Gliadin Encapsulated Procyanidolic Oligomers Nanoformulation: Aggrandized Antimicrobial, Antioxidant as Well as Anticancer Potential Makes it an Effective Therapeutic Modality for Lung Carcinoma

Abstract

Lung cancer is responsible for highest mortality rates among both men and women globally. There is a need of effective and safe treatment modality that specifically targets cancer cells and doesn’t not harm healthy cells. Procyanidolic oligomers (PCOs) is a natural antioxidant found in grape seed, acts as medicament for the treatment of various disease which are linked with oxidative stress specifically cancer. The aim of the current study was to synthesize highly stable gliadin encapsulated PCOs nanoformulation (PCO-NF). The PCOs nanoformulation (PCO-NF) showed spherical morphology (17.18–76.56 nm) with an average particle size 49.37 ± 22.17 nm diameter in electron microscopy and stable zeta potential i.e. − 23.1 mV and low polydispersity index i.e. 0.246 in dynamic light scattering (DLS). The therapeutic efficacy of PCO-NF has been evaluated through antioxidative, anticancer as well as antimicrobial studies. Antioxidant potency was assessed using DPPH scavenging assay and it was observed that IC₅₀ of PCO-NF (9.02 µg/ml) was even lower than that of Vitamin C (10.22 µg/ml) as well as pure PCOs. The MTT assay was performed for the comparative anticancer activity of pure PCOs and PCO-NF against SK-MES-1 lung cancer cell line. It was found that PCO-NF significantly inhibited the lung cancer cells at all tested concentration. Moreover at 125 µg/ml concentration the PCO-NF showed 31.54% cell viability which was even lesser than the standard anticancer drug. The antimicrobial efficacy of PCO-NF was assessed against gram-positive and gram-negative bacteria. An augmented antimicrobial, antioxidant and anticancer potential was observed due to nanoencapsulation of PCOs.

Introduction

Cancer is still a big threat to human beings all over the world, despite the significant advancements in detection, diagnosis and treatment of Cancer. In India, the second most prevalent disease is cancer, which causes the highest mortality rate, approximately 0.3 million deaths annually. In the United States, there are projected to be 611,720 cancer related deaths and 2,001,140 new cases of cancer in 2024. Among all types of cancers, lung cancer is one of the biggest threats to people’s health, because it is usually identified at later stages where treatment choices are scarce. In the US, it is expected that in 2024, there will be 125,070 lung cancer deaths and 234,580 new cases of lung and bronchus cancer among men and women [1]. Mortality rate of lung cancer is higher in Asia than Europe and USA [2].

The major types of lung carcinoma are non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC). The slowly growing NSCLC is more prevalent than SCLC [3, 4]. For different types of cancer, chemotherapy remains an important therapeutic option despite advances in surgical and radiation treatment. However, the inherent and acquired resistance of cancer cells to anticancer medications significantly restricts the effectiveness of chemotherapy. High dose, non-specific distribution, extreme toxicity to healthy cells, insufficient drug concentration at cancerous sites are the main cause of emergence of multidrug resistance [5].

Among cancer patients a major challenge to deal with is bacterial infection [6]. Bacteremia, ear, nose, throat infections and gastrointestinal tract infections are the main factors that complicate the course of lung cancer. A special symbiotic relationship exists between bacteria and cancer cells during the course of cancer development and treatment. Some bacteria like E. coli, P. aeruginosa, S. aureus etc. frequently colonize and cause infections in cancer patients. However, patients having malignant diseases can live longer when receiving appropriate cytostatic therapy along with antimicrobial therapy [7]. Antibiotics and cancer treatment go hand in hand. Thus, consideration of new and safe alternative is needed, which can be achieved by integration of natural bioactive molecule with nanotechnology.

The nanotechnology has multiple applications in broad areas including Physics, Chemistry, Biology, Engineering and Medicines [8–10]. The use of nanotechnology in the field of nanomedicine involves developing nanocarrier systems delivering therapeutic compounds in safe and controlled manner to treat various chronic diseases including cancer. Nanocarrier also improves the stability and bioavailability of the drug [11]. Bacteria and cancer cells have a symbiotic relationship during the development and treatment of cancer. With the rapid advancements in nanomedicine, antimicrobial treatment has made significant strides in cancer therapy [12].

Herbal therapy is one of the most pre-eminent types of traditional medicine in every corner of world, and approximately 70–80% of people worldwide prefer this therapy for the treatment of any kind of disease [13]. The established relationship between oxidative stress and cancer makes sense that antioxidants can dramatically lower the onset and progression of cancer [14]. Bacteria and cancer cells have a symbiotic relationship during the development and treatment of cancer [12].

A novel fusion of science and nature could result in a medicinal revolution. The bioactive polyphenols protected by nanotechnology for therapeutic purposes is the basis of this potentially ground-breaking concept. In present study we have explored plant based effective anticancer candidate that has many health benefits along with anticancer and antimicrobial property, so we can use it as supportive therapies for lung cancer. Polyphenols obtained from plants accounts not only for antioxidant but also for anticancer activity. Grape is one of the popular fruits consumed globally and the grape seeds contain around 90% proanthocyanidin (procyanidolic oligomers (PCOs)) and 7% other various types of polyphenols. Proanthocyanidin is a phenolic compound present in plant, which has recently garnered attention due to their powerful antioxidative potencies [15, 16].

Gliadin is protein derived from wheat and due to its biodegradable, biocompatible and natural origin it is used for synthesis of polymeric nanoformulation. Previous studies have shown the synthesis of gliadin nanoparticles and evaluated for drug delivery and controlled release application.

The anticancer properties of pure procyanidolic oligomers (PCOs) are widely acknowledged, PCOs causes anti-tumorigenic effect on fresh tumor tissues taken from colorectal patients. It also inhibit multiple pathways linked with cancer and suppress the growth of tumor in animal models [17]. PCOs suppress cancer stem cells and inhibit patient derived organoids as well as mouse derived organoids [18]. Similarly lots of in vitro and in vivo research on cancer has been conducted on pure procyanidolic oligomers, but there is very limited research available on nanoformulation of PCOs.

At present, there are no studies on anticancer studies of nanoformulation of Procyanidolic oligomers on Lung cancer cell line (SK-MES-1). In this research we have developed the nanoformulation of procyanidolic oligomers (PCOs) and investigate its anticancer potential on SK-MES-1: Lung cancer cell line in vitro.

Materials and Methods

Materials

Gliadin (Cat No.: 101778, MP Biomedicals India Private Limited, Mumbai, India) and Procyanidolic oligomers (Cat No.: 1298219, Sigma-Aldrich, St. Louis, MO, USA), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (#4060, HiMedia), RPMI 1640 (#AL-162S,Himedia), and Cisplatin (Cat No.: #C2210000, Sigma-Aldrich), tween-80 (Cas No. 9005-65-6, SD Fine Chemicals limited), vitamin C (Cat No.: 50-81-7, HiMedia Lab. Pvt. Ltd, Mumbai, India), trichloroacetic acid (SISCO Research Laboratories Pvt. Ltd. Mumbai), triton x-100 (Cat. No.:9002-93-1, SD Fine Chemicals limited, Mumbai) were used in the present study. All other chemicals used were of analytical grade.

Preparation of Procyanidolic Oligomers Nanoformulation (PCO-NF)

Gliadin encapsulated Procyanidolic oligomers nanoparticles were synthesized by modified desolvation method [19, 20]. Briefly, 20 mg gliadin and 5 mg PCOs were dissolved in 10 ml of 70% ethanol, and the solution was added (dropwise) in a solution containing pluronic F-68 (0.5%) and physiological saline phase (NaCl 0.9% w/v in water), where pluronic F-68 was used as stabilizer. After that, gluteraldehyde was added as cross-linker and stirred for 12 h at room temperature. Ethanol was made to evaporate by appropriate procedure and the prepared nanoparticles were purified by high-speed centrifugation (15,000 rpm) for 1 h. The resulting supernatant was decanted off, and pellet was reconstituted in sterile distilled water. The nanoparticles thus prepared were freeze dried and then used for various characterization.

Characterization

A diverse range of advanced techniques were used to characterize the synthesized PCO-NF. The size distribution, dispersity and zeta potential were measured by performing the dynamic light scattering (DLS). The surface morphology and accurate size were investigated through Field emission scanning electron microscope (FESEM), Atomic force microscopy (AFM) and transmission electron microscopy (TEM). The infrared spectrum was obtained on the FTIR spectrophotometer to study the composition and interaction within the nanoformulation.

Encapsulation Efficiency

The free drug concentration in the supernatant relative to total drug was calculated to determine the encapsulation efficiency of PCO-NF. To separate the free drug from the nanoformulation, centrifugation was done at 10,000 rpm (4 °C) by using centrifuge for 30 min. UV–visible spectrophotometer (Shimadzu Corporation, UV-2450) was used to analyze the amount of drug (PCOs) in the supernatant at 280 nm wavelength.

The following formula was used to calculate the percent encapsulation efficiency:-

$$\%\text{Encapsulation}\text{Efficiency =}\frac{\text{Total}\text{drug}-\text{Free}\text{drug}}{\text{Total}\text{drug}}\times 100$$

Swelling Behavior

The swelling experiment was conducted to evaluate how the lyophilized nanoformulation swells at room temperature over a 24 h period using three different pH solutions: 0.1 N HCl (pH 1.2), bicarbonate buffer (pH 6.8), and phosphate-buffered saline (pH 7.4) [21]. The swelling medium was used to immerse the dried test sample weighing 50 mg for 24 h at room temperature. The swollen sample was removed from the solutions in order to remove any residual swelling media from the surface and then blotted out with the help of filter paper. To calculate the equilibrium degree of swelling (EDS) in terms of percentage, the swollen sample was weighed and following formula was used.

$$\text{EDS}\left(\%\right)=\frac{\text{Swollen}\text{weight}-\text{Initial}\text{weight}}{\text{Initial}\text{weight}}\times 100$$

In Vitro Drug Release Study

The release studies of PCOs and PCO-NF were conducted at pH 7.4 utilizing the dialysis bag method. The dialysis membrane (cut off between 12 and 14 kDa, Hi-Media, India) were immersed in double distilled water for 12 h before starting the release study.

Fresh PBS (pH 7.4) was used to dissolve 50 mg of PCOs and PCO-NF (weighing equivalent to 5.5 mg of PCOs). At different time intervals, one milliliter sample were taken out and replaced with an equal volume of fresh buffer. As a reference, the absorbance of the collected sample was determined using UV–visible spectrophotometer at 280 nm. Each experiment was conducted three times.

The PCOs and PCO-NF were also examined for mechanism of drug release and release kinetics. In order to estimate the drug release behavior by fitting the drug release data into various kinetics models, and Korsmeyer Peppa’s equation was used to derive the diffusion exponent (n) [22].

Evaluation of Antioxidative Potential of PCO-NF

The antioxidative potential of the PCO-NF in comparison with pure PCOs was studied by performing in vitro DPPH (1,1-diphenyl-B-picryl-hydrazyl) free radical scavenging assay according to the protocol used by Manickam et al., with minor adaptations [23]. The reaction mixture including DPPH solution (1 ml: 3.9 mg/100 ml) and different concentrations (25–200 µg/ml) of PCOs and its nanoformulation were incubated at room temperature in dark for half an hour. The absorbance of the reaction mixture was measured at 517 nm using UV–Vis spectrophotometer. DPPH without sample was used as control and percent inhibition was measured by using the below mentioned formula:

$$\%\text{inhibition =}\frac{\left(\text{Absorbance}\text{of}\text{Control - Absorbance}\text{of}\text{Sample}\right)}{\text{Absorbance}\text{of}\text{Control}}\times 100$$

The antioxidant capacity of the PCO-NF was expressed in terms of IC₅₀ value which was calculated by plotting the graph between percentage of DPPH scavenging against nanoformulation concentration.

In Vitro Cytotoxicity MTT Assay in Vero Cell Line

For the cytotoxicity studies the Vero cell line was procured from the National Centre of Veterinary Type Cultures (NCVTC), NRCE, Hisar, India as described in earlier study [24]. These cell lines were then maintained in Eagle’s minimum essential medium (EMEM). For the assessment of cytotoxicity assay,100 µl of 1 × 10⁵/ml cell suspension was added to 96-well plate (Greiner, cell star) and incubated at 37 °C with 5% CO₂ in an incubator for 24 h. Next day, the cell density was checked microscopically for monolayer formation of Vero cell lines. Different concentrations of PCOs and PCO-NF were added over cells and incubated for 24 h. Following a PBS wash, 50 µl of 5 mg/ml MTT was added to each well and then incubated again for 4 h. Then, DMSO was added into the wells and the absorbance was recorded at 590 nm using ELISA microplate reader (SPECTRO_star Nano Microplate Reader, BMG Labtech, Germany). Untreated cells and triton-X (1%) was used as negative control and positive control, respectively. The percent cell viability was calculated by following formula.

$$\%\text{Cell viability}=\frac{\text{Absorbance}\text{of}\text{treated cells}}{\text{Absorbance}\text{of}\text{untreated cells}}\times 100$$

Evaluation of Anticancer Activity of PCO-NF

The anticancer evaluation of the PCO-NF was done using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on SK-MES-1 cell lines according to the protocol used by Aydin Acar et al., with slight modifications [25]. For performing the MTT assay, the trypsinized cell suspension was adjusted to the 5.0 × 10⁵ cells/ml. 100 µl of the cell suspension was poured in the wells of sterile 96-well plate. A partial monolayer that developed after 24 h was checked and the cells were once again washed. Then 100 µl of the samples were added in various test concentration viz. 31.25, 62.5 and 125 µg/ml [26]. After 24 h incubation at 37 °C with 5% CO₂, the test solutions were added in the wells and 100 µl of MTT (5 mg/10 ml of MTT in PBS) was added. The 96- well plates were then incubated at 37 °C in 5% CO₂ atmosphere for 4 h. After removing the supernatant, 100 µl of DMSO was added and the plates were shaken gently to dissolve the formazan that had formed. The absorbance was noted at 590 nm wavelength using a microplate reader and the percentage growth inhibition was determined using the following formula:

$$\%\text{Cell viability}=\frac{\text{Mean Absorbance}\text{of}\text{Sample - Blank}}{\text{Mean Absorbance}\text{of}\text{untreated cells - Blank}}\times 100$$

The optical microscopy The morphological changes in the PCO-NF treated SK-MES-1 cells were observed by using inverted microscope (BioLinkz, India) at 200 × magnifications.

Evaluation of Antimicrobial activity

The antimicrobial evaluation of the PCO-NF was done by agar well diffusion method. The antimicrobial potency of the PCO-NF was assessed against the bacterial strains viz. Escherichia coli (MTCC 048), Staphylococcus aureus (MTCC 6908), Pseudomonas aeruginosa (NCDC 105) and Bacillus subtilis (MTCC 441) using cefotaxime sodium antibiotic as positive control as described in earlier studies [27, 28]. The molten nutrient agar medium was poured aseptically into the sterile petri dishes and 10⁷ CFU of fresh bacterial culture was inoculated into the dishes and allowed to solidify. With the aid of a sterile cork-borer, 5 mm diameter wells were excavated on each plate at evenly spaced intervals, and the respective test and control samples were promptly added in the specified wells. Properly sonicated, 80 µl PCO-NF of each concentration was poured in the specified wells and the petri plates were incubated at 37 °C temperature for 24 h. The clear inhibition zones were obtained post incubation and measured using ruler and expressed in mm (millimetre).

Statistical Analysis

All the experiments were carried out in triplicates. Graph Pad Prism version 8.02 software (San Diego California, USA) was used to analyze the experimental data. Tukey’s multiple comparison test was employed after two-way ANOVA for the statistical analysis of data. All the findings are shown as average ± SD (standard deviation). Results are shown as mean ± standard deviation (SD). P < 0.05 was used to determine the significance of differences.

Results and Discussions

Preparation and Characterization of Nanoformulation

Modified desolvation method was used for the preparation of nanoformulation. The prepared PCO-NF was characterized using Dynamic light scattering (DLS), Field emission scanning electron microscopy (FESEM), Transmission electron microscopy (TEM), Atomic force microscopy (AFM) and Fourier transform Infrared spectroscopy (FTIR). The DLS was performed using the aqueous reaction mixture while the lyophilized nanoformulation was used for other studies.

Size distribution, Colloidal Stability and Encapsulation efficiency of PCO-NF

The prepared nanoformulation was initially characterized by using the DLS (Nano ZS-90, Malvern Instruments, UK) for its particle size distribution, polydispersity index (PDI) and (ZP) zeta potential. The average size of the nanoformulation as per the DLS examination was found to be 236.4 nm, while the PDI and zeta potential were 0.246 and − 23.1 mV respectively (Fig. 1).

Fig. 1.

a Micrograph obtained from particles size analysis of PCO-NF, b Zeta potential curve of PCO-NF

The low PDI value obtained in DLS of PCO-NF infers towards the uniform distribution of the nanoparticles, while strongly negative surface zeta potential of the nanoparticles is the measure of high colloidal stability. DLS gives rough estimation of the nanoparticle size as it provides the information about hydrodynamic diameter. Therefore, the electron microscopy viz. FESEM and TEM were performed to decipher the actual size and morphology of the nanoparticles in PCO-NF. The encapsulation efficiency of the PCO-NF was calculated and it was found to be 91.24%.

Particle Size, Shape and Surface Morphological Analysis of PCO-NF

The surface morphology of the PCO-NF was observed by using Field Emission Scanning electron microscope (FESEM: TeScan Megna). To further study the precise size and shape Transmission electron microscope (TEM, Hitachi H-7500) was used. Highly monodisperse, spherical nanoparticles having size ranging from 50 nm to 275.9 nm with an average size 160.56 ± 94.49 nm having smooth surface were observed in the FESEM micrographs of PCO-NF (Fig. 2a, b).

Fig. 2.

a, b FESEM image of (PCO-NF) PCOs nanoformulation; c, d TEM image of PCO nanoformulation (PCO-NF)

Further the TEM micrograph of the PCO-NF represented that the synthesized nanoparticles have spherical morphology having 17.18–76.56 nm size range with an average particle size 49.37 ± 22.17 nm and are in non-agglomerated form (Fig. 2c, d).

Nanoformulation of PCOs (PCO-NF) were also analyzed using Atomic force microscope (AFM, Park Systems NX10, Korea) for their morphology, size and surface topography.

AFM image (Fig. 3) of PCO-NF showed semi spherical morphology with particle sizes ranging 3.65 nm to 48.04 nm with an average particle size 11.86 ± 8.10 nm. Particle sizes obtained with AFM were closer to those obtained with the TEM results.

Fig. 3.

AFM image of (PCO-NF) PCOs nanoformulation, nanoformulation (PCO-NF), a 2 D image, b 3 D image of PCO-NF nanoformulation

FTIR Spectral Analysis

The FTIR analysis of all the samples were carried out in the range 4000–400 cm⁻¹ (Fig. 4) on a spectrophotometer (Perkin Elmer, USA).The information related to functional group as well as chemical bond can be revealed by the Fourier transform infrared spectroscopy (FTIR) spectrum of the compound. The spectra of different samples shown in Fig. 4. The spectrum of gliadin showed the stretching vibrations of –OH group at the broad band in the range 3200–3550 cm⁻¹ and centered at 3433 cm⁻¹ (16.77%). Accordingly, amide I band (C=O stretching) and amide II band (C–N stretching and C–N–H in plane bending) for gliadin were located at 1660 cm⁻¹ (18.78%) and 1546 cm⁻¹ (30.35%) respectively. The –CH₂ bending vibration exhibited the peak at 1450 cm⁻¹ (39.87%) [29, 30].

Fig. 4.

The FTIR spectra: a of pure gliadin, b Procyanidolic oligomers (PCOs), and c PCOs nanoformulation (PCO-NF)

The broad absorption band in spectrum of pure procyanidolic oligomers, which spans from 3400 to 3150 cm⁻¹ centered at 3435 cm⁻¹ (28.88%), was due to the H bond effect between the phenolic hydroxyl groups in procyanidolic oligomers. Procyanidolic oligomers (PCOs) contain functional groups at 1610 cm⁻¹ (36.89%) and 1105 cm⁻¹ (43.82%) that are characteristic of poly flavonoid moiety. The bending of CH₃ out of plane at 1387 cm⁻¹ (44.24%) and the C–O stretch at 1049 cm⁻¹ (47.17%) are associated with polysaccharide structures.

The aromatic rings’s skeletal stretching modes in the procyanidolic oligomers’ structure and the aromatic rings’s CH out of plane deformation containing two adjoining free hydrogen atoms exhibited peaks at 1523 cm⁻¹ (46.71%) and 767 cm⁻¹ (52.59%) [31, 32]. The presence of distinctive bands at 1154 cm⁻¹ (47.83%) and 827 cm⁻¹ (54.03%) accounts for the presence of PCOs, which are shifted in the nanoformulation (PCO-NF), which corroborate the interaction of PCOs with polymer.

The spectrum of nanoformulation of PCOs showed that the peaks of –OH were moved to 3402 cm⁻¹ (33.28%), which was caused by hydrogen bond formation between procyanidolic oligomers’ phenolic groups and the amide groups of glutamine in gliadin. Apart from the notable difference in the O–H stretching band, the conspicuous difference was observed in the fingerprint region at wavenumbers 1500 to 400 cm⁻¹, specially at following peaks viz. 1428 cm⁻¹ (51%), 1385 cm⁻¹ (47.32%), 1090 cm⁻¹ (48.39%), 1016 cm⁻¹ (49.52%), 710 cm⁻¹ (53.28%) and 626 cm⁻¹ (51.74%), which confirmed the presence of PCOs in PCO-NF nanoformulation.

Colloidal Stability

Since the environmental conditions has a major impact on the stability of nanoformulations, this study was carried out to understand the colloidal stability of the as synthesized PCO-NF nanoformulation in various environmental settings.

The nanoformulations of PCOs (PCO-NF) were kept at two different environmental conditions i.e. 4 °C and 25 °C for 12 weeks in order to examine the colloidal stability, the results are shown in Table 1. The PCO-NF nanoformulation didn’t show any significant changes in PS, PDI and ZP.

Table 1.

Colloidal stability of the PCO-NF under different environmental conditions and different time intervals, showing particle size, PDI and zeta potential

Time (Weeks)	At 4 °C			At 25 °C
	PS	PDI (nm)	ZP (mV)	PS (mV)	PDI (nm)	ZP (mV)
Zero weeks	228.3	0.319	− 28.2	228.3	0.319	− 28.2
Four weeks	229.3	0.311	− 27.3	235.5	0.381	− 27.1
Eight weeks	232.7	0.37	− 27.1	236.4	0.246	− 25.6
Twelve weeks	235.5	0.381	− 25.2	245	0.239	− 22.8

Swelling Behavior

The nanoformulation was tested for its swelling properties in various absorbing media, including 0.1N HCl, pH 1.2, PBS (pH 7.4) and bicarbonate ion pH 6.8. The nanoformulation exhibited a decrease in swelling at pH 1.2 (0.1 N HCl) compared to pH 6.8 and pH 7.4 (Fig. 5a). From the findings it is clear that there was a pH dependent swelling behavior in the PCOs nanoformulation. The slightly elevated EDS% value from pH 6.8 to pH 7.4 indicates that elevated pH causes more swelling of PCO-NF i.e. nanoformulation of PCOs. It is clear from the results that in alkaline pH i.e. in intestine the PCOs nanoformulation (PCO-NF) sustain more swelling, while at acidic pH in stomach the PCO-NF swells slightly. As mentioned later in the drug release study, this swelling behavior could facilitate the release of bioactive compound i.e. PCOs.

In Vitro Drug Release Study

The in vitro drug release study of PCOs nanoformulation (PCO-NF) was performed at pH 7.4 which suggests that PCOs nanoformulation exhibits sustained release of PCOs in comparison to pure PCOs. The polymer matrix that causes the drug to release typically determines the drug’s stability and release rate pattern, which change in response to the changes in temperature and pH of the releasing medium.

The percentage cumulative release of PCOs loaded nanoformulation and pure PCOs at pH 7.4 is shown in Fig. 5b. PCOs nanoformulation exhibites sustained drug release pattern. At pH 7.4 the PCOs nanoformulation exhibits drug release of 18.2%, 37.6% and 56.5% at 1, 4 and 24 h, respectively, whereas the pure PCOs demonstrates drug release of 22.8% 56.2% and 82.08% at 1, 4, and 24 h, respectively (Fig. 5b).

Fig. 5.

a Swelling behavior study of PCOs nanoformulation (PCO-NF) at various pH; b In vitro release of pure PCOs and PCOs nanoformulation (PCO-NF) which follows zero order kinetics; c Higuchi model kinetic release of PCOs from Pure PCOs and PCO-NF; d Korsmeyer Model kinetic release of PCOs from Pure PCOs and PCO-NF

It is possible that there may be non-encapsulated PCOs present at the surface of nanoformulation, causing the initial burst release of PCOs from its nanoformulation. Analogous outcomes have been previously documented for various hydrophilic drugs, whereas a delayed release of the drug was achieved through encapsulation of drug or bioactive compounds within nanocarriers [33, 34].

Drug Release Kinetics

The value of release exponent (n) is used to determine the drug release mechanism. In the case, where n = 0.43, it shows that there is fickian diffusion mechanism. A value falls into the anomalous (non-Fickian) diffusion category, when the value of release exponent falls in between this range i.e. 0.43 < n < 0.85. When n = 0.85, it displays class II transport [35]. In the present investigation, at pH 7.4, the n value obtained by applying the Korsmeyer–Peppas equation was 0.562. This indicates that anomalous or non-Fickian, diffusion was observed. This kinetics imply that multiple mechanisms are involved in the release kinetics, i.e. a combination of the two techniques- swelling controlled release and diffusion controlled release.

Antioxidant Activity

Free radical damage has been closely linked to almost all chronic degenerative diseases, such as cancer, rheumatoid arthritis, and cardiovascular disease. It is evident that Procyanidolic oligomers (PCOs) have a wide range of therapeutic applications because of their antioxidant activity. PCOs are most notable for their well-documented capacity to scavenge free radicals [36]. The DPPH antioxidant assay was employed to assess the free radical scavenging ability of PCOs and its nanoformulation i.e. PCO-NF. The results obtained in the DPPH radical scavenging assay clearly demonstrated the excellent potential of PCO-NF when compared with pure PCOs and Vitamin C (Fig. 6). The IC₅₀ of the PCO-NF, pure PCO and Vitamin C obtained from the dose dependent curve were 9.02, 14.49 and 10.22 µg/ml respectively. The IC₅₀ is a measure of antioxidant potential and inversely proportional to the scavenging potential. PCO-NF, in present research, depicted an IC₅₀ even lower than the Vitamin C, which confirmed the extraordinary therapeutic potential of the developed PCO-NF.

Fig. 6.

Graph demonstrating dose dependent DPPH radical scavenging activity of Vitamic C, Pure PCOs and (PCO-NF) nanoformulation of PCOs. *The data are displayed as Mean ± SD and the samples were analyzed in triplicates.

In Vitro Cell Cytotoxicity MTT Assay in Vero Cell Line

In vitro cytotoxicity assay in Vero cell line was assessed by using MTT assay. The percentage cell viability of different concentration and drug-treated Vero cell lines images is shown in graph (Fig. 7).

Fig. 7.

Graph showing in vitro cytotoxicity study of PCOs and its nanoformulation (PCO-NF) on Vero cell lines showing percent cell viability at different concentration

The morphological examination of Vero cell lines under an inverted microscope exhibited normal characteristics (Fig. 8). PCO-NF did not exhibit any phenotypic changes in Vero cells, suggesting that the PCO-NF nanoformulation do not affect normal cells. It is found that viability of Vero cells were found to be 104%, 91.56%, 85.03%, 76.43% and 73.60% at concentrations 15.62 µg/ml, 31.25 µg/ml, 62.5 µg/ml, 125 µg/ml and 250 µg/ml respectively, indicating that the prepared nanoformulation of PCOs (PCO-NF) is nontoxic to normal cells and hence, suitable for biological applications.

Fig. 8.

Microscopic images of Vero cells: a negative control, b PCOs-treated, c PCO-NF treated

In the present investigation, it was found that the different concentrations of PCOs and its nanoformulation have no cell toxicity, as cell viability of more than 73% was observed at their higher concentrations. The cell viability of the Vero cell line treated with nanoformulation was found to be comparable to that of negative control.

Anticancer Activity of PCO-NF Against SK MES-1 Lung Cancer Cell Line

The anticancer potential of PCO-NF and pure PCOs was examined using MTT assay. Various concentrations of the PCO-NF and PCOs viz. 31.25, 62.5 and 125 µg/ml were incubated for 24 h. The results of the assay showed that at all the tested concentrations, SK-MES-1 cells growth was significantly inhibited by PCO-NF as presented in graph (Fig. 9). The graph in Fig. 9 shows that the cytotoxic effect on SK-MES-1 cancer cell-line increases with increase in concentration of PCO-NF. PCO-NF demonstrated even more potent cytotoxic effect as compared to standard cisplatin drug at 125 µg/ml concentration.

Fig. 9.

In-vitro anticancer activity of different concentrations of PCOs and nanoformulation of PCOs (PCO-NF) on SK-MES-1 cancer cell lines

Further, the optical microscopy was used for the morphological evaluation of the treated cells (Fig. 10). The optical microscopic images showed the dose-dependent killing of the cancer cells with increasing concentration of the PCOs (Fig. 10b–d) and PCO-NF (Fig. 10f–h).

Fig. 10.

Microscopic images of the morphological alterations that PCOs (10b-d) and PCOs-NF (10f–h) at different concentrations caused in SK-MES-1 cancer cell lines following a 24 h treatment period, compared to untreated controls (10a) as well as standard drug (10e)

SK-MES-1 cells were visualized under an inverted microscope to examine their morphological changes. While observing the morphology of control SK-MES-1 cells, the presence of a nucleus indicates that the cells in the control SK-MES-1 sample were alive and no morphological alterations were observed.

In contrast, the treated cell lines exhibited irregular confluent aggregates with rounded and polygonal cell shapes (Fig. 10), and this phenomenon was more frequent at higher concentrations. Following exposure to various concentrations of samples, the cell begins to die. The nucleus in the cells was invisible at a concentration of 31.25 µg/ml. The viability of the cells decreases at a concentration of 62.5 µg/ml, due to rupture and death of some cells. If concentration exceeds 62.5 µg/ml i.e. at 125 µg/ml concentration most of the cells rupture and die, which is comparable to standard drug.

Hence the PCO-NF is significantly effective in killing the cancer cells, than pure PCOs in equivalent concentration against the cancer cells.

The graph (Fig. 9) shows percent cell viability (mean ± SD; n = 3) observed at different concentrations of PCOs and its nanoformulation (PCO-NF). Two way ANOVA was used to analyze the data and Tukey’s multiple comparison test (Graph Pad Prism version 8.02 software, San Diego California, USA) was then performed, where, ‘a’ denotes significant difference at 31.25 µg/ml concentration as compared to PCOs; “b” denotes significant difference at 62.25 µg/ml concentration as compared to PCOs; and “c” denotes significant difference at 125 µg/ml concentration as compared to PCOs. ****—p < 0.0001, ***—p < 0.001, **—p < 0.01, *—p < 0.05.

Antimicrobial Evaluation

The antibacterial activity of the PCO-NF (40 µg/ml, 100 µg/ml) was performed against S. aureus and B. subtilis (gram-positive) and E. coli and P. aeruginosa (gram-negative) bacterial pathogenic strains. The PCO-NF exhibited significant antibacterial activity against all test strains (Fig. 11). The comparative zone of inhibition has been shown in graph (Fig. 11b). The maximum zone of inhibition at 100 µg/ml (22 mm) was obtained against E. coli and minimum zone of inhibition (15 mm) was obtained against P. aeruginosa (Fig. 11a). This implies that the PCO-NF depicted broad spectrum of bactericidal activity. There is little knowledge of the mechanism of action by which PCOs act as antibacterial agent.

Fig. 11.

a Zone of inhibition of PCOs and PCO-NF against (i) B. subtilis, (ii) P. aeruginosa, (iii) S. aureus and (iv) E. coli; b Graph showing comparative size of zone of inhibition of PCOs and PCO-NF against the bacterial strains used.

However according to recent studies, the main mechanism underlying antibacterial action of plant flavonoids might be the inhibition of DNA gyrase in gram negative bacteria, while in gram positive bacteria the antibacterial activity is primarily based on their membrane action [37].

Discussion

For this study modified desolvation method was used to synthesize PCO-NF nanoformulation and their optimization was demonstrated by a low PDI, smaller size nanoparticles, high encapsulation efficiency and smooth surfaced spherical nanoparticles.

The nanoformulation was successfully synthesized having spherical shape, smooth surface with average size 158.05 ± 10.88 nm and 70.05 ± 7.86 nm confirmed by FESEM and TEM respectively. The difference in size is approximate more than double. The discrepancy in the sizes obtained in the FESEM and TEM micrograph might have arisen due to the difference in sample preparation technique and hydration due to the presence of surfactant [38, 39]. A SEM microscope shows particles to be 3% larger than a TEM microscope. Different ways of producing contrast in microscopes, as well as inconsistent calibration of magnification, may account for the difference. When the electron beam reaches the edge of particle, secondary electron emission increases, increasing the particle size in SEM, which is one of the reason behind the larger particle size in FESEM [40]. However, the morphologies and distribution obtained in FESEM and TEM complement each other. PCO-NF showed 91.24% encapsulation efficiency and peaks in the FTIR spectra also confirmed the presence of PCOs in its nanoformulation. The stability studies of the nanoformulation (PCO-NF) was performed under two different physiological conditions i.e. at 4 °C and 25 °C for 12 weeks. It is clear from the graph that the PCO-NF is highly stable at 4 °C & 25 °C, because at these conditions particle size, PDI and zeta potential was not affected too much. Therefore, the nanoformulation is highly stable.

In this study, the cumulative release of nano PCOs from the PCO-NF at specified times was determined and tested against conventional kinetic models. The key advantages of this study include the decreased burst drug release from the in situ forming system with the incorporation of gliadin nanoparticles, prolonged drug release duration and controlled release through an optimal release profile. This prolonged release effect is also confirmed by drug release study which make its prolonged bioavailability of drug in the biological environment.

One of the most crucial factors affecting the biocompatibilities and bioactivities of micro and nanoparticles is their size and shape. Shape of nanoparticles affect the cellular uptake. Spherical nanoparticles can interact strongly with cell surfaces and less affected by shear, which increases cellular uptake events and promotes clearance [41]. A large majority of nanoparticles developed for drug delivery have spherical shape [42]. Spherical polymer nanoparticles displayed higher in vivo circulation half-lives (6.2 h) than nanoworm of the same volume and surface chemistry [43]. As documented earlier that small and spherical size nanoparticle may provide higher cellular uptake than larger ones [41, 44], so our nanoformulation which have small and spherical morphology and having high stability can be good option for in vivo studies due to its higher cellular uptake. Moreover, the encapsulation efficiency was found to be 91.24%.

Pure procyanidolic oligomers (PCOs) have anticancer effects on various types of cancers [45], but very less research has been conducted on nanoformulation of Procyanidolic oligomers. In the present investigation, nanoformulation of PCOs (PCO-NF) was developed and the anticancer study on lung cancer cell line (SK-MES-1) was carried out. There is no available research till now on anticancer activity of nanoformulation of PCOs on lung cancer cell lines specially on SK-MES-1 cell line. The synthesized nanoformulation (PCO-NF) and pure PCOs was comparatively evaluated on lung cancer cell line (SK-MES-1) which showed more prominent dose-dependent effect of PCO-NF. The cellular morphology demonstrated the anti-proliferative activity of PCO-NF as evidenced by decrease in the number of SK-MES-1 cells. The treated cell lines exhibited irregular confluent aggregates with rounded and polygonal cell shapes and begin to die (Fig. 10), and this phenomenon was more frequent at higher concentrations, i.e. interaction occurs with the cells in dose dependent manner [46].

In addition to this, PCO-NF showed aggrandized antioxidant as well as antimicrobial activity as compared to pure PCOs. There were not any detrimental effects observed by normal cell lines with the nanoformulation as concluded from the in vitro cytotoxicity assays on Vero cell lines.

Procyanidolic oligomers (PCOs) have several anticancer effects, besides their antioxidant properties. Kim et al. [45] reported the mechanistic evaluation that how oligomeric proanthocyanidins induced apoptosis impacts the human colorectal cancer cell line SNUC4. Several other studies have shown that, in breast cancer cells PCOs selectively induce apoptosis via programmed cell death; in leukemia cells PCOs inhibit cell proliferation, differentiation and apoptosis and in lung, PCOs microsomes inhibit nitrosamine activation [45, 47]. Therefore, the reason behind the improved antiproliferative effect of PCO-NF over PCOs in this research against lung cancer cell line could be enhanced cell apoptosis.

Good antiproliferative activity, biocompatibility and less cytotoxicity behavior of PCO-NF make the nanoformulation a good candidate for biomedical applications. Overall, it is evident from the research that PCO-NF is a promising candidate for the treatment of lung carcinoma.

While encouraging outcomes have been achieved, additional preclinical in vivo studies would be beneficial to definitely confirm the advantages of PCOs and its nanoformulation (PCO-NF). Additional insight into the pharmacokinetic profile, safety and efficacy of PCO-NF will be gained from the in vivo studies, as a result, the potential clinical applications of PCO-NF will be strengthened.

Conclusion

In present study, PCO-NF was prepared using gliadin as a nanocarrier due to its unique properties suitable for delivering bioactive molecules. The as synthesized spherical PCO-NF nanoparticles showed an optimal and stable zeta potential and highly monodispersed distribution having an average size of 49.37 ± 22.17 nm. The therapeutic potential of the PCO-NF in lung cancer treatment was explored by MTT assay. In the current investigation, it was found that nanoformulation of PCOs (PCO-NF) exhibited significantly high anticancer activity as compared to pure PCOs. Additionally, when compared to cisplatin, PCO-NF showed significantly higher anticancer activity. While the antioxidative potential of the PCO-NF was found to be even higher than the Vitamin C in equivalent concentration. Furthermore, an excellent broad spectrum bactericidal effect of the PCO-NF was also seen on gram-positive and gram-negative bacteria.

The gliadin encapsulated PCOs nanoformulation showed potent anticancer activity, strong antioxidative potential as well as significant antimicrobial activity, which fulfill all the essential requirement to combat the cancer and bacterial infections associated with the deadly disease. Therefore, PCO-NF holds promising potential for lung carcinoma treatment due to its three in one approach i.e. Antioxidant + Antimicrobial + Anticancer.

Declarations

Conflict of interest

The authors have no conflict of interest.