Introduction

Lung cancer is a deadly disease that affects millions of people worldwide. There are various therapies available for treating lung cancer, including chemotherapy, radiation therapy, targeted therapy, and immunotherapy. However, each of these therapies has its limitations and has been shown to develop resistance, which can reduce their effectiveness over time (Harrison et al. 2022). In addition, the increased efficacy of the chemo drug combination leads to a rise in adverse effects and off-target toxicity. Therefore, there has been a lot of interest in creating new natural-based chemotherapy regimens for non-small-cell lung cancer (NSCLC). Furthermore, urrent studies are exploring the use of nanoformulations, which involve the encapsulation of drugs within nanoparticles (NPs) (Norouzi and Hardy 2021; Gavas et al. 2021). This approach can improve drug delivery to the tumor site, reduce systemic toxicity, and enhance drug efficacy. As a viable alternative, administration of NPs with chemotherapeutic combinations has demonstrated an integrative manner to both improving reactions and minimizing adverse effects. NPs can lessen exposure to off-target sites by accumulating drugs inside tumors preferentially(Gavas et al. 2021). Furthermore, carefully designed, dual-drug-loaded NPs can be used to deliver chemotherapies in a synergistic drug ratio to benefit from combined effects or combating multi-drug resistance. PLGA (poly(lactic-co-glycolic acid)) is a commonly used polymer for drug delivery systems due to its biodegradability, biocompatibility, and controlled drug release characteristics. In recent times, PLGA NPs drug delivery systems have been utilized extensively in cancer therapy. Following the unique properties of PLGA, carriers of various structures are designed to maintain the function of drugs or bioactive substances, which ensure the efficient delivery of molecules, and enhance the bioavailability of drugs in targeted tissues (Ruirui et al. 2021). Dual drug encapsulation using PLGA can be an effective strategy for lung cancer treatments. Co-delivery of PLGA allows for the controlled release of both the drugs at the same time, providing a synergistic effect and potentially reducing drug resistance. For example, in an earlier study, doxorubicin and sorafenib were combined in nanotherapeutics using PLGA, allowing the anticancer drugs to work in synergistic effects (Huang et al. 2016; Babos et al. 2018).

An appropriate composition can achieve a synergistic effect by delivering anticancer drugs to tumor cells at the same time (Wen et al. 2017). Additionally, another study has revealed that dual-drug-loaded inhalable PLGA porous NPs greatly reduced tumor development and spread in animals with in situ lung cancer (Xie et al. 2021). Considering these benefits, the current investigation focused on co-delivery of the natural compounds phytol and α-bisabolol using FDA-approved PLGA NPs as the drug delivery system. PLGA was chosen for its high biocompatibility, substantial drug-loading capacity, and potential for rapid therapeutic applications (Makadia and Siegel 2011; Rezvantalab et al. 2018).

Phytol is a diterpene alcohol found in plants such as green tea, and it has been shown to have anti-proliferative and apoptotic effects on various cancer cells, including lung cancer cells (Komiya et al. 1999; Thakor et al. 2017; Sakthivel et al. 2018). α-Bisabolol is a natural terpene alcohol found in chamomile and other plants, and it has been shown to have an anti-proliferation effect on A549 cells (Cavalieri et al. 2004; Murata et al. 2017; Wu et al. 2018). Furthermore, it has been suggested that the inclusion of α-bisabolol into PLGA NPs is a possible approach for creating novel anti-inflammatory, antipyretic, and perhaps immune therapeutic substances (Marongiu et al. 2014). In addition to that, the inclusion of both the A549 (NSCLC cell line) and L-132 cell line, a normal human alveolar cell line in the study, facilitated a comparative analysis between cancerous and normal cell lines. The utilization of L-132 cells, representing normal lung tissue, was integral for evaluating the selectivity and preferential targeting of cancer cells in lung anticancer studies.

In a prior study, the research demonstrated the synergistic induction of apoptosis and cell senescence in NSCLC cells by phytol and α-bisabolol, highlighting their effectiveness as anti-cancer agents and their potential application in the development of new therapies for lung cancer (Kiruthiga et al. 2024). The objective of the present study was to co-deliver phytol and α-bisabolol using PLGA NPs as a therapeutic approach for NSCLC. To achieve this, phytol and α-bisabolol was individually encapsulated within PLGA NPs, as well as a combination of both compounds. The NPs were characterized and their safety profile was evaluated. Furthermore, the study aimed to elucidate the apoptotic mechanisms through which the phytol and α-bisabolol-loaded PLGA NPs exerted their anti-cancer effects. The outcomes of this study have the potential to provide valuable insights into the advancement of innovative therapeutic modalities for NSCLC, particularly by exploiting the co-delivery of phytol and α-bisabolol using PLGA NPs.

Materials and methods

Cell culture and chemicals

The human adenocarcinoma lung cancer cells A549 and normal human lung epithelial cell line L-132 were obtained from the National Centre for Cell Science (NCCS) in Pune, India. The A549 and L-132 cells were cultured in DMEM medium supplemented with 10% FBS and 1× antibiotic solution (streptomycin and penicillin) at 37 °C in a humidified atmosphere with 5% CO2. The DMEM medium was purchased from Gibco in Grand Island, USA. An Apoptosis Kit, containing Annexin V-FITC (Fluorescein isothiocyanate) and PI (Propidium Iodide), was obtained from Thermo Fisher Scientific in Waltham, MA, USA.

Experimental chemicals, including phytol, (-)-α-bisabolol, poly(d,l-lactide-co-glycolide), 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and secondary antibody (anti-mouse immunoglobulin G), were obtained from Sigma in St. Louis, MO, USA. β-Actin, B cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (Bax) were purchased from Santa Cruz Biotechnology Inc. in Dallas, Texas, USA. All the chemicals used in the experiments were of high purity and high grade, obtained from HiMedia in Mumbai, MH, India.

Preparation and characterization of the formulation

Preparation of PLGA-Phy NPs, PLGA-Bis NPs, PLGA-Phy+Bis NPs

Nanoparticles containing PLGA and either phytol (PLGA-Phy), α-bisabolol (PLGA-Bis), or both phytol and α-bisabolol (PLGA-Phy+Bis) were made using the oil-in-water emulsion solvent evaporation method (Mathew et al. 2012). In detail, 40 mg of PLGA (with a ratio of 50:50 and a molecular weight range of 30,000–60,000) was dissolved in 3 mL of dichloromethane. Subsequently, 5 mg of phytol, 5 mg of α-bisabolol, and 5 mg of both compounds were added to the solution. The mixture was stirred on a magnetic stirrer for 10 min to break down the compounds. The resulting mixtures were then added drop by drop to 20 mL of 1% polyvinyl alcohol (PVA), which was used as a surfactant to make a fine emulsion. The composite suspension of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis underwent sonication at 40% amplitude for 5 min. The emulsion was left on the stirrer for 12 h to facilitate solvent evaporation. The nanoparticles were subsequently harvested through centrifugation at 13,000 rpm for 15 min at 4°C. Following this, the nanoparticle pellets underwent triple washing with water, were freeze-dried, and were stored at −20°C for future use. Blank nanoparticles were produced using an identical procedure, excluding the addition of phytol and α-bisabolol. For all analytical assessments, the nanoparticles were dispersed in milli-Q water.

Drug loading and encapsulation efficiency

After the synthesis of the NPs, the amount of phytol and α-bisabolol were measured following centrifugation at 13,000 rpm and 4°C for 15 min. The encapsulation efficiency (EE), drug loading capacity (DLC), and yield of the NPs were then quantified through UV analysis, as outlined in Mathew et al. (2012). To do so, a standard calibration curve was created using standard concentrations of phytol and α-bisabolol in dichloromethane, ranging from 1 to 5 mg/mL. The amount of each compound present in the NPs was measured by taking the absorbance at 250 nm for phytol and 212 nm for α-bisabolol. Finally, the EE, DLC, and yield of the NPs were calculated individually, based on the results obtained from the UV analysis.

Particle size and zeta potential

Dynamic light scattering (DLS) and micro-electrophoretic techniques were used for investigating the main physicochemical parameters of PLGA NPs such as polydispersity index (PDI), hydrodynamic diameter, and particle charge (zeta potential), on a Malvern Zetasizer Nano Series (Malvern Zetasizer Nano-ZS90, UK) equipped with a He–Ne laser (632 nm). DLS measurements were taken at 26°C with a detection angle of 175°. The mean diameter of the samples was measured in triplicate with 10–20 readings per sample and the results were expressed as mean size ± SD. When the PDI values ranged from 0.01 to 0.7, the particle distributions were narrow. High PDI readings (PDI > 0.7) showed relatively broad distributions.

High-resolution transmission electron microscope analysis

The morphology of the obtained NPs was studied by using high-resolution transmission electron microscopy (HR-TEM; Quanta FEG 250, FEI, Eindhoven, The Netherlands). For the TEM imaging, before observation, PLGA NPs were diluted 1:50 (v/v) and a few drops of diluted PLGA NPs were directly on a Cu–Ni grid and stained with uranyl acetate (2.5%) for 5 min followed by drying for 12 h at 26°C.

Characterization of drug-loaded PLGA NPs using FTIR spectroscopy

The FT-IR spectrophotometer (FTIR Thermo Nicolet 380 spectrometer) was used for the study, and the spectra were recorded in the region of 3800–500 cm−1 with a resolution of 2 cm−1. To make the pellets, samples were mixed individually with potassium bromide (300 mg) and compressed in a hydraulic press at a pressure of 200 kg/cm2 for 3 min. The pellets containing drug-loaded PLGA NPs were analyzed by placing the compressed pellet in the light path and recording the spectral data.

GC-MS drug release study

To evaluate the active drug content available in post-processed nanoparticle samples, each sample (PLGA-Phy, PLGA-Bis, and PLGA-Phy-Bis) was analyzed. Agilent gas chromatography (GC-MS/MS, Agilent Tech 7890B GC System) Q with a fused GC column (DB-624 [Agilent]; 30 m × 0.53 mm, 3.00 m) was used to analyze NPs. The components of the NPs were identified probably by matching peaks using NIST 17.0 libraries, and the results were verified by comparison with peaks of mass spectra reported in the published literature.

In vitro drug release kinetics

The in vitro drug release profile of PLGA-Phy+Bis NPs was studied by the dialysis method. Briefly, 1 mg of nanoparticle suspension was placed in a dialysis bag (molecular weight cutoff 10,000–12,000, Himedia, Mumbai, India), which was then sealed at both ends. The dialysis bag was soaked in 5 mL of 0.1 M phosphate-buffered saline (pH 7.4), which was stirred constantly at 100 rpm and maintained at 37°C in a shaker. At various time intervals, a sample of the solution was withdrawn, and the same volume of fresh dissolution medium was replaced in a beaker. The absorbance of the solutions was determined in triplicate using a UV spectrophotometer at 250 nm for phytol and 212 nm for α-bisabolol (Ray et al. 2017).

Biocompatibility studies

Cell viability

The cytotoxic effects of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs against lung cancer cell line A549 and normal human lung alveolar epithelial cell L-132 were determined by MTT assay. Cells (1 × 105/mL) were seeded per well in 96-well plate containing DMEM and incubated for 24 h. The medium was then removed and the cells were treated for 24 h with PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs at various concentrations (0 to 100 µg/mL, 0 to 50 µg/mL, and 0 to 50 µg/mL, respectively). After removing the medium from the wells, 100 µL of MTT (1mg/mL) was added. Then, the formazan products were dissolved with dimethyl sulfoxide (DMSO) and incubated in the dark for 1 h at ambient temperature. At 570 nm, UV absorbance was measured using a multi-well plate reader (Molecular Device Spectromax M3 with Softmax Pro V5 5.4.1 software). The IC50 values for PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs were determined by using graphpad prism.

AnnexinV-FITC/PI staining

The translocation of phosphatidylserine in cells treated with PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs was assessed using AnnexinV-FITC and PI labeling according to the manufacturer’s instructions (Invitrogen, Catalog no. V13242). Following treatment with IC50 concentration of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs, the cells were washed with ice-cold PBS and suspended in 1× AnnexinV binding buffer. In addition, the cells were stained with 5 µL of AnnexinV-FITC and 100 µl of the PI stain (50 µg/mL) and incubated in the dark for 15 min. After the incubation period, the cells were rinsed and resuspended in 0.5 mL of 1× Annexin V binding buffer. Subsequently, apoptotic cells were examined using fluorescence microscopy (Nikon ECLIPSE, Ti-E, Japan) with AnnexinV and PI labeling to identify positively stained cells.

Western blot

After treatment with PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs, A549 cells per well were trypsinized and washed with PBS for analysis of the apoptotic mechanism using western blot. The pelleted cells were mixed with RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mMTris, pH 8.0) and 1 mM PMSF, and then incubated for 2 h at −20°C. After incubation, the cells were vortexed and centrifuged for 30 min at 13,000 rpm and 4°C. The cell-free supernatant was transferred to new tubes, and the protein content was measured using the Bradford technique. On a 12% SDS-PAGE gel, about 100g of protein was separated and then transferred to a PVDF membrane. After transformation, membranes were blocked with 5% skim milk at 4°C for 3 h, followed by overnight incubation at 4°C with appropriate primary antibodies (Bcl-2, Bax, and β-actin (Internal control) at a dilution of 1:1000). After incubation, membranes are washed with TBST (Tris-buffered saline with Tween 20) for 5 min, then incubated for 3 h at 4°C with an anti-mouse ALP-conjugated secondary antibody (1:1000). The proteins were visualized using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), and pictures were acquired using a gel documentation system. Quantification of the protein bands was performed using ImageJ software.

In vivo toxicity assessment

The toxicity assessment was conducted using zebrafish embryos (D. rerio), following ethical approval from the Institutional Animal Ethics Committee (IAEC/AU/OCT/2021/Fish-6). The embryos were harvested from the zebrafish aquarium tank at 3 h post-fertilization (hpf) and transferred to 24-well plates, with each well containing 30 embryos and an exposure medium. The toxicity assessment of the NPs was carried out following the standard protocol (Lammer et al. 2009). The zebrafish embryos were treated with different concentrations of NPs including PLGA-Phy NPs (50 and 100 μg/mL), PLGA-Bis NPs (50 and 100 μg/mL), and PLGA-Phy+Bis NPs (50 and 100 μg/mL). Each test concentration was performed in triplicate at various time intervals (24, 48, 72, and 96 hpf), and morphological abnormalities were examined using an OLYMPUS CH 20i inverted-phase contrast light microscope. The mortality results were then calculated.

Statistics

The experiments were performed in triplicate (n = 3) and statistical analysis was conducted using IBM SPSS Statistics (version 25). To determine the IC50 value, GraphPad Prism 7.04 software (La Jolla, USA) was employed. One-way ANOVA was used to analyze the statistical variance between the control and treated groups. To identify significant differences between the control and treatment groups, the Dunnett T3 post hoc test was applied, with a p-value less than 0.05 being considered significant.

Results

Preparation and characterization of phytol, α-bisabolol, and their combination in PLGA NPs

In the current study, phytol, α-bisabolol, and their combinations were successfully encapsulated into PLGA NPs by the oil-in-water immersion, solvent evaporation method (Fig. 1). The results showed that the drug/polymer ratio of 1:4 for phytol and α-bisabolol resulted in nearly 83.30 ± 0.81% and 77.17 ± 0.181% encapsulation efficiency (EE), respectively. PLGA-Phy+Bis NPs with a drug/polymer ratio of 0.5:0.5:4 (phytol:α-bisabolol:PLGA) resulted in nearly 82.64 ± 0.09% and 78.71 ± 0.103% EE, respectively. These findings suggest that the encapsulation process improves the solubility of phytol and influences its physicochemical and pharmacokinetic properties (Sah et al. 2017). The high EE of Phy-PLGA, Bis-PLGA, and PLGA-Phy+Bis NPs observed in the study can be attributed to the ionic interactions between the drug and the polymer. Moreover, it reduces the wastage of drugs during the encapsulation process.

Fig. 1
figure 1

A diagram illustrating the encapsulation of bioactive compounds within PLGA nanoparticles, including PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs 

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Furthermore, the loading efficiency of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs were found to be 8.49 ± 0.08%, 8.64 ± 0.02%, and 8.43 ± 0.01% (phytol), 8.026 ± 0.005% (α-bisabolol), respectively, depending on the drug/polymer ratio. During the formulation process, an increase in the drug concentration was observed to increase the loading efficiency. This effect might be due to the ability of the polymer matrix to accommodate a higher amount of phytol and α-bisabolol molecules in the polymeric matrix, thus ensuring the ability of the polymeric matrix to release higher drug concentrations at the specific target site. The yield of the NPs was around 80%. Notably, the high EE, loading efficiency, and yield of the NPs achieved in this study (Table 1) are similar to those reported in previous studies on PLGA NPs (Fornaguera et al. 2015).

Table 1 Encapsulation efficiency, Loading efficiency, and Yield
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Nanocarrier size, polydispersity, and particle charge

Photon correlation spectroscopy (PCS) was employed to determine the mean particle size of drug-loaded PLGA NPs. The analysis revealed that the NPs, which were prepared with three different drug compositions (phytol, α-bisabolol, and phytol+α-bisabolol) and incorporated into PLGA, fell within the nanometer range. The mean size of PLGA-Phy NPs, PLGA-Bis, PLGA-Phy+Bis NPs, and PLGA NPs alone was found to be 162.5 ± 12.33 nm, 137.3 ± 7.79 nm, 189.3 ± 5 nm, and 91.1 ± 1.9 nm, respectively, as confirmed by DLS measurements. The size distributions of all nanoparticle formulations ranged from 0.224 to 0.348, as shown in Table 2 and Fig. 2a. The mean zeta potential of PLGA-Phy NPs, PLGA-Bis, and PLGA-Phy+Bis NPs was determined by Malvern zeta-sizer and it was found to be −38.2mV, −0.3 mV, and 0.1 mV, respectively.

Table 2 Particle size, polydispersity index, charge and electrical mobility mean of PLGA-loaded nanoparticles
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Fig. 2
figure 2

(a) High-resolution transmission electron microscopy image of (A) PLGA-Phy NPs, (B) PLGA-Bis NPs, (C) PLGA-Phy+Bis NPs and the size distributions of all NPs by DLS measurements, (D) PLGA-Phy NPs, (E) PLGA-Bis NPs, (F) PLGA-Phy+Bis NPs. (b) FT-IR spectra of phytol, α-bisabolol, PLGA NPs alone, PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs. (c) In vitro drug release profile of phytol, α-bisabolol, and PLGA-Phy+Bis NPs

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Shape and morphology

The study used high-resolution transmission electron microscopy (HR-TEM) to characterize the PLGA-stabilized NPs. HR-TEM is a rapid, efficient, and generally non-invasive approach that may offer evidence on the form, morphology, and size distribution of polymeric nano-systems. The TEM imaging revealed oval-shaped particles of equally uniform size across the sampling region. The darkest area corresponds to the thicker PLGA outer shell, suggesting that these shells were created effectively. The size obtained was 318±27 nm, 144±25 nm, and 268 ±54 nm for PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs respectively (Fig. 2a).

FTIR spectroscopy analysis of drug-loaded PLGA NPs

The chemistry of drug-loaded PLGA NPs was assessed using Fourier transform infrared (FTIR) vibrational spectroscopy. As shown in Fig. 2b, the FTIR spectra of PLGA, phytol, α-bisobolol, and NPs including PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis depict spectra derived from the IR studies, which correspond to wavelengths of 3800 cm−1 to 500 cm−1, respectively. The FTIR spectra of PLGA reveal distinctive peaks due to the functional groups included in it. The carbonyl stretching vibration in the –COO– group, which is present in both the lactic and glycolic acid monomers, generates a peak close to 1723 cm−1. The C–H bending vibration in the –CH3 found in the lactic acid monomer and glycolic acid monomer causes the peak at about 1402 cm−1 and 1187 cm−1, respectively (Soni et al. 2010). The C–O–C stretching vibration in the COO group is responsible for the peak near 1085 cm−1. The C–H bending vibration in the CH2 group found in the PLGA backbone essentially creates the peak around 1450 cm−1 (Soni et al. 2010).

Free drugs Phy and Bis exhibit absorption peaks at 2928, 1469, 1375, and 1000 cm−1 (Wang et al. 2013) and 3431, 2912, 1447, 1370, and 1011 cm−1 (Picquart and Lefèvre 2003), respectively. All the groups on Phy and Bis are highly related to the presence of –OH, C–H, and C=O groups. The main peaks of PLGA at 2914, 1723, and 1085 cm−1 can be attributed to –COO, –CH3, and C–O groups, respectively. The spectral analysis for PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis indicates that the functional groups on the surface of the PLGA NPs have chemical characteristics similar to the PLGA. As seen on the FTIR spectra, there was no chemical interaction between functional groups of PLGA and drug actives, indicating the structural stability upon nanoparticle synthesis with Phy and Bis. The interaction study between the drug and polymer was evaluated. The absence of distinctive spectra in the PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis indicates that drug actives and PLGA were not involved in intermolecular interaction. However, the intensity of the peak at 2914, 1723, and 1085 cm−1 was decreased in the FTIR spectrum of the PLGA-Phy+Bis NPs (Anwer et al. 2019). The decrease in peak intensity following nano-PLGA encapsulation is an instant of encapsulation efficiency. Upon encapsulation within a polymeric matrix, both molecules and the surrounding polymer modify their vibrational modes, resulting in a diminished intensity of characteristic peaks in FTIR spectra. Furthermore, the encapsulation process can prompt alterations in the molecular environment of the encapsulated compound, potentially introducing steric hindrance or modifying the electrostatic surroundings. These changes can impact the vibrational properties of the compound, consequently influencing the observed intensity of FTIR peaks (Hadjiivanov et al. 2021).

GC-MS drug release study

The chemical composition of PLGA nanocarriers loaded with drugs was analyzed using GC-MS, by measuring peak areas and retention times (Fig. 3 and Table 3). The retention time for phytol was found to be the highest at 20.7373 s, while α-bisabolol was detected at 16.53 s in all NPs. The GC-MS analysis showed that PLGA-Phy contained 57.22% phytol, while PLGA-Bis contained 87.54% α-bisabolol. The higher drug encapsulation efficiency of α-bisabolol in PLGA-Bis led to an increase in particle size compared to PLGA-Phy. In PLGA-Phy+Bis, 38.02% of phytol and 64.72% of α-bisobolol were found to be encapsulated when compared individually. α-Bisabolol was identified as the major drug encapsulated in both PLGA-Bis and PLGA-Phy+Bis.

Fig. 3
figure 3

GC-MS drug release analysis of all NPs

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Table 3 Drug release profile of NPs analyzed by GC-MS
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In vitro drug release kinetics

The in vitro drug release kinetics of PLGA-Phy+Bis NPs were evaluated using the dialysis method, where the percentage of phytol and α-bisabolol released from the PLGA NPs at various time intervals was analyzed. The release rate of a drug from a nanocarrier depends on several factors such as the concentration of PLGA, size of the nanoparticle, solubility, molecular weight, and biodegradable properties of the polymer matrix (Jinhyun Hannah Lee and Yeo 2016). The resulting PLGA-Phy+Bis NPs showed sustained release behavior, with a slow cumulative drug release of up to 98.4% from the NPs (drug 1: drug 2:polymer ratio, 0.5:0.5:4) observed over 120 h (Fig. 2c). The encapsulated NPs were completely released within this time frame. Similar to other loaded PLGA NPs, a biphasic release pattern with an initial burst effect followed by sustained drug release was observed (Fornaguera et al. 2015). Overall, the in vitro drug release study demonstrated that the delivery action of PLGA-Phy+Bis NPs sustained the release of phytol and α-bisabolol molecules, making them a promising candidate for the treatment of NSCLC.

Anti-cancer activity assessement through MTT assay

The PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs significantly inhibited the proliferation of A549 cells in a concentration dependent manner. In PLGA alone, NPs do not exhibit any inhibition on A549 cells. The IC50 values of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis against A549 cells were found to be 25 µg/mL, 14.56μg/mL, and 14 μg/mL at 24 h respectively (Fig. 4a). Overall, the MTT results confirmed that the PLGA-Phy+Bis NPs have a significant anti-proliferative effect against A549 cells.

Fig. 4
figure 4

PLGA-Phy+Bis NPs bioactivity against A549 cells. a Antiproliferative effect of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs against A549 cells. b Representative microscopic images of A549 cells for apoptosis study using Annexin V-FITC/PI staining. c Quantitative measurement of Annexin V/PI positive apoptotic cells after treatment with all NPs for 24 h. Data are presented as the mean ± SD of three samples per group. d Western blot analysis of Bax and Bcl-2 expression in A549 cells after 24 h of incubation with all NPs at IC50 concentrations. The gel blots were cropped from different gel and full length of blot is given in supplementary file. e Changes in the protein expression levels of apoptotic regulators in A549 cells treated with all NPs. The protein levels were normalized with β-actin. (*P<0.05 & **P<0.01 vs control group)

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Apoptotic assessment through AnnexinV-FITC/PI staining and western blot analysis

Apoptotic cells display a signal that presumably exposes phosphatidylserine to the outer cell membrane. In addition, apoptotic cells lose their membrane integrity because proteolytic enzymes such as caspase are activated. To examine the apoptosis induced by PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs, AnnexinV-FITC, and PI staining were performed and analyzed by fluorescence microscopy. A549 cells were treated for 24 h with IC50 concentrations of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs, and the percentage of apoptotic cells was quantified using Annexin V-FITC and PI. Under fluorescence microscopy, AnnexinV-FITC and PI-positive cells upon exposure to PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs were initially qualitatively observed. The increase of Annexin V and PI-positive cells was confirmed by fluorescence images shown in Fig. 4b.

Furthermore, the quantitative analysis revealed a significant and substantial increase in both early and late apoptotic cells following exposure to PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs (Fig. 4c). Notably, PLGA-Phy+Bis NPs exhibited a higher proportion of late apoptotic cells compared to PLGA-Phy NPs and PLGA-Bis NPs. These findings strongly support the notion that PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs actively promote apoptotic cell death in A549 cells.

To investigate the underlying biochemical processes associated with the induction of apoptosis, western blot was employed to analyze the expression levels of key apoptotic proteins. In particular, the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were assessed. The results revealed a significant reduction in Bcl-2 expression and a concurrent increase in Bax expression following treatment with PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs. Importantly, PLGA-Phy+Bis NPs exhibited a particularly pronounced reduction in Bcl-2 expression and upregulation of Bax, as depicted in Fig. 4d and e. This alteration in apoptotic protein expression strongly suggests that these NPs induce apoptotic cell death as the predominant mode of cell death.

In vitro and in vivo safety assessment

The effects of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis on the safety of human L-132 cells were investigated using the MTT test. The tested NPs did not affect the growth and viability of normal human lung L-132 cells after exposure for 24 h. However, mild toxicity was observed at the maximal dose of 200 μg/mL in PLGA-Phy and PLGA-Bis at 24 h, as shown in Fig. 5a, compared to PLGA-Phy+Bis. Cytotoxic studies conducted on normal human cells revealed that the combination of Phy and Bis selectively induced cell death in A549 cells alone without affecting the normal cells.

Fig. 5
figure 5

Toxicity assessment of all NPs: a Microscopic images of normal lung L-132 cells treated with specified concentrations (25, 50, 100, and 200 μg/mL) of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs after 24 h. b Toxic effects of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs on the development of zebrafish (Danio rerio) embryos. Representative pictures of each stage of embryo development at various NPs concentrations (50 and 100 μg/mL). Data are presented as the mean ± SD of three samples per group

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The in vivo safety of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs was evaluated by observing the morphology and mortality of zebrafish embryos at different concentrations (50 μg/mL and 100 μg/mL) and time intervals (24, 48, 72, and 96 hpf). No mortality or morphological abnormalities were observed even after 96 hpf at a higher concentration of 100 μg/mL in PLGA-Phy NPs, PLGA-Bis NPs, or PLGA-Phy+Bis NPs, and their morphology resembled that of the control group (Fig. 5b). These results indicate that concentrations of up to 100 mg/L of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs are safe for use.

Discussion

Phytol and α-bisabolol are natural compounds with numerous pharmacological properties. However, the utility of phytol is greatly restricted due to its hydrophobic nature, which results in poor solubility in biological fluids. This problem can be overcome through encapsulation. The present study evaluated the potential of PLGA-Phy+Bis NPs as a drug delivery system for the treatment of NSCLC. Phytol, α-bisabolol, and their combination were successfully encapsulated into PLGA NPs by the oil-in-water immersion, solvent evaporation method. The high encapsulation efficiency of PLGA-Phy+Bis NPs could be attributed to the ionic interactions between the drug and the polymer. Therefore, the utilization of ionic interactions for encapsulating phytol and α-bisabolol proves to be an effective strategy in minimizing drug wastage during the encapsulation process (Singh and Tiwari 2010). Previous research has highlighted the significance of this ratio in optimizing the encapsulation efficiency of drugs within the PLGA NPs. By carefully adjusting the drug/polymer ratio, researchers were able to maximize the amount of drug incorporated into the NPs. Additionally, the drug/polymer ratio has a direct impact on the drug release characteristics of the PLGA NPs (Bala et al. 2004; Makadia and Siegel 2011). The loading efficiency of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs depended on the drug/polymer ratio, with an increase in the drug concentration leading to higher loading efficiency. This effect might be due to the ability of the polymer matrix to accommodate a higher amount of phytol and α-bisabolol molecules in the polymeric matrix, thus ensuring the ability of the polymeric matrix to release higher drug concentrations at the specific target site. The results notably highlight that the preferential encapsulation of α-bisabolol over phytol, even at equal concentrations, is attributed to its distinctive hydroxyl group arrangement, fostering a more favorable interaction with the PLGA polymer. Due to the high polarity and presence of hydroxyl groups, α-Bisabolol is highly soluble in the PLGA solution, which enhances its effective dissolution and subsequent encapsulation. Consistent computational predictions from ESOL Prediction, Ali Prediction, and SILICOS-IT reinforce this observation, affirming α-bisabolol’s superior solubility compared to phytol. Furthermore, the observed differential diffusion rates in the PLGA solution suggest that α-bisabolol may exhibit a greater ability to rapidly diffuse into the PLGA polymer matrix compared to phytol, thereby contributing to the observed higher encapsulation efficiency.

The size of all nanoparticle formulations were in the nanometer range with a very narrow PDI, and the hydrodynamic diameter of the NPs fell into the region of NPs. The particle size distribution analysis for PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis revealed a narrow size distribution profile with very low PDI and high stability, which are important characteristics for effective drug delivery and passive targeting of cancer tumors, including NSCLC (Anwer et al. 2019). The small size of the NPs allows them to passively accumulate in the tumor tissues via the enhanced permeability and retention (EPR) effect, which is a common phenomenon observed in most solid tumors (Varani et al. 2020; Ibrahim et al. 2020). The high stability of the NPs ensures that they remain intact in the bloodstream and can effectively deliver the drug payload to the tumor site (Wei et al. 2016). Furthermore, the ability of the NPs to encapsulate and deliver drugs such as phytol and α-bisabolol to the tumor site has potential therapeutic benefits for NSCLC treatment. Studies have shown that both phytol and α-bisabolol have anti-cancer properties and can inhibit the growth and proliferation of NSCLC cells in multiple pathways. In a previous report, it was demonstrated that phytol and α-bisabolol synergistically inhibit A549 cells through several mechanisms, including an increase in ROS, changes in [Ca2+]i, induction of G0/G1 cell cycle arrest and cellular senescence, and intrinsic mode of apoptosis. Additionally, they may interact with the actin cytoskeleton to impede cell migration, demonstrating their potential as anti-metastatic agents (Kiruthiga et al. 2024). The zeta potential of the drug-loaded NPs exhibited negative values for PLGA-Phy (highly negative) and PLGA-Bis (near neutral), and neutral for PLGA-Phy+Bis NPs. Because of the neutral charge of PLGA-Phy+Bis, there is no electrostatic hindrance in drug-cell interactions, and it can be further used as a potent drug delivery system for multiple cancer targets. Additionally, unlike cationic and anionic nano-emulsions, neutral NPs will not accumulate in the liver and cause further toxicity. Furthermore, neutrally charged NPs do not undergo electrostatic moments (electrical mobility mean, 0.000001 cm/Vs) inside tissues, and thus can be used as a targeted delivery system via injections on the site of a tumor (Yao et al. 2019). However, some studies have shown that negatively charged NPs can improve cellular uptake and drug delivery to cancer cells due to the interaction with positively charged cell membranes (Clogston and Patri 2011). On the other hand, the near-neutral zeta potential of PLGA-Bis NPs and the neutral zeta potential of PLGA-Phy+Bis NPs may provide a balanced approach, with improved stability and reduced aggregation, while also facilitating cellular uptake and drug delivery to cancer cells due to a lack of electrostatic repulsion or attraction (Huang et al. 2016).

Although all PLGA NPs have a similar oval shape, variations in size were observed within the nanoparticles. The largest size nanoparticle on average mean size was observed in the case of individual nano-carrier PLGA-Phy, whereas PLGA-Bis had the lowest average mean size. This could be attributed to different parameters such as drug encapsulation efficiency of PLGA, molecule accepting groups in the PLGA, and chemical composition/molecular weight of phytol (C20H40O/296.5) and α-bisabolol (C15H26O/222.37) (Asal et al. 2022).

The study did not observe any form of aggregation in any of the NPs, indicating the stability and enhanced shelf life of NPs at room temperature. However, it should be noted that since TEM was performed in dry circumstances, the processed NPs had a propensity to shrink, resulting in the loss of their main shape and size, and the nanoparticle size range was less than the size distribution obtained by the DLS as provided in Table 2. Overall, the HR-TEM analysis provided valuable insights into the morphology and size distribution of the PLGA-stabilized NPs.

The dialysis method is a widely used technique to study drug release from NPs as it mimics the diffusion process that occurs in vivo (Patra et al. 2018; Weng et al. 2020). The results showed that sustained release behavior exhibited by the PLGA-Phy+Bis NPs is an important characteristic of a drug delivery system, as it ensures that the drug is released over a longer period time, thereby increasing its efficacy. The sustained release behavior of PLGA-Phy+Bis NPs is due to the biodegradable and biocompatible properties of PLGA, which degrades gradually to release the drug at a controlled rate. The addition of phytol and α-bisabolol to PLGA NPs could have enhanced this behavior by interacting with the polymer and influencing the degradation rate (Rezvantalab et al. 2018). The anti-proliferative effect of the NPs against A549 cells suggests that these NPs have potential as therapeutic agents for NSCLC. Furthermore, the induction of apoptotic cell death by PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs confirms their potential as a treatment option for NSCLC. The upregulation of Bax and downregulation of Bcl-2 in A549 cells treated with PLGA-Phy+Bis NPs suggests that this combination of NPs may induce apoptosis in cancer cells via the mitochondrial pathway(Balan et al. 2021). Induction of apoptosis holds significant importance in cancer therapies as it aims to eliminate cancer cells by triggering their programmed cell death (Hanahan and Robert A 2017; Chirumbolo et al. 2018). The ability of PLGA-Phy NPs, PLGA-Bis NPs, and PLGA-Phy+Bis NPs to induce apoptosis in cancer cells, as evidenced by their anti-proliferative effects and modulation of apoptotic proteins, highlights their potential as therapeutic agents for combating NSCLC. The study demonstrates that concentrations of PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis NPs up to 100 µg/mL are safe for use, as tested on L-132 cells. Notably, at a maximal concentration (200 µg/mL), both PLGA-Phy and PLGA-Bis exhibited mild toxicity at the 24 h time point, as depicted in Fig. 5a. This observed mild toxicity was significantly alleviated in the case of PLGA-Phy+Bis, underscoring the potential synergistic or protective effects arising from the combination of phytol and α-bisabolol. Furthermore, the PLGA-Phy+Bis NPs showcased a selective anti-proliferation effect in A549 cancer cells, with a notable sparing effect on normal L-132 cells. This substantiates that the safety profile of the NPs aligns with the non-toxic nature of phytol and α-bisabolol, particularly with a higher affinity for L-132 cells over A549 cells (Sakthivel et al. 2018). This finding is an important aspect of drug development as it suggests that these NPs have a low toxicity profile and can be a potential strategy for inducing apoptosis in cancer cells without affecting normal cells. These findings are consistent with previous studies reported in the literature (Rajavel et al. 2017, 2018). This is a promising finding for further development of these NPs as a drug delivery system for NSCLC, as it suggests that they may have a low toxicity profile in vivo. However, further research is needed to determine the molecular mechanism of these NPs in cancer therapy. Overall, these findings suggest that PLGA-Phy+Bis NPs hold promise as an effective and safe drug delivery system for the treatment of NSCLC.

Conclusion

In conclusion, the co-delivery of biocompatible PLGA NPs offers a promising strategy to enhance the effectiveness of therapy and minimize side effects in the treatment of NSCLC. The successful encapsulation of phytol, α-bisabolol, and their combination within PLGA NPs demonstrates their remarkable efficiency in terms of encapsulation, loading, and yield, as well as their uniform size distribution and stability. PLGA NPs exhibit significant potential as drug-delivery vehicles for lung cancer treatment, offering advantages such as improved therapeutic efficacy and reduced toxicity. By utilizing the capabilities of PLGA NPs, the delivery, and targeting of anti-cancer agents can be optimized, potentially leading to more effective and safer treatment outcomes for NSCLC patients. However, further research and development are needed to optimize the performance and application of PLGA NPs in the field of lung cancer therapy.