FT-IR analysis

Figure 1 exhibited the FT-IR spectrum. AITC spectrum shows various protuberant peaks, likewise the OH stretching vibration was observed at 3582 cm−1, C–H stretching was around 3101 cm−1, and the broad peak around 1726 corresponds to the C = O group present in the AITC, whereas C = C and C–H deformation vibration were depicted around 1622 and 1191 cm−1 (Fig. 1A). In FT-IR spectra of CS-TPP-NPs, IR band observed at 3419 cm−1 was ascribed to the O–H stretching vibration, whereas bands observed at 2852, 2378, 1710, and 1656 cm−1 are attributed to the C–H stretching, C–O–C (epoxy), NH2, C = C stretching vibrations, respectively. The broad band around 750 cm−1 is ascribed to the C–H bending vibrations (Fig. 1B). The FT-IR spectra of AITC@CS-TPP-NPs, the various peaks in AITC, and cross-linked CS-TPP-NPs were retained in the final composite of AITC@CS-TPP-NPs which shows the successful deposition of the AITC on the surface of CS-TPP-NPs. The various peaks were observed in the region of the IR spectrum namely, the peak around 3429 cm−1 was assigned to the N–N aliphatic amine vibration in the composite. The peak at 1550 cm−1 was ascribed to the nitro (N–O) group, and the peak around 1656 arises due to the N–H bending. The broad peak at 1031 arose due to the sulfinyl S = O stretching vibration. The C = C and C = H bending were observed in the 843 and 750 cm−1, respectively (Fig. 1C). Overall, the FT-IR spectra of AITC, CS-TPP-NPs, and AITC@CS-TPP-NPs support the data obtained. The specific peaks observed in the spectra provide evidence of the presence of functional groups in each sample. The successful deposition of AITC on the surface of CS-TPP-NPs is indicated by the retention of peaks from both components in the composite spectrum. These results suggest that the functional groups in AITC and CS-TPP-NPs are preserved and contribute to the properties and potential applications of AITC@CS-TPP-NPs.

Fig. 1

FT-IR spectra of A AITC, B CS-TPP-NPs, and C AITC@CS-TPP-NPs. (AITC—allyl isothiocyanate, CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles, and AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

Differential scanning calorimetry analysis (DSC)

Figure 2 shows the thermogram of CS-TPP-NPs and AITC@CS-TPP-NPs. In brief, the thermogram of CS-TPP-NPs shows an endothermic peak at 57.74 °C which may be because of the presence of degradation of polymeric chain on CS (Fig. 2A). In the case of the thermogram of AITC@CS-TPP-NPs, it displayed the broad endothermic peak around 219.31 °C confirming the presence of AITC in designed AITC@CS-TPP-NPs (Fig. 2B). As well, the endothermic peak at 75.79 °C may be because of the incorporation of AITC in CS-TPP-NPs. Overall, the DSC analysis supports the successful construction of AITC@CS-TPP-NPs. The distinct thermal behaviors exhibited by AITC@CS-TPP-NPs compared to the CS-TPP-NPs suggest that the incorporation of AITC has altered the thermal properties of the nanoparticles. The presence of multiple endothermic peaks and the broadening of the melting transition peak in AITC@CS-TPP-NPs indicate the presence of additional components or factors influencing the thermal behavior of the composite. These findings further support the successful formation of AITC@CS-TPP-NPs and provide valuable insights into their thermal characteristics.

Fig. 2

Thermogram of A CS-TPP-NPs and B AITC@CS-TPP-NPs. (CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles and AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

X-ray diffraction analysis (XRD)

Figure 3 presented a diffractogram for the CS-TPP-NPs and AITC@CS-TPP-NPs. The XRD spectrum of CS-TPP-NPs shows the characteristics peaks at 2θ = 10.73°A, 12.836°A, and 39.120°A (Fig. 3A). This peak indicates the crystal-like nature of the CS. However, the diffractogram of the AITC@CS-TPP-NPs exhibits broader peaks at 16.9°A, 18.4°A, and 22.68°A (Fig. 3B). These peaks resemble the prominent peaks of AITC. The reduction in the peak intensities in AITC@CS-TPP-NPs may be an outcome of the successful deposition of the AITC in the CS-TPP-NPs. The diffractograms confirm the successful deposition of AITC in the CS-TPP-NPs. The resemblance of the peaks in AITC@CS-TPP-NPs to those of AITC suggests the presence of AITC in the composite material. The changes in peak characteristics, such as broadening and intensity reduction, indicate the modifications in the crystal structure or arrangement of CS caused by the incorporation of AITC. These findings provide evidence for the interaction and successful integration of AITC into the CS-TPP-NPs matrix.

Fig. 3

Diffractogram of A CS-TPP-NPs and B AITC@CS-TPP-NPs. (CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles and AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

Zeta potential and particle size analysis

Figures 4 and 5 revealed the results for particle size and zeta potential measurement of CS-TPP-NPs and AITC@CS-TPP-NPs, respectively. The size of a particle of CS-TPP-NPs was obtained to be 256.9 nm whereas the polydispersity index (PDI) was found to be 0.221. As a result, it confirmed the design of nanosized particles of CS-TPP-NPs with even distribution in an aqueous system (Fig. 4A). In this case, the zeta potentials value of CS-TPP-NPs was found to be + 19.79 mV which assured the good stability of the CS-TPP-NPs (Fig. 4B) in an aqueous system. Similarly, the particle size of AITC@CS-TPP-NPs was found to be 356.9 nm whereas the PDI value was obtained to be 0.444. Herein, the increase in the size of the AITC@CS-TPP-NPs was found that it may be due to the deposition of the AITC in CS-TPP-NPs (Fig. 5A). Overall, the particle size analysis confirmed the design of AITC@CS-TPP-NPs. The zeta potential value of the prepared AITC@CS-TPP-NPs was found to be + 35.83 mV. Herein, the zeta potential indicates the good stability of designed NPs in solvent (Fig. 5B). Importantly, in this synthesis, the AITC interacts with the free functional groups such as COOH and OH of the CS. As per the previous agreements, CS-based NPs formed provided the positive surface charge that is beneficial for mucoadhesion, allowing them to adhere to mucous membranes and release the therapeutic payload over time [8, 34]. Overall, the particle size, PDI, and zeta potential analysis ensured the synthesis of nanosized, uniformly distributed, and stable AITC@CS-TPP-NPs.

Fig. 4

A Particle size and B zeta potential of CS-TPP-NPs. (CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles)

Fig. 5

A Particle size and B zeta potential of AITC@CS-TPP-NPs. (AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

Scanning electron microscopy and elemental analysis (SEM–EDX)

Figure 6 shows the scanning electron images of the CS-TPP-NPs and AITC@CS-TPP-NPs. The surface morphology of the CS-TPP-NPs exhibits a spherical shape and to some extent, aggregation of the particles was observed which might be due to the storage conditions and lyophilization conditions of the NPs (Fig. 6A). The size of these NPs is slightly lower than the size exhibited by the DLS method. Most likely, the shrinkage of polymeric chains during drying for microscope visualization is to blame. The AITC@CS-TPP-NPs show a non-spherical surface morphology with amorphous nature (Fig. 6B). This might be due to the result of polymer encapsulation on the NPs. Herein, the results indicated that the particle size determined by DLS and the size reported by SEM were comparable and show slight modifications. Figure 7 illustrates the elemental makeup of the prepared CS-TPP-NPs and AITC@CS-TPP-NPs. The EDX spectrum of CS-TPP-NPs shows the abundance and plenty of presence of carbon (35.09% C), nitrogen (0.77% N), and oxygen (64.14% O) (Fig. 7A). These are the characteristic element that makes bare NPs, which confirm the synthesis of CS-TPP-NPs. The evidence of some impurities in the sample of CS-TPP-NPs can also be reported by the appearance of the smaller peaks in the EDX spectra of CS-TPP-NPs. The EDX spectrum of AITC@CS-TPP-NPs shows the presence of various elements namely, C (64.17%), N (34.70%), and sulfur (1.13%S) as same as per the earlier hypothesis (Fig. 7B) [18, 35]. The presence of sulfur in the EDX spectrum indicates the successful presence of the AITC in the final composite. The results obtained from scanning electron microscopy and elemental analysis (SEM–EDX) provide important insights into the surface morphology and elemental composition of the CS-TPP-NPs and AITC@CS-TPP-NPs.

Fig. 6

SEM micrograph images of A CS-TPP-NPs and B AITC@CS-TPP-NPs. (CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles and AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

Fig. 7

EDX spectrum of A CS-TPP-NPs and B AITC@CS-TPP-NPs. (CS-TPP-NPs—tripolyphosphate-modified chitosan nanoparticles and AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

Drug entrapment efficiency (% EE)

The important parameters for describing NPs are their entrapment effectiveness and drug loading capacity. Here, UV spectrophotometric analysis was used to evaluate the entrapment effectiveness of all formulations. All formulations demonstrated good entrapment efficiency, ranging from 86 to 91%, according to the results. Overall the mean value for the %EE was found to be 88.33 ± 2.25% showing a decent amount of AITC was entrapped within the polymeric matrix. The drug loading capacity of the prepared formulation was determined using UV spectroscopically. In the preceding work, AITC entrapped up to 75 to 82% but herein it promisingly entrapped up to 89% [36, 37]. This improvement in drug loading capacity indicates the optimization and effectiveness of the developed formulation, leading to a higher amount of AITC being incorporated into the NPs. The enhanced entrapment effectiveness and drug loading capacity observed in this study are significant as they contribute to the overall efficacy and efficiency of the drug delivery system. Higher entrapment efficiency ensures that a larger fraction of the drug is encapsulated, reducing wastage and improving the therapeutic outcome. Similarly, increased drug loading capacity allows for the delivery of a higher dose of the drug, which can be beneficial in cases where higher concentrations are required to achieve the desired therapeutic effect.

In vitro drug release study

The in vitro release pattern of the AITC@CS-TPP-NPs was performed using the dialysis bag method using 0.05% SLS phosphate buffer (having pH 7.4). The usual percent release of the different formulations having a different ratio of chitosan:STTP ranged from 60 to 90% (Fig. 8). Releases of F1 to F4 were performed. Among these batches, batch F4 shows 90.14% drug release within 60 h. It may be due to the optimized concentration of polymer that offers sufficient drug release. Given that this combination was used for the formulations in the current study, these results might have had a maximum influence on the release behavior. There are many ways to regulate how much drug is released from the CS-TPP-NPs, including polymer swelling, drug diffusion across the polymeric medium, diffusion of the drug that has been adsorbed, and polymer attrition, with degradation. Swelling often starts as soon as the polymer comes into contact with the surrounding dissolving liquid. As a result, either the drug diffuses from the polymer’s surface or the polymer swells, creating holes, causing the initial burst release from the CS-TPP-NPs. When bonds break, it can occasionally lead to further physical deterioration of the polymer. Smaller particle sizes increase the surface area of the matrix, increasing the rate of NP osmosis and diffusion as per the previous rationale [20, 31]. The in vitro release study provides valuable insights into the drug release behavior of the AITC@CS-TPP-NPs. The results suggest that the polymer concentration in the formulation significantly impacts the release profile, with the optimized concentration leading to a higher drug release percentage within the specified time frame. Understanding the release mechanisms and optimizing the formulation parameters are crucial for tailoring the drug delivery system to achieve the desired release kinetics and therapeutic efficacy.

Fig. 8

In vitro drug profile of AITC@CS-TPP-NPs. (AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles)

In vitro cytotoxicity activity—SRB assay

The in vitro cytotoxicity study for AITC, AITC@CS-TPP-NPs, and adriamycin, positive control compound (ADR) was determined using the sulforhodamine B (SRB) assay using human breast cancer cell line MCF-7. As well, the compatibility with normal cell line MCF-10A was performed to ensure the cytotoxicity of designed NPs as compared to the AITC. Here, the cytotoxicity investigation showed that concentrations of AITC, AITC@CS-TPP-NPs, and normal saline solution ranging from 10 μg/mL to 80 μg/mL were effective for the cell viability study. In summary, the cell viability of the MCF-10A cell line was confirmed as there was no relationship with concentrations of AITC@CS-TPP-NPs. Herein, it shows 94.4% to 90.4% of cell viability (10 μg/mL to 80 μg/mL). Probably, it is because of the incorporation of AITC in CS-based NPs. Therefore, it confirmed the good biocompatibility of designed AITC@CS-TPP-NPs to normal cells. On the contrary, the AITC concentrations showed substantial cell cytotoxicity to MCF-10A cells (Fig. 9). In this, the % cell viability for bare AITC was found to be 60.5% to 37.5%. Similarly, the use of normal saline solution (10 μg/mL to 80 μg/mL) shows a 98.0% to 98.4% of cell viability. Hence, it confirmed the non-toxic behavior of the saline solution to normal cells. After this study, different concentrations (10 μg/mL to 80 μg/mL) of AITC, AITC@CS-TPP-NPs, and adriamycin (positive control compound, ADR) were tested for cytotoxicity against the MCF-7 cell lines (Figs. 10 and 11). In this case, the AITC exhibited concentration-dependent anticancer efficacy against MCF-7 cells. In brief, it showed 59.2% to 28.1% of cell viability. Therefore, this performed study confirmed that the AITC cytotoxicity of cancerous cells is similar to that of normal cells. In the case of % cell viability of AITC@CS-TPP-NPs, it was found to be 48.4% to 10.8%. It confirmed the concentration-dependant cell viability whereas it improved the cell cytotoxicity than the bare AITC. Importantly, it is because of the targeted delivery of AITC@CS-TPP-NPs in cancerous cells of the MCF-7 cell line. Overall, the designed AITC@CS-TPP-NPs provide the targeted delivery of AITC to cancerous cells followed by improved cytotoxicity activity [38, 39].

Fig. 9

Percentage of normal cell growth inhibition by AITC, AITC@CS-TPP-NPs and NSS. (AITC—allyl isothiocyanate, AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles and NSS—normal saline solution)

Fig. 10

Percentage of cancer cell growth inhibition by AITC, AITC@CS-TPP-NPs, and ADR. (AITC—allyl isothiocyanate, AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles and ADR—adriamycin, positive control compound)

Fig. 11

Inhibition of MCF-7 cell growth A AITC, B AITC@CS-TPP-NPs, and C ADR. (AITC—allyl isothiocyanate, AITC@CS-TPP-NPs—AITC-embedded tripolyphosphate-modified chitosan nanoparticles and ADR—adriamycin, positive control compound)