence of cholesterol on the encapsulation of drug in niosomes has been studied previously. Shilakari et al. reported that increase in concentration of cholesterol increases the bilayer hydrophobicity, vesicle stability and drug permeability which may be the reason for the increase in drug entrapment observed with F2 [70]. On the contrary, a higher amount of cholesterol may also compete with the drug for packing space in the bilayer, thereby deposing the drug from vesicles and resulting into lower drug loading [71]. Additionally, increased concentration of cholesterol beyond a limit is reported to disrupt the linear structure of vesicle bilayer, thereby causing leakage of drug [72]. This can explain the reduction in nint encapsulation in formulation upon decreasing the cholesterol content.

The addition of DOTAP in the formulation resulted in increased nint encapsulation in niosomes. The enhanced encapsulation can be explained due to the hydrogen bond interaction occurring between the nint molecules and the lipid polar group present in the unsaturated alkyl chain of DOTAP which helps in enhanced retention of drug in the niosomes vesicles [73]. Moreover, incorporation of charged lipids tends to increase the lamellar distance and impart greater overall entrapped aqueous volume and thereby increase encapsulation of hydrophilic drug [74]. This increased encapsulation of nint can also be corroborated by the increased vesicle size of the formulation which imparts increased bilayer surface and aqueous core for increased incorporation of drug in the vesicles, thereby resulting in increased encapsulation efficiency. These findings suggest that concentration of cholesterol and charge-inducing agent DOTAP played an important role in encapsulation of nint in the niosomes' vesicles. The drug encapsulation efficiency of the final optimized formulation nint DO nio was found to be ∼75%, while only 1 mg of drug was added to the niosomal vesicle for loading. Since we wanted to evaluate the efficiency of niosomes as delivery carriers and the influence of nint upon encapsulation in niosomes, we have used a small amount of drug for loading. Percentage drug loading of niosomes can be increased with the increase in amount of drug being loaded, as the encapsulation efficiency of the niosomes is high. Thus, high-drug-loaded nint niosomes can be prepared if needed for any future studies.

In vitro studies suggest that DOTAP influences the release of drug by reducing the initial burst release as well as providing slow and controlled release of drug. This can be attributed to the fact that DOTAP causes stringent molecular arrangement of the bilayer packaging resulting in slow drug release [75]. Moreover, DOTAP is also reported to form hydrogen bonds between the polar head group of the lipid alkyl chain and the hydrogen donor groups of drug molecules [73]. Additionally, higher phase transition temperature of DOTAP (∼60°C) may help to maintain the integrity of niosomal vesicles at physiological temperature (∼37°C) and prevent drug leakage [52]. Thus, DOTAP helps in providing enhanced retention of drug molecules in the vesicles and hence the slower drug release.

The results of cellular uptake studies exhibited increased internalization of DO nio as compared with reg nio. The highly charged niosome vesicles can undergo several mechanisms such as clarithin-mediated endocytosis, micropinocytosis and/or passive uptake via direct fusion with cell membrane during the cellular internalization process [2]. The increased uptake of DO nio can be attributed to the reduced surface charge of the vesicles due to incorporation of positively charged DOTAP in niosomes: this reduced surface charge would allow the niosomes to stay in closer proximity of the cell membrane which is negatively charged due to phospholipid groups, resulting in increased probability of being internalized [56].

Cytotoxicity studies confirmed the effectiveness of nint DO nio among all the NSCLC cell lines as illustrated by significant reduction in IC₅₀ values compared with nint. The results of the uptake study and cytotoxic study suggest that nint DO nio can be used as potential delivery carriers for lung cancer treatment. The safety of highly charged delivery carriers has been an apprehension. Positively charged lipids are reported to demonstrate enhanced cytotoxicity and cellular damage, which hinder their therapeutic applicability [52]. Therefore, cytotoxicity of blank niosomes was performed against HEK and A549 cells. Results demonstrated no significant difference in the cell viability when compared with control, thereby establishing the safety profile of the niosomes and suggesting that the enhanced cytotoxicity of nint-loaded niosomes is due to increased internalization and prolonged release of nint intracellularly. Next, aerosolization study was performed using nint DO nio, which displayed superior deposition of the niosomes in the broncho-alveolar region. From the results obtained, it can be confirmed that nint DO nio can serve as effective inhalable delivery carriers and thus provide increased localized accumulation of nint in the lungs.

NSCLC is reported to endure distant metastases to organs such as bones, liver, adrenal glands and extra-thoracic lymph nodes as a frequent clinical challenge [76–78]. Metastasis is known to impact the treatment efficacy and further escalate the disease progression, thus adding to the increased mortality rate [79]. We evaluated the efficacy of developed niosomes against metastasis of NSCLC cells using scratch assay, wherein the nint DO nio displayed superior inhibitory action against metastasis. Clonogenic assay was also performed to investigate the influence of developed niosomes against metastasis. Results demonstrated enhanced inhibitory activity of nint DO nio against colony formation as against nint. Thus, the findings confirm the inhibitory effect of nint DO nio against metastasis of NSCLC cells.

In vitro cellular assays performed on the monolayer or 2D cell culture do not truly reflect the physiological tumor conditions, nor do they reflect the penetrability and resistance of the therapeutic agents against the tumor cells [80]. This results in inaccurate assessment of therapeutic activity of drugs against tumors. 3D cell culture models have been used to bridge the gap between the in vitro and in vivo models due to unique characteristics resembling solid tumors [81]. 3D spheroids mimic several features of human tumors such as spatial arrangement, enhanced cell interactions, nutrient gradients and cellular layered assembling [82]. These features enable 3D spheroids to be used as reliable models for evaluating the antitumorigenic activity of several chemotherapeutic agents. We examined the therapeutic effectiveness of the developed niosomes using 3D spheroids. Results from the optical imaging of spheroids illustrated that treatment with nint DO nio resulted in significant inhibition in the tumor volume as compared with nint. Optical imaging provides valuable information about the tumor size/volume by estimating the mass of the spheroids. However, as 3D spheroids represent cellular mass grown exponentially in three dimensions to mimic the physiological tumor conditions, optical imaging may not be able to predict the influence of treatment in the tumoral core [47]. Therefore, we also evaluated the effectiveness of niosomes on 3D spheroids using cell viability assay, wherein nint DO nio resulted in significant difference in cell viability of the spheroids. Furthermore, we also performed live–dead cell assay to evaluate the niosomal properties in inhibiting the tumor growth. Results of the study were in accordance with the optical imaging and cell viability assay studies, suggesting that nint DO nio displayed enhanced antitumorigenic activity as compared with nint. Studies have reported the importance of physicochemical properties of delivery carriers such as particle size, surface charge and several surface modifications which govern the penetrability of carriers in the tumor [70,83]. Thus, understanding this interplay between several biophysical parameters will help to design effective delivery carriers possessing desired physicochemical properties. Our findings have established that cationically modified nint DO nio can be a promising approach for delivering nint for treatment of lung cancer.

Conclusion

In the present study, we discuss development of cationically modified nint-loaded niosomes as inhalable delivery carriers against NSCLC. Incorporation of cationically charged lipid results into increased drug encapsulation in niosomes along with optimum vesicle size and size distribution. Optimized niosomes displayed sustained and prolonged release of nint from the vesicles. The cationically modified niosomes displayed enhanced internalization and pronounced cytotoxic effects against NSCLC cells resulting into dose reduction for chemotherapeutic effect. The results demonstrated that developed niosomes possess appropriate aerosolization properties for efficient pulmonary delivery which could provide increased localized accumulation and thereby enhance therapeutic activity. The optimized niosomes displayed significant inhibitory action on the metastatic property of NSCLC cells. Furthermore, we exhibited superior antitumor activity of optimized niosomes using in vitro tumor simulation studies which can be further translated to preclinical application. To the authors' knowledge, this is one of first reports to explore nint-loaded niosomes in NSCLC treatment by noninvasive inhalation route.

Future perspective

Nint has poor solubility and poor absorption, which limits its oral bioavailability and thereby therapeutic applications. The developed inhalable cationically modified niosomes for delivery of nint is a promising strategy to overcome the existing limitations of nint and improve the effectiveness for application to several cancers including lung cancer.

The developed inhalable delivery carrier would be a feasible platform to deliver any drug to the respirable region of the lungs, resulting in improved efficacy and reduced off-target side effects. Other drugs aimed for treatment of respiratory diseases can be assessed for encapsulation in the developed cationically modified niosomes and delivery efficiency at the desired site of action. Future studies may involve surface modification of the niosomes to improve tumor specificity. Moving forward, pharmacokinetic studies supplemented with biodistribution studies may help to establish the platform for further clinical settings.