Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2025 Nov 1.
Published in final edited form as: Int J Biol Macromol. 2025 Mar 4;305(Pt 2):141747. doi: 10.1016/j.ijbiomac.2025.141747

Apoptotic and anti-inflammatory effect of nisin-loaded sodium alginate-gum arabic nanoparticles against colon cancer cells

Sanya Hazel Soans a, Muzaffar Jahangir Chonche c, Kunal Sharan c, Asha Srinivasan b, Ann Catherine Archer a,*
PMCID: PMC7617643  EMSID: EMS205029  PMID: 40049503

Abstract

Colon cancer is one of the leading causes of mortality and morbidity worldwide. Nisin, a polycyclic antibacterial peptide and food preservative has shown potential to combat cancer. However, it is susceptible to proteolytic cleavage in the gut. The current study investigates the protective and cytotoxic effects of nisin loaded sodium alginate gum arabic nanoparticles (Nis/ALG-GA NPs) in Caco2 cells. The physicochemical properties, loading efficiency and release kinetics were studied. Cytotoxicity (MTT assay), apoptotic effect (Ethidium bromide and acridine orange staining) and internalisation (FITC tagging) were evaluated. Gene expression of apoptotic markers and IL-10 were analysed by qPCR. The Nis/ALG-GA NPs were spherical, small with a smooth outer surface and mean size of 193 ±4 nm. The loading efficacy was 88 ± 2 % exhibiting slow sustained release of the peptide under different gut pH conditions. The IC50 value obtained was 500 μg for 48 h and 80 μg for 72 h of incubation. The Nis/ALG-GA NPs were internalised into Caco2 cells and induced apoptosis with an increased expression of bax gene and converse decrease of bcl-2 gene. Anti-inflammatory gene IL10 was upregulated upon treatment with NPs. Thus, the Nis/ALG-GA NPs may be promising oral drug delivery systems against colon cancers.

Keywords: Nisin, Alginate-gum arabic nanoparticles, anti-cancer activity

1. Introduction

In 2024, colon or colorectal cancer (CRC) was projected to account for 152,810 new cancer cases and 53,010 deaths in the United States [1]. The annual incidence rate (AAR) for colon cancer in India is 5.36 per 100,000 men and 4.3 per 100,000 women. The northeast region of India has the highest risk of CRC, followed by south India [2]. While CRC is more frequently diagnosed among people aged 65-74, its incidence is increasing in younger demographics since 2000 as they seem to increasingly adopt a sedentary lifestyle [3]. Surgery is the primary treatment, which results in only 50% of the patient’s recovery and the risk of recurrence is high. Lack of access to curative surgery and adjuvant therapies in many countries also leads to higher recurrence rates. Despite recent advances in treatment modalities, many less developed countries continue to have limited access to screening, early diagnosis, and advanced therapies [4]. Therefore, there is a need to develop novel anticancer agents to overcome side-effects and limitations of current treatments.

Nisin is an antimicrobial peptide (AMP) produced by the probiotic strain Lactococcus lactis. The peptide is composed of 34 amino acids and has a molecular weight of 3.5 kDa. Nisin has been approved by the FDA as a food preservative for human use and assigned a Generally-Regarded -As -Safe (GRAS) status [5]. Recent studies have demonstrated anti-cancer and cytotoxic effects of nisin on different cancer cells including breast, cervical, head and neck, colorectal and hepatocellular carcinoma cells [6–13]. The effects are mediated by induction of cell cycle arrest, reduced cell proliferation and activation of apoptotic pathway. The cationic moieties of nisin interact with the negatively charged phospholipids of the cell membrane, while the hydrophobic region interacts with the core of the nucleus [14]. Thus, nisin has the ability to integrate and create pores on the cell membranes of cancer cells leading to increased release of lactate dehydrogenase (LDH) [15]. Further, the presence of AMPs like nisin elicits increased reactive oxygen species (ROS) generation triggering the intrinsic mitochondria mediated apoptotic pathway by altering the bax/bcl2 ratio. Nisin enhances the levels of pro-apototic bax while decreasing the anti-apoptotic bcl2 [16,17]. In head and neck squamous carcinoma cells (HNSCC), nisin induced apoptosis via the calpain dependent pathway inhibiting angiogenesis, cell cycle arrest and decreasing cell proliferation [18].

However, the major drawback with nisin and related AMPs is their proteolytic cleavage during gastro-intestinal transit, thereby limiting its clinical application as an oral drug [7]. Although their peptidic nature may preclude oral delivery, yet oral delivery remains the most preferred mode of administration for a variety of therapeutic agents due to greater patient compliance and ease of administration [19]. Nanoparticle drug delivery system is one of the approaches employed to safely deliver peptide drugs and improve their half-life in vivo [20]. AMPs do not function by directly linking to any specific receptor on cancer cells, hence chances of developing resistance against them are less [21]. Hence, due to selective toxicity to cancer cells and lower drug resistance, AMPs like nisin are being employed to combat cancer [22].

The aim of the present study was to encapsulate nisin in a nanocarrier made up of natural polymers such as sodium alginate and gum arabic as an oral drug delivery system, which can protect nisin from proteolytic degradation during gastrointestinal transit and effectively deliver nisin to the target colonic site. Sodium alginate is a biocompatible and biodegradable hydrophilic polymer obtained from the brown seaweed. It has been used in micro- and nano-particle formulations for controlled delivery of drugs due to its remarkable properties such as acid-resistance, pH sensitivity, and crosslinking capability best suitable for oral delivery to the colon [23]. Carboxylic groups present in alginate provide sensitivity to external pH stimuli. At pH < 3.4, the carboxylic acid groups are in the non-ionized form (-COOH) leading to an insoluble structure. However, at pH > 4.4 the carboxylic groups become ionized, resulting in an increase in electrostatic repulsion of negative charges causing polymer chain expansion and swelling of the hydrophilic matrix, which is maximum at around pH 7.4 [24]. Gum arabic is largely used in food and pharmaceutical industries. It is considered as a safe food ingredient by the European Food safety authority (EFSA). The gel layer of the gum arabic acts as a barrier and retards the rate of diffusion of the drug providing high ratio of entrapment of drug due to its emulsification and gelling properties [25,26]. Gum arabic also acts as a prebiotic, been approved by the FDA. It is indigestible to both humans and animals, but fermented in the colon to release short-chain fatty acids adding to potential health benefits of the encapsulated polymer. The outer coating of sodium alginate thus provides protection during gastrointestinal transit expanding and releasing at alkaline pH while the inner coating of gum arabic ensures sustained release of the nisin in the colon [27]. A schematic representation of the encapsulation, delivery and mechanism is presented in Fig.1.

Fig. 1. Schematic representation of formulation and release of nisin from Nis/ALG-GA NPs at the colon region.

Fig. 1

Open in a new tab

The dual-polymer system swells at colonic pH, enabling colon-specific nisin release.

This study investigates the stability of nisin-loaded sodium alginate nanoparticles (Nis/ALG-GA NPs) for oral administration and the anticancer efficacy of the formulation in comparison with free nisin. To the best of our knowledge, this is the first report where a dual polymer encapsulation method is used to encapsulate nisin as an oral drug delivery system for potential therapy of colorectal cancer.

2. Materials and Methods

2.1. Chemicals and Reagents

Gum arabic (GA), sodium alginate (SA) (nominal viscosity = 300 mPa.s), Polysorbate 80 and trehalose used in this study was purchased from Sisco Research Laboratories (SRL, Bengaluru, India). Nisin (purity, >98%, Mw = 368.36 g/moL, C12H20O6) and Dulbecco’s modified Eagle’s medium (DMEM) high glucose was purchased from Sigma Aldrich Pvt. Ltd., Bengaluru, India. Fetal Bovine Serum (FBS) was purchased from Gibco, Bengaluru, India.

2.2. Synthesis of sodium alginate and gum arabic loaded nisin nanoparticles

Sodium alginate and Gum arabic nanoparticles were synthesised using the ionotropic gelation technique according to the method described by Hassani et al. [28] with a few modifications. Sodium alginate and Gum arabic (7 mg/ml each) was dissolved in deionised water and stirred for 10 mins at 1000 rpm. Nisin (1mg/ml) was added drop wise to this solution and the mixture was allowed to stir for 30 mins. Surfactants trehalose and polysorbate 80 at 0.1% were added to the dispersion and stirred for 10 mins. Finally, cross-linker CaCl2 (10%) was added drop wise to the mixture and stirred for 2 h. The suspension was centrifuged at 15000 rpm for 15 mins and the obtained pellet was lyophilised. The lyophilised nanoparticles were subsequently stored in vials for further characterization.

2.3. Measurement of biological activity of the encapsulated nisin

The effect of encapsulation on the biological activity (antibacterial effect) of nisin was tested by agar well diffusion method against indicator bacterial strain Kocuria rhizophila ATCC 9341. The Nis/ALG-GA NPs were centrifuged at 15000 rpm for 15 mins and the pellet was suspended in phosphate buffer saline (PBS) and loaded into the wells bored in 0.8% BHI (Brain heart infusion) agar plates (Himedia, Mumbai, India). The plates were incubated at 37°C for 24 h. After 24 h, the plates were observed for zone of inhibition.

2.4. Establishment of standard curve of nisin

Standard curve was obtained by UV-Vis spectrophotometer. Acetic acid solution pH 4.0 was taken as the blank reagent. Nisin at different concentrations (0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg/ml) dissolved in acetic acid solution (pH 4.0) was read at 200-300 nm wavelength in UV-Vis Scanning Spectrophotometer (Shimadzu UV-1800, Mumbai, India). The readings were plotted to obtain a standard curve, which was used to determine the encapsulation efficacy of the Nis/ALG-GA NPs [29].

2.5. Particle Size and surface charge (Zeta potential)

The particle size and charge of the synthesised ALG-GA and Nis/ALG-GA NPs were determined using a Dynamic light scattering (DLS) instrument, Zetasizer Lab (Malvern Panalytical Ltd., Malvern, UK). A fixed amount (2mg) of the Nis/ALG-GA NPs was diluted with deionized water and dispersed adequately by sonication. All measurements were made at 25°C. Surface charge analysis of the Nis/ALG-GA NPs was determined by electromotive force (mV) measurements. The Nis/ALG-GA NPs were dispersed in distilled water and the charge was developed over the surface.

2.6. Determination of Encapsulation Efficacy and Drug Loading Capacity of the Nis/ALG-GA NPs

The encapsulation efficacy of the Nis/ALG-GA NPs was determined by centrifugation at 15000 rpm at 4°C for 15 mins to separate nanoparticles from free nisin. The supernatant was collected and protein concentration in the supernatant was determined using Bradford’s reagent. The protein-dye complex had a maximum absorbance at 595 nm. The encapsulation efficiency (%EE) and drug loading capacity (%DL) was calculated according to the following equation [30].

Encapsulation Efficiency (% EE) = Total drug (mg) − Free drug (mg)/ Total drug (mg) × 100 Drug Loading (%DL) = Total drug (mg) − Free drug (mg)/ Total amount of NPs (mg) × 100

2.7. Characterisation of Nis/ALG-GA NPs

2.7.1. Surface morphology study by SEM and EDX

Morphology of the Nis/ALG-GA NPs were studied using the Scanning Electron Microscopy (SEM) and Laser Scanning Confocal Microscopy (Zeiss LSM710, Germany) equipped with an Energy dispersive X-Ray (EDX) system. The nanoparticle suspension was mounted onto aluminium stabs and coated with gold-palladium under vacuum. The samples were observed under 15000x magnification.

2.7.2. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

To study the interaction of the various ingredients of the Nis/ALG-GA NPs to form the nanoparticles and to identify their respective functional groups, FTIR spectrometer JASCO (4100 model) was employed. Nis/ALG-GA NPs, ALG-GA NPs and free nisin were analysed. The specimens were blended with dry Potassium Bromide (KBr) at a ratio of 2% (w/w). Infrared (IR) absorbance was recorded on each KBr disc at 4 mm/s with a resolution of 2 cm over a wavenumber range of 400−4000 cm−1 [31].

2.7.3. X-ray Diffraction (XRD) studies

To assess the crystallinity of the free nisin before and after encapsulation X-ray, diffraction patterns of Nis/ALG-GA NPs, ALG-GA NPs and free nisin were carried out using the Single Crystal X-ray Diffractometer (Bruker X8 Proteum, Bruker, Germany).

2.7.4. Differential Scanning Calorimetry (DSC)

DSC analysis was done using the DuPont 943 thermal analyser. Nis/ALG-GA NPs, ALG-GA NPs and free nisin weighing 5−7 mg in aluminium pans were heated at a constant rate of 10 °C/min from 40°C to 350 °C under a 25 ml/min flow of nitrogen [32].

2.8. pH Responsive study

The pH sensitivity study of Nis/ALG-GA NPs was studied in eight separate sets (pH 1, 2, 3, 4, 5, 6, 7 and 8). The pH value of each set was adjusted with 0.1 M HCl and 0.1 M NaOH accordingly. The pH sensitivities of the nanoparticles were studied by measurements of the mean particle diameter using DLS in aqueous media at 25°C [33].

2.9. In vitro nisin release study

The drug release profile for Nis/ALG-GA NPs was studied by mimicking the pH environments of the stomach (pH 2.5, hydrochloric acid buffer), small intestine (pH 6.0, PBS) and colon (pH 7.4, PBS). This study was performed at 37 ± 0.1 °C in a thermostatic rotary shaker at 90 rpm. Nis/ALG-GA NPs (30 mg suspended in PBS) were placed in a dialysis bag (MWCO of 12-14 kDa) and immersed in simulated stomach, small intestine and colon buffer for 300 mins. At predetermined time intervals, 1.0 ml of buffer was withdrawn from the respective pH buffers, and an equal volume of fresh buffer was replenished. Bradford assay was performed to quantify the amount of nisin in the buffer. Release kinetic models such as Zero order, first-order and Higuchi release equations were used to determine the release pattern and applied to interpret the release data [34].

2.10. Storage studies

The shelf-life of the nanoparticles was determined during a 30 day storage period at -20°C and 4°C temperature conditions using the polydispersity index (PDI) of the Nis/ALG-GA NPs with DLS [35].

2.11. Cell culture assays

Caco2 cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin streptomycin solution in an incubator at 37°C containing 5% CO2.

2.11.1. MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenly Tetrazolium Bromide) Assay

MTT assay was used to determine the cell viability of Caco2 cells in the presence of nisin and Nis/ALG-GA NPs. Caco2 cells were seeded at a density of 1.5×104/well in a 96 well plate and incubated until 80% confluency was obtained. They were later exposed to different concentrations of nisin and Nis/ALG-GA NPs and incubated for 24, 48 and 72 h. Post incubation, the cells were carefully rinsed with 1X PBS followed by addition of fresh serum-free medium (100 μl) containing 0.5mg/ml MTT solution. The cells were incubated for 4 h at 37°C, rinsed and resuspended in 100 μl of dimethyl sulfoxide (DMSO). The absorbance was measured at 630 nm with DMSO as blank. The viable cells were calculated based on optical density (OD) values of the treated and untreated cells using the formula % cell viability = [OD of treated cells/OD of control cells] ×100 [36].

2.11.2. Determination of Apoptosis by Ethidium Bromide/Acridine Orange (EB/AO) staining

Caco2 cells were seeded in a 96-well plate (2×106 cells/well) and incubated for 24 h. Thereafter, cells were treated with 1mg/ml each of free nisin and Nis/ALG-GA NPs while control consisted of untreated cells. The cells were collected from each well, centrifuged at 1000 rpm at 4°C for 5 min and washed with PBS to completely remove the media. Ten μl of EB/AO dye mix (10:10 ratio in PBS) was added to each pellet and incubated for 5- 10 mins at room temperature in the dark. Excess dye was washed with PBS. An aliquot of 10 μl was mounted on a clean glass slide and examined under an inverted fluorescence microscope [37].

2.11.3. Nanoparticle internalisation assay

To detect the uptake and internalization of the ALG-GA NPs into the Caco2 cells, the nanoparticles were labelled with Fluorescein isothiocyanate (FITC). Briefly, 20 mg of ALG-GA were suspended in milliQ water and mixed with 5 ml of 0.3 mg/ml of FITC-ethanol solution. The mixture was stirred for 1 h and centrifuged at 15000 rpm for 5 mins. The pellet was collected and washed with PBS twice to remove unbound FITC. The cells were treated with the FITC tagged ALG-GA NPs for 4 h. Cell nuclei were stained with 4′,6-diamidino-2-phenylindol (DAPI). The cells were washed with PBS and incubated for 15 min at 37 °C with DAPI solution. The media was removed, the cells were washed with PBS, fixed with fixation solution (0.2% glutaraldehyde and 2% formaldehyde in PBS) for 10 mins and observed under fluorescence microscope [38].

2.11.4. Gene Expression Analysis

The expression of apoptotic genes (bax and bcl-2) and anti-inflammatory cytokine IL10 was analysed by qPCR. Briefly, Caco2 cells were treated with 1 mg of free nisin and Nis/ALG-GA NPs for 24 h. RNA was isolated with Trizol reagent as per manufacturer’s instructions. RNA obtained was reverse transcribed into cDNA using a Transcriptor high fidelity cDNA synthesis kit (Verso, Thermo Fischer, India) as per the manufacturer’s instructions. Diluted cDNA was used as template for real-time qPCR analysis with specific primers (Supplementary Table 1). The samples were analysed in triplicates with non-template control (NTC). Primer specificity and efficiency was deduced from the melting curves. Cycle threshold (Ct) values obtained were normalised with endogenous control gene (GAPDH-glyceraldehyde 3-phosphate) and expressed as relative normalised expression (2^-ΔΔCt) [39,40].

2.12. Statistical Analysis

Data were statistically analysed using GraphPad Prism version 5.00. All data were expressed as mean ± SD of triplicates. Paired student’s t-test was performed between two experimental groups, and one-way ANOVA was performed between more than two experimental groups followed by Tukey’s posthoc test with p < 0.05 and p < 0.001 being considered significant, respectively. All equipment used in the study were calibrated before use.

3. Results

3.1. Biological activity of the encapsulated Nisin

Nisin released from the Nis/ALG-GA NPs were found to possess antibacterial activity against Kocuria rhizophila ATCC 9341 comparable to that of nisin indicating that the encapsulation process did not affect the biological activity of nisin (Supplementary Fig. 1a).

3.2. Calibration curve of Nisin

The maximum absorption peak of nisin was observed at 212 nm at a concentration of 0.5 mg/ml and the calibration curve showed linearity (y = 1.2418x + 0.0234) corresponding to the concentration (Supplementary Fig. 1b). The correlation coefficient was found to be 0.996.

3.3. Encapsulation efficacy and drug loading capacity

Based on a previously generated calibration plot (Supplementary Fig. 1b), the encapsulation efficiency of Nis/ALG-GA NPs was found to be 88 ± 2% and the drug loading capacity was found to be 19 ± 0.5%.

3.4. Characteristics of Nis/ALG-GA NPs

3.4.1. Surface morphological characteristics

SEM images of Nis/ALG-GA NPs showed the formation of small spherical alginate capsules with a smooth outer surface (Fig 2a-b). EDX of the Nis/ALG-GA NPs and ALG-GA (control) was performed to determine the elemental characteristics of the capsules, which revealed characteristic peaks of calcium (Ca), carbon (C), oxygen (O) and chlorine (Cl) which are the major components of calcium alginate (Fig. 2c-d). This indicates that the outermost coating is formed by calcium alginate as expected, with nisin encapsulated within the capsule.

Fig. 2. Surface morphology of Nis/ALG-GA NPs by SEM (a) at 1μm scale and 15000X magnification, (b) at 200 nm scale and 50000X magnification.

Fig. 2

Open in a new tab

The nanoparticles were visualised at 15000x magnification with a spherical shape and a smooth surface. EDX spectra showing elemental composition of (c) ALG-GA, (d) Nis/ALG-GA.

3.4.2. Size and zeta potential

The Nis/ALG-GA NPs had a mean size of 193 ±4 nm while that of the ALG-GA was 174 ±7 nm (Fig. 3a-b). The PDI of the Nis/ALG-GA NPs and ALG-GA was 0.19 and 0.25, respectively indicating the homogeneity of the particles. The zeta potential was -13 ±0.3 mv and -12 ±2 mv for Nis/ALG-GA NPs and ALG-GA, respectively (Fig. 3c-d). The physicochemical characteristics of Nis/ALG-GA NPs are summarised in Table 1.

Fig. 3. (a-b) DLS images of ALG-GA and Nis/ALG-GA, (c-d) Zeta potential of ALG-GA and Nis/ALG-GA.

Fig. 3

Open in a new tab
Table 1. Summary of physicochemical characteristics of Nis/ALG GA NPs.
Parameters ALG-GA NPs Nis/ALG-GA NPs
Size 174 ±7 nm 193 ±4 nm
Zeta potential -12 ±2 mv -13 ±0.3 mv
PDI 0.25 0.19
Encapsulation efficiency - 88 ± 2%
Drug loading capacity - 19 ± 0.5%.
Open in a new tab

3.4.3. Fourier transforms infrared (FTIR) spectrum

FTIR spectroscopy tests were performed to detect either the appearance of new chemical bonds or the modification of existing ones, which can be attributed to possible interactions between the polymers and nisin. Fig. 4a illustrates the FTIR spectrum of nisin, ALG-GA and Nis/ALG-GA. The FTIR spectrum of free nisin showed two peaks at 1557 cm-1 and 3283 cm-1. The 1557 peak corresponds to amide II while the 3283 cm−1 peak is due to the O-H stretching vibrations of the hydroxyl groups, C-H symmetrical stretching, and amide group bending of the primary amines [41]. The peaks obtained with ALG-GA NPs were at 3364 cm−1, 1604 cm−1, 1080 cm−1 and 1023 cm−1. The 3364 cm−1 and 1604 cm−1 peaks correspond to the O-H stretching vibrations of hydroxyl group and C-O stretching vibrations of the glycosidic bonds in the polysaccharides, respectively while the remaining two peaks are due to the C-O-C stretching vibrations indicating that the glycoside bonds of the polysaccharides are highly ordered and aligned. In the case of Nis/ALG-GA NPs, the 3420 cm−1 and 1690 cm−1 peaks are attributed to the hydroxyl groups and carboxyl groups of the alginate polymer. The peak at 1402 cm−1 corresponds to the C-O bending vibrations of gum arabic while the 1601 cm−1 and 1422 cm−1 peaks indicate the amide I and C-H stretching vibrations of the alkyl chains of nisin [42].

Fig. 4. (a) FTIR Spectra of ALG-GA, Nis/ALG-GA and Nisin. (b) XRD pattern of Nis/ALG-GA, ALG-GA and Nisin.

Fig. 4

Open in a new tab

3.4.4. X-ray Diffraction (XRD) pattern

The presence of distinct peaks at 2θ angles of 16.18°, 21.23°, 27.54°, and 28.79° suggests that ALG-GA are of a crystalline structure. The Nis/ALG-GA NP exhibits similar diffraction patterns to that of ALG-GA, with peaks at 19.21°, 25.43°, 28.01°, and 32.07°. The slight change and degree of crystallisation between ALG-GA and Nis/ALG-GA indicates there is strong interaction between the two polymers. The peaks centred at 19.21° and 28.01° show the increased aggregation degree of the molecules of the two biopolymers. No characteristic peaks of gum arabic were seen in the nanoparticles which indicates uniform mixture of gum arabic and sodium alginate [43,44]. The diffraction pattern obtained in this study resembles that of Hosseini et al. [45] where nisin showed crystalline structure with peaks at 13.91°, 20.05°, 31.65°, 45.36°, and 56.44° related to the presence of its crystalline phase (Fig. 4b) [44]. The peak obtained at 31.65° is a characteristic peak for nisin which is in line with Zheng et al. [46].

3.4.5. DSC thermogram

The heating thermograms of (a) free nisin (b) ALG-GA and (c) Nis/ALG-GA NPs are presented in Fig 5. Nisin showed an endothermic peak at 112°C. The endothermic peak observed for ALG-GA shifted from 134°C to 122°C for Nis/ALG-GA NPs indicating a dehydration process. The decomposition temperature slightly decreased after the encapsulation. In addition, no peak related to direct degradation of nisin was observed.

Fig. 5. DSC thermograms of Nis/ALG-GA, ALG-GA and Nisin.

Fig. 5

Open in a new tab

3.5. pH -sensitivity of the Nis/ALG-GA NPs

The pH-sensitivity of the Nis/ALG-GA NPs was studied by measurements of the mean particle diameters within aqueous media of different pH values. As shown in Fig 6a, the diameters of all samples increased with increasing pH value. The particle diameter was smaller at low pH value and had a maximum at pH 8.0 exhibiting a pH responsive swelling of the NPs.

Fig. 6. (a) pH induced size distribution of Nis/ALG-GA, (b) Effect of different pH media on invitro nisin release from Nis/ALG-GA NPs.

Fig. 6

Open in a new tab

3.6. In vitro release profile of Nisin from the ALG-GA NPs

The anti-cancer activity of Nis/ALG-GA NPs is dependent on the release of nisin from the polymeric matrix into the environment. Fig. 6b shows the release of entrapped nisin from the Nis/ALG-GA NP. At pH 2.5 the release of nisin is relatively slow and gradual. The cumulative drug release reaches only 20% after 300 mins. At pH 6 the release is significantly faster when compared with pH 2.5. The cumulative drug release reached 68% after 300 mins. At pH 7.5 the cumulative drug release reaches 98% after 300 mins. Controlled release of the peptide was observed in all tested pH values.

When subjected to kinetic model fitting (zero order, first order and Higuchi kinetics) to determine the type of diffusion responsible for release of nisin from the NP, it was found that Nis/ALG-GA NPs followed the zero-order model with correlation value R2 reaching close to 0.999 in all pH values (Supplementary Table 2).

3.7. Shelf-life of Nis/ALG-GA NPs

The storage studies demonstrated that Nis/ALG-GA increased in size during a 30 day storage period in both the temperatures. The size showed a gradual increase from 187 nm to 246 nm at 4°C and from 187 nm to 255 nm at -20 °C. However, the PDI remained below the range of 0.4 - 1 displaying a polydisperse system over storage period (Supplementary Fig. 2). Overall, Nis/ALG-GA NPs appeared to be stable during the storage duration.

3.8. Cell viability of Caco2 cells treated with Nis/ALG-GA NPs

The cell viability of Caco2 treated with free nisin and Nis/ALG-GA NPs was evaluated by MTT assay (Fig. 7). The results showed that the cell viability was affected in a dose dependent manner. The cell viability was significantly inhibited at 48 and 72 h with Nis/ALG-GA NP treatment giving an IC50 value of 500 μg and 80 μg, respectively. Nis/ALG-GA gave a lower percentage of viable cells (43.7%) at 72 h compared to cells treated with free nisin (61.4%).

Fig. 7. (a) Effects Nis/ALG-GA on the percent cell viability of Caco2 cells in vitro for 24, 48 and 72 hours.

Fig. 7

Open in a new tab

Data shown are representative of 6 replicates and values are given as mean ± SD. Significance level p <0.0001.

3.9. Apoptotic effect on Caco2 cells

The results of the ethidium bromide and acridine orange (EB/AO) staining for apoptosis detection show significant differences between the untreated and treated cells. A number of apoptotic and necrotic cells were observed in the Nis/ALG-GA NPs treated cells compared to free nisin indicating that the Nis/ALG-GA NPs has enhanced apoptotic effect on the cancer cells (Fig. 8a). While the percentage of apoptotic cells upon treatment with Nis/ALG-GA NPs (47%) was significantly higher compared to free nisin (22%), the percentage of necrotic cells were not statistically significant between free nisin and NP treated cells (Fig. 8b).

Fig. 8.

Fig. 8

Open in a new tab

(a) Apoptotic effect of Control, 1mg nisin, Nis/ALG-GA NPs on Caco2 cells. The apoptotic (yellow arrow), necrotic (red arrow), live cells (green arrow) are indicated, (b) Graphical representation of percentage apoptotic, necrotic and live cells. There was no statistically significant difference in the percentage of necrotic cells between the cells treated with free nisin and the cells treated with Nis/ALG-GA NPs (* = <0.05, **= <0.01, ****<0.0001), (c) Internalisation of FITC tagged Nis/ALG-GA, (d) Effect of Nisin and Nis/ALG-GA treatment on IL 10, bax and bcl-2 gene expression in caco2 cells. Statistical Significance level p <0.0001 was achieved.

3.10. Internalisation of Nis/ALG-GA NPs

Internalisation or intracellular uptake of Nis/ALG-GA NPs was analysed by FITC labelling of the NPs and staining of the Caco2 cells with DAPI. The fluorescence microscopic images showing green fluorescence confirmed the internalisation and intracellular presence of the FITC loaded Nis/ALG-GA NPs in live Caco2 cells (Fig. 8c).

3.11. Gene Expression of immune and apoptotic markers

The gene expression of immune and apoptotic markers are presented in Fig. 8d. At 24 h, the Nis/ALG-GA NPs showed a significant increase of IL10 expression in Caco2 cells compared to the control and free nisin treatments suggesting that the Nis/ALG-GA NP may be more effective in inducing IL10 production. The Nis/ALG-GA NP also induced a significant increase in bax expression compared to free nisin, indicating apoptosis induction. Conversely, the Nis/ALG-GA NPs showed a significant decrease in bcl-2 expression, which is an anti-apoptotic gene (Fig. 8d).

4. Discussion

Peptides and proteins exhibiting biological activity are increasingly being explored for their anti-cancer and therapeutic effect. However, challenges associated with their gastrointestinal transit and proteolytic cleavage remains [47]. Colon cancer is a condition which necessitates the drug to be delivered at the site of colon without being degraded or altered during transit. Hence, use of nanoparticles as drug delivery agents have shown promise to overcome this challenge and effectively deliver the drug in cancer treatment [48]. Nisin which is a cationic peptide enables it to induce apoptotic effect in cancer cells and has shown to be a potential agent for treatment of cancer [7]. In this study, sodium alginate-gum arabic dual polymer seemed to be a promising carrier, which effectively protected the nisin against degradation and demonstrated controlled release of the peptide at colonic pH. The encapsulation of nisin in sodium alginate and gum arabic did not affect its antibacterial effect indicating that the peptide retained its biological activity during the formulation process. The absorption spectrum obtained in the study was consistent with a previous report, which reported the characteristic absorption spectrum of nisin with prominent peak around 210-215 nm [49]. The absorption spectra between 180 and 230 nm are dominated by π → π* transitions in peptide bonds. The spectral band from 190 to 210 nm is attributed to secondary structural features such as α-helix, β-sheets, and random coils. Most peptide antibiotics have characteristic absorbance patterns around 210-230 nm and 270-280 nm [49]. The nanoparticles developed in the study had a high encapsulation efficiency, which could be attributed to the gelling property of gum arabic [25]. Our findings revealed that the nanoparticles formed were capable of developing spherical shaped particles with a smooth surface. The difference in the particle size of Nis/ALG-GA NPs and ALG-GA indicated the successful loading of the peptide into the formulation. The polydispersity index of less than 1 obtained for both of these particles indicates that the nanoparticles are all monodispersed [50]. The zeta potential measured indicates a negative charge, which could be attributed to the negatively charged groups of sodium alginate [51]. The decrease in negative zeta potential from -12 ±0.3 mV for the ALG-GA to -13 ±2 mV upon nisin encapsulation can be attributed to the interaction between the cationic nisin and the anionic carboxyl groups of alginate [52]. Similar zeta potential values were observed in the Hassani et al. study [27]. The pH sensitivity of the Nis/ALG-GA NPs demonstrated decreased size at lower pH and increased size at higher pH, which might be attributed to the following reasons. The protonation of alginate carboxyl groups in pH 2-4 solutions causes a large number of hydrogen bonds to form in the hydrophilic shells of nanospheres. Because of the compact structure caused by the hydrogen bonds, the particle size is reduced. In pH 5-7 environments, unbound carboxylic acid groups would be ionized, breaking hydrogen bonds and causing electrostatic repulsion between polymer hydrophilic chain segments. As a result, bigger particle sizes were seen with increase in pH. A reduction in particle diameter at pH 7 can be attributed to the charge screening effect of the counter ions [53]. Thus, maximum increase in the size of the NP at pH 8 as seen in Fig 6a suggests the dissociation of the alginate network leading to the controlled release of nisin [23]. Nanoparticles are designed to provide a controlled release of the payloads which allows for the sustained release of the drug from the carrier for a long period of time leading to a more effective therapeutic window. Cells when exposed to the drug for an extended period of time enhances the cytotoxicity of the drug [54].

The FTIR spectra of alginate and gum arabic nanoparticles showed differences in the intensity and wave number of the stretching vibrations of the hydroxyl and carboxyl groups. Furthermore, the addition of nisin resulted in variations in peak intensity, which might be attributed to electrostatic interaction between the polymers, indicating successful nisin encapsulation. DSC thermographs reveal the absence of the characteristic peak of nisin in the Nis/ALG-GA NPs suggesting the molecular interaction of the two components, forming a new material with a difference in structure and, therefore, different thermal behaviour. The stability data shows that the Nis/ALG-GA increased in size during the storage period in both the temperatures, this could be because of excess Ca2+ present in the media inducing intermolecular crosslinking [55]. The stability of pharmaceuticals are usually studied at 4°C and 37°C for a period of 6 months (accelerated study) to 12 months (long-term stability) [56]. However, peptides and proteins show moderate stability under storage conditions being stable mostly at 4°C and -20°C [57]. DLS is a common method that measures changes in the physicochemical parameters such as size of polymeric nanoparticles [56]. Hence, our study shows stability of the NP during 30 day period. However, long-term storage studies are required to assess the stability of the peptide and polymer. In our study 61.4% cell viability was obtained upon treatment with free nisin for 72 h compared to Nis/ALG-GA NPs (43.7%) suggesting that the NPs augmented the cytoxicity effect of nisin. Encapsulated drugs may take up a different cellular pathway or mechanism than free drugs which could increase the amount of cell death [58]. The Nis/ALG-GA NPs gave an IC50 value of 500 μg at 48 h, identical to Ahmadi et al. study [7]. An IC50 value of 80 μg after 72 h of incubation was identical to Zainodini et al. study [8]. It was observed that cell death was proportional to the concentration and incubation period. The EB/AO staining of Caco2 cells treated with Nis/ALG-GA NPs revealed that there were more cells undergoing apoptosis than necrosis. This was further confirmed in the research findings of Ahmadi et al. [7] and Joo et al. [6] who reported more apoptotic cells than necrotic cells. FITC-labelled nanoparticles successfully entered the cells, demonstrating the intracellular uptake of nisin into the Caco2 cells [31]. Gene expression analysis revealed that the Nis/ALG-GA NPs were more effective than nisin in inducing apoptosis by upregulating the bax gene and downregulating the bcl-2 gene. Apoptosis can occur either via intrinsic or extrinsic pathways. In our case, the intrinsic pathway was involved, which relies on the mitochondrial proteins (BAX and BCL-2) to mediate apoptosis. BAX functions as a pro-apoptotic protein while BCL-2 is an anti-apoptotic protein. Our findings align with previous study demonstrating that nisin induces apoptosis in cervical cancer cells by upregulating the bax/bcl2 ratio which stimulates ROS generation leading to increased membrane permeability [16]. Similar results have been reported in CRC cells [10,17]. The upregulation of IL10 following Nis/ALG-GA treatment is consistent with nisin’s and gum arabic’s known anti-inflammatory and immunomodulatory effects [59–61].

5. Conclusion

This study demonstrated the effective encapsulation of nisin into Nis/ALG-GA nanoparticles by the ionotropic gelation process. SEM, EDX, FT-IR, XRD, and DSC analysis of Nis/ALG-GA nanoparticles confirm their shape and incorporation of nisin into ALG-GA nanocavities with a mean size of 193 ±4 nm and loading efficacy of 88 ± 2 %. Nis/ALG-GA NPs exhibited higher Caco2 cell death compared to free nisin with an IC50 value of 500 μg for 48 h and 80 μg for 72 h. The synthesized nanoparticles showed successful internalisation, indicating that the nanoparticles were able to transport nisin inside the cells and exhibit its anticancer activity. The EB/OA staining visibly confirms the apoptosis triggered by Nis/ALG-GA and free nisin. Gene expression studies revealed that the formulation upregulated apoptotic gene bax by 22.78 fold and anti-inflammatory gene IL10 by 8.12 fold in Caco2 cells. The nanoparticles were found to be stable over different pH ranges and displayed controlled release of nisin in the colonic pH. Overall, ALG-GA formulation enhanced the therapeutic potential of nisin. These findings suggest that the formulation could be used as an effective peptide drug delivery system overcoming the limitations of peptidic degradation and potential application as an oral therapeutic agent in the management of colon cancer. Although this study explored the oral delivery of nisin by using Nis/ALG-GA NPs in Caco2 cells, its anti-cancer effects could potentially be demonstrated in other carcinoma cells. Moreover, the effect and behaviour of the Nis/ALG-GA NPs needs to be evaluated in an appropriate in vivo animal model of CRC to validate the in vitro data. Further, the encapsulation of combination of nisin with 5-FU or other anti-cancer drug by using this method could also be explored as a combination treatment strategy. Additional inflammatory markers must be evaluated to support the anti-inflammatory effect.

Supplementary Material

Table 1, Table 2, Fig 1, Fig 2
Supplementary Figure

Acknowledgements

The authors thank JSS Academy of Higher Education and Research (AHER) for the support extended toward this work. We also thank Dr. Gopinath, Scientist, Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysuru, India, for his technical inputs and suggestions.

Funding

This work was supported by JSS Academy of Higher Education & Research Junior Research Fellowship (JSSAHER/REG/RES/JSSURF/29(1)/2010-11) and DBT Wellcome Trust Early Career Fellowship (IA/E/20/1/505689).

Footnotes

Conflict of Interest

The authors declare no conflict of interest.

CRediT statement

Sanya Hazel Soans: Conceptualization, Methodology, Visualization, Investigation, Data curation, Writing-Original draft preparation. Muzaffar Jahangir Chonche: Investigation. Kunal Sharan: Supervision, Writing-reviewing and editing. Asha Srinivasan: Supervision, Writing − reviewing and editing. Ann Catherine Archer: Conceptualization, Methodology, Supervision, Writing − reviewing and editing.

References

  • [1].Siegel RL, Giaquinto AN, Jemal A. Cancer statistics. CA Cancer J Clin. 2024;74:12–49. doi: 10.3322/caac.21820. [DOI] [PubMed] [Google Scholar]
  • [2].Asthana S, Khenchi R, Labani S. Incidence of colorectal cancers in India: A review from population-based cancer registries. Curr Med Res Pract. 2021;11:91–96. doi: 10.4103/cmrp.cmrp_65_20. [DOI] [Google Scholar]
  • [3].Vuik FE, Nieuwenburg SA, Bardou M, Lansdorp-Vogelaar I, Dinis-Ribeiro M, Bento MJ, Zadnik V, Pellisé M, Esteban L, Kaminski MF, Suchanek S. Increasing incidence of colorectal cancer in young adults in Europe over the last 25 years. Gut. 2019;68:1820–1826. doi: 10.1136/gutjnl-2018-317592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Shah SC, Kayamba V, Peek RM, Heimburger D. Cancer control in low-and middle-income countries: Is it time to consider screening? J Glob Oncol. 2019;5:1–8. doi: 10.1200/JGO.18.00200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Özel B, Şimşek Ö, Akçelik M, Saris PE. Innovative approaches to nisin production. Appl Microbiol Biotechnol. 2018;102:6299–6307. doi: 10.1007/s00253-018-9098-y. [DOI] [PubMed] [Google Scholar]
  • [6].Joo NE, Ritchie K, Kamarajan P, Miao D, Kapila YL. Nisin, an apoptogenic bacteriocin and food preservative, attenuates HNSCC tumorigenesis via CHAC 1. Cancer Med. 2012;1:295–305. doi: 10.1002/cam4.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Ahmadi S, Ghollasi M, Hosseini HM. The apoptotic impact of nisin as a potent bacteriocin on the colon cancer cells. Microb Pathog. 2017;111:193–197. doi: 10.1016/j.micpath.2017.08.037. [DOI] [PubMed] [Google Scholar]
  • [8].Zainodini N, Hassanshahi G, Hajizadeh M, Falahati-Pour SK, Mahmoodi M, Mirzaei MR. Nisin induces cytotoxicity and apoptosis in human asterocytoma cell line (SW1088) Asian Pac J Cancer Prev. 2018;19:2217–2222. doi: 10.22034/APJCP.2018.19.8.2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Avand A, Akbari V, Shafizadegan S. In vitro cytotoxic activity of a Lactococcus lactis antimicrobial peptide against breast cancer cells. Iran J Biotechnol. 2018;16 doi: 10.15171/ijb.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Hosseini SS, Hajikhani B, Faghihloo E, Goudarzi H. Increased expression of caspase genes in colorectal cancer cell line by nisin. Arch Clin Infect Dis. 2020;15 doi: 10.1007/s10989-021-10281-1. [DOI] [Google Scholar]
  • [11].Rana K, Pandey SK, Chauhan S, Preet S. Anticancer therapeutic potential of 5-fluorouracil and nisin co-loaded chitosan coated silver nanoparticles against murine skin cancer. Int J Pharm. 2022;620:121744. doi: 10.1016/j.ijpharm.2022.121744. [DOI] [PubMed] [Google Scholar]
  • [12].Balcik-Ercin P, Sever B. An investigation of bacteriocin nisin anti-cancer effects and FZD7 protein interactions in liver cancer cells. Chem Biol Interact. 2022;366:110152. doi: 10.1016/j.cbi.2022.110152. [DOI] [PubMed] [Google Scholar]
  • [13].Patil SM, Kunda NK, Nisin ZP. Nisin ZP, an antimicrobial peptide, induces cell death and inhibits non-small cell lung cancer (NSCLC) progression in vitro in 2D and 3D cell culture. Pharm Res. 2022;39:2859–2870. doi: 10.1007/s11095-022-03220-2. [DOI] [PubMed] [Google Scholar]
  • [14].Niamah KA, Al-Sahlany ST, Verma DK, Shukla RM, Patel AR, Tripathy S, Singh S, Baranwal D, Singh AK, Utama GL, González ML. Emerging lactic acid bacteria bacteriocins as anti-cancer and anti-tumor agents for human health. Heliyon. 2024;15:17. doi: 10.1016/j.heliyon.2024.e37054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Wiedemann I, Breukink E, Van Kraaij C, Kuipers OP, Bierbaum G, De Kruijff B, Sahl HG. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J Biol Chem. 2001;276:1772–1779. doi: 10.1074/jbc.M006770200. [DOI] [PubMed] [Google Scholar]
  • [16].Sadri H, Aghaei M, Akbari V. Nisin induces apoptosis in cervical cancer cells via reactive oxygen species generation and mitochondrial membrane potential changes. Biochem Cell Biol. 2022;100:136–141. doi: 10.1139/bcb-2021-0225. http://hdl.handle.net/1807/110115 . [DOI] [PubMed] [Google Scholar]
  • [17].Abdoullahi S, Jahangiri A, Shahali M, Rashmezad MA, Halabian R. Combination of Nisin and Glabridin enhanced apoptosis in colorectal cancer cells. Int J Peptide Res Ther. 2023;29:44. doi: 10.1007/s10989-023-10514-5. [DOI] [Google Scholar]
  • [18].Kamarajan P, Hayami T, Matte B, Liu Y, Danciu T, Ramamoorthy A, Worden F, Kapila S, Kapila Y. Nisin ZP, a bacteriocin and food preservative, inhibits head and neck cancer tumorigenesis and prolongs survival. PLoS One. 2015;10:e0131008. doi: 10.1371/journal.pone.0131008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Deshayes C, Arafath MN, Apaire-Marchais V, Roger E. Drug delivery systems for the oral administration of antimicrobial peptides: Promising tools to treat infectious diseases. Front Med Technol. 2022;3:e778645. doi: 10.3389/fmedt.2021.778645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Nordström R, Malmsten M. Delivery systems for antimicrobial peptides. Adv Colloid Interface Sci. 2017;242:17–34. doi: 10.1016/j.cis.2017.01.005. [DOI] [PubMed] [Google Scholar]
  • [21].Yaghoubi A, Khazaei M, Avan A, Hasanian SM, Soleimanpour S. The bacterial instrument as a promising therapy for colon cancer. Int J Colorectal Dis. 2020;35:595–606. doi: 10.1007/s00384-020-03535-9. [DOI] [PubMed] [Google Scholar]
  • [22].Hosseini SS, Hajikhani B, Goudarzi H, Rommasi F, Nasiri MJ. Cytotoxic activity of nisin on human cancer cell lines: A systematic review. Nov Biomed. 2022;10:184–191. doi: 10.22037/nbm.v10i3.36826. [DOI] [Google Scholar]
  • [23].Ahmad A, Mubarak NM, Jannat FT, Ashfaq T, Santulli C, Rizwan M, Najda A, Bin-Jumah M, Abdel-Daim MM, Hussain S, Ali S. A critical review on the synthesis of natural sodium alginate based composite materials: An innovative biological polymer for biomedical delivery applications. Processes. 2021;9:e137. doi: 10.3390/pr9010137. [DOI] [Google Scholar]
  • [24].Agüero L, Zaldivar-Silva D, Peña L, Dias ML. Alginate microparticles as oral colon drug delivery device: A review. Carbohydr Polym. 2017;168:32–43. doi: 10.1016/j.carbpol.2017.03.033. [DOI] [PubMed] [Google Scholar]
  • [25].Sharifi-Rad J, Rayess YE, Rizk AA, Sadaka C, Zgheib R, Zam W, Sestito S, Rapposelli S, Neffe-Skocińska K, Zielińska D, Salehi B. Turmeric and its major compound curcumin on health: Bioactive effects and safety profiles for food, pharmaceutical, biotechnological and medicinal applications. Front Pharmacol. 2020;11:e01021. doi: 10.3389/fphar.2020.01021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Patel S, Goyal A. Applications of natural polymer gum arabic: A review. Int J Food Prop. 2015;18:986–998. doi: 10.1080/10942912.2013.809541. [DOI] [Google Scholar]
  • [27].Rawi MH, Abdullah A, Ismail A, Sarbini SR. Manipulation of gut microbiota using acacia gum polysaccharide. ACS Omega. 2021;6:17782–17797. doi: 10.1021/acsomega.1c00302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Hassani A, Mahmood S, Enezei HH, Hussain SA, Hamad HA, Aldoghachi AF, Hagar A, Doolaanea AA, Ibrahim WN. Formulation, characterization and biological activity screening of sodium alginate-gum arabic nanoparticles loaded with curcumin. Molecules. 2020;25:e2244. doi: 10.3390/molecules25092244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Luo L, Wu Y, Liu C, Huang L, Zou Y, Shen Y, Lin Q. Designing soluble soybean polysaccharides-based nanoparticles to improve sustained antimicrobial activity of nisin. Carbohydr Polym. 2019;225:e115251. doi: 10.1016/j.carbpol.2019.115251. [DOI] [PubMed] [Google Scholar]
  • [30].Khazaei Monfared Y, Mahmoudian M, Cecone C, Caldera F, Zakeri-Milani P, Matencio A, Trotta F. Stabilization and anticancer enhancing activity of the peptide nisin by cyclodextrin-based nanosponges against colon and breast cancer cells. Polymers. 2022;14:e594. doi: 10.3390/polym14030594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Rade PP, Garnaik B. Synthesis and characterization of biocompatible poly(L-lactide) using zinc (II) salen complex. Int J Polym Anal Charact. 2020;25:283–299. doi: 10.1080/1023666X.2020.1783496. [DOI] [Google Scholar]
  • [32].Mahcene Z, Khelil A, Hasni S, Akman PK, Bozkurt F, Birech K, Goudjil MB, Tornuk F. Development and characterization of sodium alginate based active edible films incorporated with essential oils of some medicinal plants. Int J Biol Macromol. 2020;145:124–132. doi: 10.1016/j.ijbiomac.2019.12.093. [DOI] [PubMed] [Google Scholar]
  • [33].Chiu HI, Ayub AD, Mat Yusuf SNA, Yahaya N, Abd Kadir E, Lim V. Docetaxel-loaded disulfide cross-linked nanoparticles derived from thiolated sodium alginate for colon cancer drug delivery. Pharmaceutics. 2020;12:e38. doi: 10.3390/pharmaceutics12010038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Chang D, Lei J, Cui H, Lu N, Sun Y, Zhang X, Gao C, Zheng H, Yin Y. Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: Preparation, characterization, and in vitro drug release behavior. Carbohydr Polym. 2012;88:663–669. doi: 10.1016/j.carbpol.2012.01.020. [DOI] [Google Scholar]
  • [35].Haider T, Pandey V, Behera C, Kumar P, Gupta PN, Soni V. Spectrin conjugated PLGA nanoparticles for potential membrane phospholipid interactions: Development, optimization and in vitro studies. J Drug Deliv Sci Technol. 2020;60:e102087. doi: 10.1016/j.jddst.2020.102087. [DOI] [Google Scholar]
  • [36].De Oliveira JL, Campos EVR, Pereira AE, Nunes LE, Da Silva CC, Pasquoto T, Lima R, Smaniotto G, Polanczyk RA, Fraceto LF. Geraniol encapsulated in chitosan/gum arabic nanoparticles: A promising system for pest management in sustainable agriculture. J Agric Food Chem. 2018;66:5325–5334. doi: 10.1021/acs.jafc.8b00331. [DOI] [PubMed] [Google Scholar]
  • [37].Vincer B, Sindya J, Rajanathadurai J, et al. Exploring the cytotoxic and anticancer potential of naringin on oral cancer cell line. Cureus. 2024;16:e64739. doi: 10.7759/cureus.64739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Liu K, Liu PC, Liu R, Wu X. Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res. 2015;21:15–20. doi: 10.12659/MSMBR.893327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Rashidi L, Ganji F, Vasheghani-Farahani E. Fluorescein isothiocyanate-dyed mesoporous silica nanoparticles for tracking antioxidant delivery. IET Nanobiotechnol. 2017;11:454–462. doi: 10.1049/iet-nbt.2016.0120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Sharan K, Lewis K, Furukawa T, Yadav VK. Regulation of bone mass through pineal-derived melatonin-MT 2 receptor pathway. J Pineal Res. 2017;63:e12423. doi: 10.1111/jpi.12423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Sharan K, Mishra JS, Swarnkar G, Siddiqui JA, Khan K, Kumari R, Rawat P, Maurya R, Sanyal S, Chattopadhyay N. A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action. J Bone Miner Res. 2011;26:2096–2111. doi: 10.1002/jbmr.434. [DOI] [PubMed] [Google Scholar]
  • [42].El-Din AMS, Sayed MA, Monir TM, Sami NM, Aly AM. Sponge-like Ca-alginate/Lix-84 beads for selective separation of Mo (VI) from some rare earth elements. Int J Biol Macromol. 2021;184:689–700. doi: 10.1016/j.ijbiomac.2021.06.138. [DOI] [PubMed] [Google Scholar]
  • [43].Webber JL, Namivandi-Zangeneh R, Drozdek S, Wilk KA, Boyer C, Wong EH, Bradshaw-Hajek BH, Krasowska M, Beattie DA. Incorporation and antimicrobial activity of nisin Z within carrageenan/chitosan multilayers. Sci Rep. 2021;11:e1690. doi: 10.1038/s41598-020-79702-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Homem NC, Tavares TD, Miranda CS, Antunes JC, Amorim MTP, Felgueiras HP. Functionalization of crosslinked sodium alginate/gelatin wet-spun porous fibers with nisin Z for the inhibition of Staphylococcus aureus-induced infections. Int J Mol Sci. 2021;22:e1930. doi: 10.3390/ijms22041930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Cheng T, Xu J, Li Y, Zhao Y, Bai Y, Fu X, Gao X, Mao X. Effect of gum ghatti on physicochemical and microstructural properties of biodegradable sodium alginate edible films. J Food Meas Charact. 2021;15:107–118. doi: 10.1007/s11694-020-00605-y. [DOI] [Google Scholar]
  • [46].Hosseini SM, Tavakolipour H, Mokhtarian M, Armin M. Co-encapsulation of Shirazi thyme (Zataria multiflora) essential oil and nisin using caffeic acid grafted chitosan nanogel and the effect of this nanogel as a bio-preservative in Iranian white cheese. Food Sci Nutr. 2024;12:4385–4398. doi: 10.1002/fsn3.4105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Zheng X, Zhang X, Xiong C. Effects of chitosan oligosaccharide-nisin conjugates formed by Maillard reaction on the intestinal microbiota of high-fat diet-induced obesity mice model. Food Qual Saf. 2019;3:169–177. doi: 10.1093/fqsafe/fyz016. [DOI] [Google Scholar]
  • [48].Tryfon A, Siafarika P, Kouderis C, Kaziannis S, Boghosian S, Kalampounias AG. Evidence of self-association and conformational change in nisin antimicrobial polypeptide solutions: a combined Raman and ultrasonic relaxation spectroscopic and theoretical study. Antibiotics. 2023;12:e221. doi: 10.3390/antibiotics12020221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Xie M, Liu D, Yang Y. Anti-cancer peptides: classification, mechanism of action, reconstruction and modification. Open Biol. 2020;10:e200004. doi: 10.1098/rsob.200004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Caputo F, Clogston J, Calzolai L, Rösslein M, Prina-Mello A. Measuring particle size distribution of nanoparticle enabled medicinal products, the joint view of EUNCL and NCI-NCL. A step by step approach combining orthogonal measurements with increasing complexity. J Control Release. 2019;299:31–43. doi: 10.1016/j.jconrel.2019.02.030. [DOI] [PubMed] [Google Scholar]
  • [51].Wathoni N, Nguyen AN, Rusdin A, Umar AK, Mohammed AFA, Motoyama K, Joni IM, Muchtaridi M. Enteric-coated strategies in colorectal cancer nanoparticle drug delivery system. Drug Des Devel Ther. 2020;14:4387–4405. doi: 10.2147/DDDT.S273612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Yilmaz T, Maldonado L, Turasan H, Kokini J. Thermodynamic mechanism of particulation of sodium alginate and chitosan polyelectrolyte complexes as a function of charge ratio and order of addition. J Food Eng. 2019;254:42–50. doi: 10.1016/j.jfoodeng.2019.03.002. [DOI] [Google Scholar]
  • [53].Reczyńska-Kolman K, Hartman K, Kwiecień K, Brzychczy-Włoch M, Pamuła E. Composites based on gellan gum, alginate and nisin-enriched lipid nanoparticles for the treatment of infected wounds. Int J Mol Sci. 2021;23:e321. doi: 10.3390/ijms23010321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Kumarasamy RV, Natarajan PM, Umapathy VR, Roy JR, Mironescu M, Palanisamy CP. Clinical applications and therapeutic potentials of advanced nanoparticles: A comprehensive review on completed human clinical trials. Front Nanotechnol. 2024;6:1479993. doi: 10.3389/fnano.2024.1479993. [DOI] [Google Scholar]
  • [55].Chang D, Lei J, Cui H, Lu N, Sun Y, Zhang X, Gao C, Zheng H, Yin Y. Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: Preparation, characterization, and in vitro drug release behavior. Carbohydr Polym. 2012;88:663–669. doi: 10.1016/j.carbpol.2012.01.020. [DOI] [Google Scholar]
  • [56].Hu M, Zheng G, Zhao D, Yu W. Characterization of the structure and diffusion behavior of calcium alginate gel beads. J Appl Polym Sci. 2020;137:e48923. doi: 10.1002/app.48923. [DOI] [Google Scholar]
  • [57].Dikpati A, Maio VD, Ates E, Greffard K, Bertrand N. Studying the stability of polymer nanoparticles by size exclusion chromatography of radioactive polymers. J Contr Release. 2024;369:394–403. doi: 10.1016/j.jconrel.2024.03.053. [DOI] [PubMed] [Google Scholar]
  • [58].Cheng X, Xie Q, Sun Y. Advances in nanomaterial-based targeted drug delivery systems. Front Bioeng Biotechnol. 2023;11:1177151. doi: 10.3389/fbioe.2023.1177151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [59].Salatin S, Farjami A, Siahi-Shadbad M, Hamidi S. Biological stability of peptides/proteins therapeutic agents. Int J Peptide Res Ther. 2023;29:77. doi: 10.1007/s10989-023-10549-8. [DOI] [Google Scholar]
  • [60].Huang F, Teng K, Liu Y, Wang T, Xia T, Yun F, Zhong J. Nisin Z attenuates lipopolysaccharide-induced mastitis by inhibiting the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways. J Dairy Sci. 2022;105:3530–3543. doi: 10.3168/jds.2021-21356. [DOI] [PubMed] [Google Scholar]
  • [61].Lin CY, Chen WL, Huang YC, Lim CL, Yang CH. Gum Arabic in combination with IFN-γ promotes the M1 polarization in macrophage. Int J Biol Macromol. 2022;209:506–512. doi: 10.1016/j.ijbiomac.2022.04.024. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table 1, Table 2, Fig 1, Fig 2
EMS205029-supplement-Table_1__Table_2__Fig_1__Fig_2.pdf (88.7KB, pdf)
Supplementary Figure
EMS205029-supplement-Supplementary_Figure.docx (14.9KB, docx)

RESOURCES