Characterization of CS-NTsUV spectra of CS-NTs

In the UV spectra of the CS hydroalcoholic extracts, two optimal absorptions were expressly noted at 204.0 and 224 nm. However, in the UV spectra of CS-NTs 25, CS-NTs 50, and CS-NTs 100, the maximum absorption peaks remained consistent after loading of CS extract, occurring at 204.0, 204.8, and 205.0 nm.

FTIR spectrum of CS-NTs

The FTIR spectra of the CS hydroalcoholic extract and CS-NTs 25, CS-NTs 50, and CS-NTs 100 were utilized to identify potential functional groups in the synthesis of NTs. Figure 1a depicts that the FTIR of CS extract with identified functional group viz. C=O (carbonyl), O–H (stretch), C–Ocyclic, C–Haliphatic, C/O/Cstretch, and C–Haromatic can be identified at 1595.15, 3321.79, 1239.02, 2881.86, 1481.97, and 2930.32 cm−1, respectively. In CS-NTs 25 with C=Ocarbonyl, O–Hstretch, C/O/Cstretch, C–Ocyclic, C–Haromatic, and C–Haliphatic can be identified at specific vibrations underwent a shift to new peaks of 1735.73, 3383.80, 1454.76, 1055.87, 2922.67, and 2856.39 cm−1, respectively, as illustrated in Fig. 1b. In the spectrum of CS-NTs 50, distinct peaks corresponding to the vibrations of C=Ocarbonyl, O–Hstretch, C–Ocyclic, C/O/Cstretch, C–Haromatic, and C–Haliphatic were observed. These peaks experienced a shift to new positions at 1733.92, 3348.90, 1055.83, 1454.99, 2922.64, and 2856.18 cm−1, respectively, as depicted in Fig. 1c. In the analysis of CS-NTs 100, distinctive bands corresponding to O–H stretching, carbonyl (C=O), cyclic C–O, C/O/C stretching, aliphatic C–H, and aromatic C–H vibrations were discerned. These bands shifted to different positions, appearing at 3394.95, 1735.05, 1082.43, 1455.82, 2859.19, and 2922.10 cm−1, respectively, as visualized in Fig. 1d.

Fig. 1

IR spectra of a CS extract, b CS-NTs 25, c CS-NTs 50, and d CS-NTs 100

Particle size of the CS-NTs

The particle size analysis is crucial for assessing and defining vesicle size distribution within CS-NTs. Dynamic light scattering (DLS) is employed to analyse particle size, covering a dynamic range from 0.8 to 8 µm. The average vesicle diameter (Z-average) and polydispersity index (PDI) of the CS-NTs were confirmed by dynamic light scattering (DLS). The average sizes of CS-NTs 25, CS-NTs 50, and CS-NTs 100, which were determined to be 463.6 ± 100.5, 265.5 ± 61.6, and 409.6 ± 106.2 nm, resided within the predicted range in terms of their average vesicle size using DLS analysis. The outcomes of the vesicle size are depicted in Fig. 2a–c. The average Z values of the prepared CS-NTs 25, CS-NTs 50, and CS-NTs 100 were determined to be 492.6 nm, 301.9 nm, and 751.7 nm, respectively. Additionally, the PDI values associated with each sample were found to be 0.558, 0.362, and 1.002, respectively. The Z-average values obtained for the CS-NTs 25, CS-NTs 50, and CS-NTs 100 indicate the mean hydrodynamic diameter of the NTs in each sample. The observed values reflect the effective particle size distribution within the samples, considering both the size and abundance of particles present. The PDI provides insights into the uniformity or heterogeneity of particle sizes within each sample. Lower PDI values indicate a narrower particle size distribution, suggesting a more homogeneous sample. In contrast, higher PDI values suggest a broader distribution of particle sizes within the sample. The PDI values associated with CS-NTs 25 (0.558) and CS-NTs 50 (0.362) indicate relatively narrow size distributions, implying a higher degree of uniformity in particle size within these samples. However, the PDI value for CS-NTs 100 (1.002) exceeds unity, suggesting a broader size distribution with higher degree of heterogeneity than the other samples.

Fig. 2

Particle size of a CS-NTs 25, b CS-NTs 50, and c CS-NTs 100 and zeta potential of d CS extract, e CS-NTs 25, f CS-NTs 50, and g CS-NTs 100

Zeta potential of the CS-NTs

The instrument utilizes electrophoretic light scattering (ELS) to determine zeta potential (mV) measurements, allowing for assessing the charge on the vesicle surface across CS, CS-NTs 25, CS-NTs 50, and CS-NTs 100 samples. This is a critical attribute in ensuring the physical stability of both CS and CS-NTs. The studies revealed a negative value (− 56.9 mV) of CS (Fig. 2d), whereas, CS-NTs 25 exhibited a lower negative value noted to be − 46.1, − 44.1, and − 39.4 mV, respectively, owing to the electrostatic interaction and introduction of coating of lipid layer upon modification (Shejawal et al., 2021) (Fig. 2e–g). Upon modification to form CS-NTs 25, the zeta potential values exhibited a further decrease in the negative surface charge. The observed reduction in the magnitude of the negative zeta potential can be attributed to several factors associated with the surface modification process. One major factor contributing to the reduction of zeta potential is the electrostatic interaction between the lipid layer and the surface of CS nanoparticles. Introducing a lipid coating alters the surface chemistry of the nanomaterial, leading to changes in the distribution of surface charges and a reduction of the negative surface potential. The lipid molecules may contain polar functional groups interacting with surface ions or functional groups on the nanomaterial, thereby neutralizing some negative charges [32].

XRD spectrum of CS-NTs

The extent of the diffraction band enables deductions about the crystalline form of the prepared system. Figure 3 depicts the XRD pattern of the CS-NTs 25, CS-NTs 50, and CS-NTs 100. The identified broadened diffraction pattern at about 18.200°, 32.032°, 45.601°, and 57.542° in the XRD spectrum denoted the presence of CS-NTs 25. The appearance of conspicuous diffraction peaks around 18.301°, 25.564°, 31.891°, 45.521°, and 59.002° in the XRD spectra unequivocally indicates the existence of crystalline form of CS-NTs 50. Moreover, the broadened diffraction pattern at about 19.176° and 32.541° in the XRD patterns revealed the presence of CS-NTs 100. The appearance of broad diffraction peaks in the XRD patterns indicates the nanocrystalline nature of the CS-NTs 25, CS-NTs 50, and CS-NTs 100. The broadening of peaks suggests a lack of long-range order in the crystal structure, which is typical for nanomaterials due to their large surface area-to-volume ratio and smallar size.

Fig. 3

XRD spectra of a CS-NTs 25, b CS-NTs 50, and c CS-NTs 100

DSC analysis of CS-NTs

DSC analysis explores the thermal characteristics, stability, and phase transitions exhibited by CS-NTs 25, CS-NTs 50, and CS-NTs 100. DSC curves for CS-NTs are depicted in Fig. 4. The CS-NTs 25 noted sharp exothermic peaks at 123.50, 411.06, 480.45, 564.96, and 633.07 °C, consistent with the melting temperature and decomposition of CS-NTs 25. The CS-NTs 50 noted sharp exothermic peaks at 124.76, 384.59, 472.88, 518.29, and 626.76 °C, consistent with the melting temperature and decomposition of CS-NTs 50. The CS-NTs 100 noted sharp exothermic peaks at 400.99 and 481.71, consistent with the melting temperature and degradiation of CS-NTs 100. Sharp endothermic notation implies that CS-NTs 25, CS-NTs 50, and CS-NTs 100 were in a crystalline state.

Fig. 4

Thermal analysis a DSC of CS-NTs 25, b TGA spectra of CS-NTs 25, c DSC spectra of CS-NTs 50, d TGA spectra of CS-NTs 50, c DSC spectra of CS-NTs 100, and d TGA spectra of CS-NTs 100

TGA analysis of CS-NTs

TGA analysis is a versatile technique used to investigate the thermal properties, composition, and stability of materials. It quantifies weight changes in a material as it undergoes thermal processes such as decomposition, oxidation, or dehydration. This information is valuable for understanding the thermal behaviour and stability of prepared CS-NTs. TGA/DTA analysis was performed at a checking span from 25 to 1000 °C with N2 gas by applying a temperature range of 10 °C min−1. The inflexion heating rates of CS-NTs 25 were 410 and 721 °C. Their corresponding mass lost at − 62.38 and − 26.16 wt %, respectively, as depicted in the TGA graph (Fig. 4). The inflection heating rates of CS-NTs 50 were 412 and 625 °C. Their corresponding mass lost at − 64.16 and − 21.74 wt %, respectively, as depicted in the TGA graph (Fig. 4). The inflection heating rates of CS-NTs 100 were 512 °C. Their corresponding mass was lost at − 93.81, as depicted in the TGA graph (Fig. 4). DTA and TGA results showed good stability due to the proper thermal transitions and phase changes in the prepared CS-NTs.

SEM of CS-NTs

CS-NTs formulation was found to be in clustered shapes with soft surfaces as observed under SEM, and the vesicle size of the CS-NTs 25, CS-NTs 50, and CS-NTs 100 revealed the better size of particles in SEM (Fig. 5).

Fig. 5

SEM analysis of CS-NTs 25, CS-NTs 50, and CS-NTs 100

The observation of clustered shapes with soft surfaces in the SEM images of the CS-NTs 25, CS-NTs 50, and CS-NTs 100 formulations suggests intriguing morphological characteristics of the NTs. The clustering phenomenon may arise due to various factors, such as aggregation during synthesis or sample preparation and the inherent tendency of NTs to form clusters due to attractive interparticle forces. Moreover, the soft surfaces observed on the clustered shapes indicate a certain degree of surface irregularity or smoothness, which could be attributed to the occurrence of lipid bilayer coatings or surface modifications, which could impart a layer of softness or flexibility to the NTs surfaces.

TEM of CS-NTs

TEM was employed to confirm the morphological attributes, such as the size and shape of the vesicle, specifically of CS-NTs 25, with the obtained TEM images representing the circular vesicle (Fig. 6). SAED pattern in TEM of CS-NTs does not exhibit rings or spots, suggesting that the material may lack long-range order or crystallinity. Instead, the absence of distinct diffraction features could indicate an amorphous or disordered structure within the CS-NTs sample. This phenomenon is typical for materials that are either non-crystalline or possess very small crystallite sizes, where the scattering of electrons occurs randomly due to the lack of periodicity in the atomic arrangement. The absence of discernible diffraction features in the SAED pattern implies that the CS-NTs may exhibit a partially disordered structure at the nanoscale. This could be attributed to factors such as the influence of surface coatings of the lipid layer.

Fig. 6

TEM analysis of nanotransferosome

% EE

The % EE of the optimized CS-NTs 25, CS-NTs 50, and CS-NTs 100 batches were noted to be 86.89 ± 1.43, 80.33 ± 1.09, and 83.32 ± 1.76%, which is remarkably high. This result indicates that the formulation has successfully encapsulated a substantial amount of CS, making it a promising candidate and potential use in drug delivery systems. The findings suggest that the optimized formulations can potentially improve the bioavailability and stability of CS, thereby enhancing its therapeutic effects. Moreover, the % drug loading capacity of the optimized batch CS-NTs 25, CS-NTs 50, and CS-NTs 100 were recorded as 6.54, 5.99, and 5.67%, respectively.

Altin et al. [33] conducted experiments illustrating a notable enhancement in the bioavailability of cocoa hull waste crude extract when encapsulated, showcasing an over sixfold increase. Concurrently, Zhou et al. [34] demonstrated stability and significant enhancement in the bioavailability of acteoside through encapsulation with liposome and chitosan. The findings underscore liposome delivery systems' efficacy in facilitating the in vivo distribution of bioactive compounds. A notable EE of CS-NTs 25, CS-NTs 50, and CS-NTs 100 underscore their proficient ability to encapsulate CS efficiently. This high EE holds promise for facilitating enhanced stability, improved bioavailability, and optimized therapeutic efficacy. Effective encapsulating CS offers potential benefits such as prolonged shelf life through protection against degradation, enhanced absorption leading to increased bioavailability, and precise dosing for optimized therapeutic outcomes. The observed values align with the characteristics of the loaded transferosomes, providing insights into the successful encapsulation of CS. Moreover, the DLC of CS-NTs 25, CS-NTs 50, and CS-NTs 100 suggest that a substantial amount of CS is effectively incorporated into the NTs, ensuring a sufficient drug payload for therapeutic efficacy.

In vitro % bioactive release

The % CS release from CS-NTs was studied using a membrane dialysis tube kept in a beaker with a buffer solution of pH 5.5. The investigation was continued for 240 min, and % CS release was determined at specified intervals. After the analysis of % CS release, all the prepared batches showed sustained-release action, amongst which the CS-NTs 25 batch noted sustained drug release (84.32%). From batches CS-NTs 50 and CS-NTs 100, the in vitro CS release from CS-NTs was noted between 94.34 and 95.43% at 240 min (Fig. 7). Kinetic models were utilized, and the zero-order model was the best-fitted model for the CS-NTs, 25, CS-NTs 50, and CS-NTs 100 prepared formulation. The sustained-release profile is desirable for prolonging drug action, minimizing dosing frequency, and improving patient compliance. The drug release from CS-NTs 25, CS-NTs 50, and CS-NTs 100 was prolonged for up to 240 min in a simulated buffer solution of pH 5.5. All prepared batches (CS-NTs 25, CS-NTs 50, and CS-NTs 100) follow zero-order kinetics for % drug release data (Supplementary Table), which implies that the release of the drug occurs at a uniform rate over time, independent of the initial drug content. This finding suggests a controlled and sustained-release profile, which could be advantageous for achieving prolonged therapeutic effects and maintaining drug concentrations within a desired range.

Fig. 7

% drug release study and anticancer activity results of NTs

Short-term stability

The CS-NTs 25, CS-NTs 50, and CS-NTs 100 exhibited exceptional stability for 60 days in terms of %EE, vesicle size, zeta potential, and PDI value at defined temperatures (25 ± 3 °C with relative humidity of 65 ± 2%) and may serve as an appropriate carrier for CS.

MTT assay method

Figure 8 illustrates the findings from the MTT assay, evaluating cytotoxicity against the selected cancer cell line B10F16. The concentrations of CS, CS-NTs 25, CS-NTs 50, and CS-NTs 100 were examined alongside their respective inhibition percentages, comparing their effectiveness against a standard. The data show varying levels of inhibition at different concentrations. For instance, at a strength of 10, 40, and 100 µg mL−1, CS exhibited per cent inhibition of 34.73, 41.29, and 49.52, respectively, while the corresponding values for CS-NTs 25 were found to be 45.38, 50.21, and 56.85%, respectively. CS-NTs 50, CS-NTs 100, and standard exhibited per cent inhibition at concentration 100 µg mL−1 were found to be 60.23, 52.66, and 75.03%, respectively. At a lower concentration of 10 µg mL−1, standard, CS, CS-NTs 25, CS-NTs 40, and CS-NTs 100 exhibited a cell inhibition rate of 66.81, 34.73, 45.38, 48.38, and 35.56% with the IC50 value found to be 41.7077, 41.56, 38.40, 40.78, and 33.56, respectively, in the B10F16 cell line (Fig. 8 and Table 2). As per the literature mentioned by Ashley et al., 2011, nanoparticle-based drug delivery systems, such as liposomes or nanoparticles, a narrow size distribution (low PDI) is often desirable for efficient delivery of pharmacotherapeutic agents to cancer cells. Uniformly sized particles can exhibit better circulation time, enhanced tumour accumulation, and improved cellular uptake compared to formulations with a wide size distribution [35]. Hence, the present study reveals our conclusion that an increased PDI may lead to reduced drug delivery efficiency and a decline in anticancer effectiveness. As an illustration, in the CS-NTs 100 formulation, featuring a higher concentration (100 mg) of soya phosphatidylcholine, the elevated PDI (1.002) and larger particle size (751.7 nm) directly influence the in vitro anticancer activity when compared to CS-NTs 25 (soya phosphatidylcholine—25 mg) and CS-NTs 50 (soya phosphatidylcholine—50 mg), which exhibited lower PDI and particle size. So, the CS-NTs 25 and CS-NTs 50 have shown good anticancer activity against the B10F16 cell line due to the lower particle size and good PDI value.

Table 2 MTT assay results