Bioactive silver nanoparticles derived from Carica papaya floral extract and its dual-functioning biomedical application

Plant polyphenols, especially the phytochemicals found in different parts of the papaya plant, have attracted considerable interest for their capacity to chelate metal ions and their diverse applications, including antibacterial and anticancer properties^32,33. These extracts function as competitive reducing and stabilizing agents. The bonding of phenolic compounds present in KQE with Ag⁺ ions was monitored by noting the changes in the absorption spectra of KQE before and after the addition of Ag⁺ ions. Figure 2a shows the UV–Vis absorption spectrum of KQE (Kaempferol-Quercetin Enriched extract) in an aqueous solution before its reaction with Ag⁺ ions.

The spectrum exhibits two notable absorption features: a sharp peak at 272 nm and a shoulder around 330 nm. The sharp peak at 272 nm corresponds to Band II, which is associated with the benzoyl system. This band arises due to the n → π* electronic transition localized between the A and C rings of kaempferol and quercetin molecules. The shoulder at around 330 nm corresponds to Band I, attributed to the cinnamoyl system. Band I is located in the range of 300–415 nm and serves as evidence of the π → π* electronic transition within the B-ring, which is conjugated with the carbonyl group present in the C-ring³⁸.

Further we have investigated the reaction between KQE with aqueous AgNO₃ solution at three different pH conditions such as acidic, alkaline and neutral and corresponding absorption characteristics were noticed (Fig. 2b). In acidic conditions, the mixture showed agglomeration. In contrast, under basic conditions, the pale yellow solution turned dark coffee brown within 3 min, indicating rapid NP formation. For the neutral medium, the reaction proceeded very slowly, which normally give rise to larger sized NPs. Speisky et al. experimentally confirmed that at alkaline pH, both the oxidation rate and antioxidant activity of kaempferol, quercetin, and their derivatives are significantly higher³⁷. From the Fig. 2b, it can be evident that compared to neutral pH at which reaction is quiet slow, the basic medium (pH = 9) is favourable for rapid formation of KQ-AgNPs with small size. Since KQE is abundant with flavonoids a large number of hydroxyl (-OH) groups which can deprotonate under alkaline conditions and enhances the formation of negatively charged ions. These increased electron density of free electrons further lowers the energy barrier for its oxidation by reducing the Gibbs free energy (ΔG). This reduction in ΔG facilitates the faster and efficient conversion of Ag⁺ ions into AgNPs (e.g.,\({Ag}^{+}+ {e}^{-}\to {Ag}^{0})\) of reduced size so that aggregation is prevented. The experimental evidence presented by Chen et al. substantiates the hypothesis regarding the accelerated redox mechanisms of kaempferol in the presence of aminoacid residues³⁹. However around physiological pH, the electrochemical intermediates produced by kaempferol and quercetin were unstable and proceed to form a stable final product in a quick manner. Since for metal ion reduction the availability reactive quinones are highly dependent, formation of KQ-AgNPs are slow at neutral pH^37,40,41.

To optimize the quantity of KQE required for the synthesis of KQ-AgNPs or to know the involvement of bioactive components present in KQE for the formation of KQ-AgNPs a series of reactions were carried out using a constant volume of aqueous AgNO₃ (10 ml) and varying the volume percentage of KQE (1%, 2%, 5%, 10%, 15%, and 20%). It was observed that higher concentrations of KQE accelerated the reaction rate and resulted in more symmetric SPR peaks, as illustrated in Fig. 2c. Notably, when 20% KQE was added to the aqueous solution of AgNO₃, the reaction mixture turned brown within 8 min, providing visible confirmation of KQ-AgNPs formation. Elemental analysis of the corresponding sample (Fig. 2d) revealed a composition of biogenic elements (C and O) alongside the Ag metal core, with 31.55%, 33.80%, and 34.65%, respectively. Interestingly, our results contrast with previous studies conducted by Alyami et al., where kaempferol, when chemically incorporated into AgNPs, resulted in carbon dominance over silver. This disparity may be attributed to the higher concentration of kaempferol, which could reduce the silver content in those AgNPs and increase the carbon composition due to the structural features of kaempferol¹⁷. On the other hand, in another study where quercetin-functionalized AgNPs were synthesized by Maghsoodloo et.al, the carbon content was significantly low, below 10%, indicating that the synthesized AgNPs had less bioactive coverage¹⁵. Therefore, our KQ-AgNPs exhibit a balanced elemental composition, ensuring adequate bioactive coverage and stability. From Fig. 2c, it is evident that as the concentration of KQE increases from 5 to 20%, the SPR peak intensity of KQ-AgNPs rises significantly, indicating enhanced NP synthesis. This improvement is attributed to the higher availability of bioactive compounds in KQE, which act as efficient reducing and capping agents. However, at higher KQE concentrations, a broadening and slight shift in the SPR band were observed, suggesting potential NP agglomeration and polydispersity^42,43. Consequently, further increases in KQE concentration were avoided to maintain NP stability. The KQ-AgNPs produced at 20% KQE displayed a well-balanced elemental composition (Fig. 2d) and attained an impressive yield of 78.76 ± 0.35%, notably surpassing previously documented yields. In optimized conditions, a yield of 53.31% was achieved with 29.22% of Rubus discolor leaf extract for the synthesis of AgNPs, necessitating elevated temperatures and extended reaction durations⁴⁴. The effectiveness and sustainability of the current approach are highlighted by this comparison, which also highlights its potential as a time- and energy-efficient green synthesis technique.

Figure 2e represents the FTIR spectra of KQE (black line) and KQ-AgNPs (blue line) which deliver vital information about the surface chemistry of our KQ-AgNPs. Since KQE is enriched with flavonoids, it is very probable that the interactions of those moieties with surroundings will be effectively reflects as spectral changes related to hydroxyl and carbonyl vibrations¹⁵. The complex character of biological materials present in the control KQE reflects in the vibrational spectrum with dominated bands at 3300, 1740, 1635, 1445, 1370 and 1215 cm⁻¹. The first broad and dominant band at around 3300 cm⁻¹ corresponds to stretching vibrations of hydroxyl moieties. The second band is due to C=O stretching vibrations of esters^1,17. Similar band at 1740 cm⁻¹ can also see from onion peel extract that contains superior quantity of quercetin and kampferol groups in it⁴⁵. The third band can be associated with the C–C stretching vibrations of A-ring of both kampferol and quercetin^5,46,47. The presence of aromatic benzene ring vibrations of kampferol and quercetin like flavonoids evident from the band at 1445 cm⁻¹.The asymmetric deformations of CH₃ groups can also associated with this band. C–OH bending in the B-ring of flavonoid moieties can be visible at 1370 cm^−146,47. The band at 1215 cm⁻¹ can be attributed to the stretching vibrations of C-rings⁴⁷. Figure 2f shows that KQ-AgNPs exhibit a remarkable difference in the vibrational bands at 1740 and 1635 cm⁻¹ to that of KQE. After the formation of KQ-AgNPs, the intensity of 1740 cm⁻¹ get increased but a decrement is evident for the band at 1635 cm⁻¹ (as depicted in the Fig. 2e (i & ii). Notably, the C=O/OH groups and catechol moieties in kaempferol-like flavonoids are known to be the most sensitive sites for metal interactions, as validated by Simunkova et al. in their study on Kaempferol-Cu²⁺ interactions⁴⁸. These vibrational characteristics of KQ-AgNPs indicate the active role of dominant flavonoids in KQE during NPs synthesis. Additionally, the observed spectral changes before and after NP formation align with previous reports on AgNPs synthesized via green methods using extracts of tomato, onion, acacia catechu, and combined extracts as reducing and stabilizing agents, confirming effective flavonol interactions in the coordination redox reaction with metal ions⁴⁹.

Moreover, our method demonstrates uniform NP morphology, as evidenced by the TEM results (Fig. 3a, b), with a narrow size distribution and an average diameter of 12.3 ± 3.0 nm (Fig. 3c). Assuming all nanoparticles formed are uniform spheres, the approximate number of KQ-AgNPs produced in 1 ml of our sample is \(4.88\times {10}^{12}\) (calculation is given in Supplementary Information). The HR-TEM highlights the crystalline nature of the KQ-AgNPs, with lattice fringes corresponding to the (111) plane of Ag (Fig. 3b)^5,10. The surface charge of the KQ-AgNPs was measured at different concentrations of KQE (10%, 15%, and 20%) and over various time durations (1 day, 1.5 months, and 3 months). A higher surface charge was observed at 20% KQE, indicating greater stability, with the zeta potential remaining consistent over three months (Fig. 3d)¹⁰. These findings align with the surface charge characteristics of NaBH₄ reduced AgNPs synthesized by Khatoon et al. for antimicrobial and cytotoxicity studies⁷. The size distribution obtained by DLS shows an average hydrodynamic diameter of 66.5 ± 1.6 nm, with a polydispersity index (PDI) of 0.445 (Fig. 3e) (DLS analysis of KQ-AgNPs prepared by using 10 and 15% of KQE is given in Figure S1 of SI). This larger size compared to the TEM analysis is attributed to the hydrodynamic shell formed by the capping agents (KQE) around the NPs⁴³. Our results are align with previous studies conducted by Deng et al.¹⁰.This highlights the potential of KQE in green nanotechnology for producing stable KQ-AgNPs with minimal environmental impact. Our methodology therefore, presents a significant advantage, as AgNPs are traditionally unstable and particularly prone to oxidation. However, to maintain consistent biochemical interactions and prevent aggregation or morphological changes during scale-up, careful process standardization and optimization are required.

(a) TEM and (b) HR-TEM (inset: lattice fringes) images of KQ-AgNPs. (c) size distribution histogram (d) surface charge analysis for time durations 1 day, 1.5 month & 3 month. (e) DLS spectrum and (f) XRD pattern for KQ-AgNPs.

The XRD pattern (Fig. 3f) exhibits prominent diffraction peaks at 2θ values of 38.1°, 44.3°, 64.5°, and 77.5°, corresponding to the (111), (200), (220), and (311) planes, respectively further validate the formation of highly crystalline AgNPs with prominent peaks indicative of the FCC structure^5,43. These findings collectively establish that the use of 20% KQE optimizes the synthesis process and highest yields KQ-AgNPs with improved structural and stability characteristics, making it a promising method for future applications.

Mechanism of formation of KQ-AgNPs

The synthesis of MNPs induced by free radicals is well-documented^5,43,50. In this study, we propose that the in-situ generation of free radicals, such as ortho-quinones formed during the oxidation of polyphenols in KQE, facilitates the reduction of Ag salts to form KQ-AgNPs. Figure 4 illustrates the schematic representation for the plausible mechanism of formation as well as stabilization of KQ-AgNPs by the surface functional groups present in KQE. According to Van et al., KQE contains bioactive flavonoids, including kaempferol, quercetin, myricetin, and their glycosides (e.g., Kaempferol 3-O-α-L-rhamnopyranoside and Quercetin 3-O-β-D-galactopyranoside)²⁷. These flavonoids are structured with three cyclic carbon rings: two benzene rings (A and B) linked by a heterocyclic pyran ring (C). Kaempferol and quercetin, are polyhydroxyflavonols, differ in their B-ring substituents: kaempferol has a phenol group, while quercetin contains a catechol group, which provides stronger antioxidant properties¹¹. The electrochemical oxidation potential of kaempferol, quercetin and Ag are in the range of ~ 0.3–0.5, ~ 0.2–0.4 and ~ 0.7996 V respectively^11,37,43. Since quercetin contains one additional hydroxyl group than kaempferol, it may be acting as stronger reducing agent in the KQ-AgNPs formation process³⁹. The alkaline medium further facilitates the deportation of hydroxyl moieties to form highly reactive and nucleophilic phenoxide ions which reduce Ag⁺³⁹. Existing studies supports that usually catechol-containing molecules reduce metal ions to form NPs while simultaneously forming robust interfaces with metal surfaces⁴³. These molecules coordinate strongly through adjacent hydroxyl groups and undergo oxidation to quinones, enabling covalent cross-linking via Michael addition and Schiff base reactions⁵⁰. This redox coupling between metal ion reduction and catechol oxidation results in a stable polymeric shell around the NPs preventing them from aggregation. As reported by Gan et al. in the formation of Ag-lignin NPs, the mechanism involves the oxidation of catechol groups to o-quinones, which reduce Ag ions and stabilize the NPs through hydrogen bonding⁵⁰. Inspired by these findings which are resembles to the phenol-metal-catechol redox chemistry observed in marine mussels and plant hydrogels, we hypothesize that similar interactions underlie the formation and exceptional stability of KQ-AgNPs. This dual action of reduction and stabilization provides a robust, green method for synthesizing highly stable AgNPs.

Schematic representation of plausible mechanism of formation of KQ-AgNPs mediated by kaempferol and quercetin enriched KQE.

Antibacterial activity

Different parts of the papaya plant extracts have been widely reported for their antibacterial activity, primarily attributed to the rich phytochemical profile^23,24,33. Numerous studies have documented the bactericidal properties of papaya-derived metabolites, highlighting their potential for biomedical applications. According to Dwivedi et al., statistical analysis revealed a significant difference in the flavonoid content across various extracts of papaya flowers²⁶. These findings suggest that papaya flowers are abundant in flavonoid and phenolic compounds, which are the key contributors to their medicinal properties. Building on this knowledge, our study aimed to explore the synergistic action of KQE and AgNPs against four different bacterial isolates (Detailed procedure provided in Supplementary Information). In this study, we tested a comparative evaluation of antibacterial activity of KQE, KQ-AgNPs and AgNPs-Chem against four bacterial models: Fig. 5a–e represents the inhibition activity against various microorganisms: S. aureus, B. subtilis, P. aeruginosa, and E. coli. Figure 5e provides a comparative analysis of the inhibition activity of the samples against these tested microorganisms. The corresponding zone of inhibition diameters are recorded in Table 1.

The diameter of zone of inhibition show by of 16 µg/mL of KQE, KQ-AgNPs, AgNPs-Chem and streptomycine on bacterial samples (a) S. aureus, (b) B. Subtillus, (c) P. aeuruginosa and (d) E. coli and corresponding (e) comparative graphical representation.

The antibacterial activity of the samples at 16 µg/mL revealed that KQ-AgNPs demonstrated superior performance against all tested bacterial strains compared to KQE and Streptomycin. Notably, AgNPs-Chem showed significant activity against S. aureus (9.84 mm), surpassing KQ-AgNPs (9.26 mm), although the latter exhibited comparable efficacy. KQ-AgNPs showed the highest zones of inhibition against E. coli (12.45 mm) and P. aeruginosa (11.76 mm), indicating enhanced efficacy, especially against gram-negative bacteria. This superior action can be attributed to the unique cell wall chemistry of gram-negative bacteria, which facilitates interactions between the NPs and the bacterial membrane, leading to increased disruption^17,26,34. AgNPs synthesized using Gmelina arborea also shown similar kind of enhanced antibacterial activity against gram-negative bacteria, whereas in the case of Smilax Chenensis and Moringa oleifera the NPs were more resistant towards gram-positive strains^51,52,53.While KQE alone exhibited limited antibacterial activity, its combination with AgNPs suggesting a synergistic effect of its bioactive compounds with silver in KQ-AgNPs. Our findings align with previous antibacterial research on AgNPs derived from three distinct varieties of Tulsi plants, demonstrating greater efficacy compared to their crude extracts⁵⁴. Compared to AgNPs-Chem, KQ-AgNPs consistently outperformed in most cases, highlighting the advantages of green synthesis. Streptomycin, used as a standard, showed moderate activity but was surpassed by KQ-AgNPs in all the cases, underscoring the potential of these NPs as effective antibacterial agents. Similar studies by Jahangir et al. reported the synthesis of AgNPs using six different substrates of papaya, including leaf, seed, callus, peel, fruit juice, and bark. Among these, AgNPs derived from callus extract exhibited the most potent antimicrobial activity, with an inhibition zone of up to 19 mm against Xanthomonas campestris and 14 mm against Aspergillus niger³³. These findings align with our observations, suggesting that the bioactive compounds in extracts prepared from different part of papaya plant play a critical role in enhancing the antimicrobial potential of AgNPs³¹.

Figure 6a, b presents the SEM images of E. coli cells before and after treatment with KQ-AgNPs. The images clearly demonstrate significant physical damage to the bacterial cells, including cell wall disruption, shrinkage, aggregation, and increased surface roughness. Compared to chemically synthesized AgNPs generally NPs synthesized from biogenic sources exhibit a greater surface area due to the phytochemical capping around their surface^26,34. This enhanced surface area facilitates more effective interactions with bacterial cells, contributing to the increased antibacterial impact observed. These interactions are schematically illustrated in Fig. 6c, which shows the sequence of events following bacterial exposure to KQ-AgNPs with biogenic capping provided from KQE. The NPs induce cell wall disruption, oxidative stress, leakage of intracellular contents, damage to internal cellular components, and inhibition of biofilm formation, ultimately leading to bacterial cell death. This comprehensive mechanistic depiction underscores the superior antibacterial efficacy of KQ-AgNPs compared to their chemically synthesized NPs.

SEM images of E. coli cells before (a) and after (b) treating with KQ-AgNPs. (c) Proposed antibacterial mechanism shown by KQ-AgNPs with biological capping of phytochemicals from KQE.

As our KQ-AgNPs exhibit superior in vitro antibacterial activity against both gram-negative and gram-positive bacteria, they hold great potential for incorporation into antibacterial coatings, medical formulations, or packaging materials. However, compliance with industry standards and safety regulations must be evaluated, along with comprehensive in vivo testing to ensure safety and efficacy for real-world applications.

Anticancer activity

Research shows that combining papaya plant extracts with metallic components significantly enhances their anticancer properties^{44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60}. Metal-polyphenolic networks, including those formed with Ag, Au, Cu, Ni, Se, and other metals, have been synthesized using efficient, rapid, and eco-friendly methods^35,55,56,60. These methods utilize extracts derived from various parts of the papaya tree, such as leaves, peels, bark, oil, fruit, and latex, all of which demonstrate promising anticancer activities.

Table 2 presents a comparative analysis of IC₅₀ values reported in various studies for metallic NPs synthesized from different parts of the papaya plant. Inspired by these findings, this study investigates the anti-proliferative properties of KQ-AgNPs on MCF-7cells and compares them with AgNPs-Chem (Detailed procedure provided in Supplementary Information). The microscopic images Fig. 7a–e represent the inhibitory effect of KQ-AgNPs, while Fig. 7g–k depict the effects of AgNPs-Chem, showing progressive morphological changes in MCF-7 cells with increasing concentrations. Whereas, both Fig. 7f, l are control groups. The results clearly demonstrate that the anticancer activity of both KQ-AgNPs and AgNPs-Chem are concentration-dependent. As shown in the microscopic images, the morphological changes in MCF-7 cells become progressively severe with increasing NP concentrations and highlighting cellular shrinkage, loss of adherence, and cell death at higher concentrations. This trend is further supported by the quantitative analysis in the bar graph Fig. 6m, which reveals a dose-dependent reduction in cell viability. The IC₅₀ value for KQ-AgNPs was calculated to be 21.25 ± 1.14 µg/mL, significantly lower than the IC₅₀ value of 33.05 ± 3.13 µg/mL for AgNPs-Chem, indicating that KQ-AgNPs exhibit stronger cytotoxic effects against MCF-7 cells. Existing in-vivo experimental results by Nainggolan et al. and Sianipar et al. support the superior anticancer activity of papaya male flowers and align with our findings^28,57. In alignment with our research, Qamar et al. demonstrated that AgNPs derived from Ocimum basilicum L. tea display enhanced anticancer efficacy against cervical immortalized (HeLa) cancer cells, yielding an IC₅₀ value of 21.78 ± 0.68 μg/ml⁶¹. Another study looked into the exceptional anticancer activity of AgNPs made from Vaccinium myrtillus extract against human umbilical vein endothelial cells (HUVEC) and breast adenocarcinoma epithelial cells (MDA-MB-231)⁶². These studies provide evidence that AgNPs produced using more environmentally friendly methods have significant anticancer properties.

Microscopic images representing the dose dependent inhibitory effect of KQ-AgNPs (a–e) and AgNPs-Chem (g–k) against MCF-7 cell lines with concentrations 6.25, 12.5, 25, 50 and 100 µg/mL respectively. (f, l) Control cells. (m) Viability (%) of KQ-AgNPs & AgNPs-Chem against MCF-7 for 24 h incubation period at concentrations 6.25, 12.5, 25, 50 and 100 µg/mL. ** refers to significant difference where p < 0.01.

The lower IC₅₀ value of KQ-AgNPs underscores their superior anticancer potency, further highlighting their potential for targeted anticancer therapy, where optimal dosing can achieve maximum therapeutic effects with minimal toxicity to healthy cells. In comparison, the study by Devanesan et al. on dimethyl flubendazole-extracted papaya leaf-derived AgNPs reported an IC₅₀ value of 51.37 µg/mL against MCF-7 cells⁵⁸. This is notably higher than the IC₅₀ values observed in our work, suggesting that KQ-AgNPs are more effective at inducing cell death at lower concentrations. Papaya latex-reduced AgNPs on MCF-7 cells have also demonstrated comparable toxicity but required a longer incubation period, indicating a time-dependent effect⁵⁹. While several studies have highlighted the strong anticancer activity of papaya-mediated AgNPs, a notable limitation is that effective concentrations against cancer cells often exhibit toxicity towards normal cells. In contrast, the IC₅₀ value of our KQ-AgNPs against the HEK-293 cell line is 169.96 ± 2.3 µg/mL (Biocompatibility test results are provided in Figure S2 & S3 of SI), underscoring their selective nature and favorable safety profile. This selective toxicity towards cancer cells while sparing normal cells is consistent with the findings of Vundela et al. and Elderdery et al., who synthesized NPs using papaya fruit extracts^55,56.

The selective cytotoxicity of KQ-AgNPs can be attributed to a combination of factors. The inherent flavonoid enrich surface modification making them more biocompatible nature towards normal cells and toxic towards cancer cells by exhibiting antioxidant actions. On the other hand the negative surface charge of KQ-AgNPs, which influences cellular internalization and subcellular localization differently in normal and cancer cells. According to Asati et al., Cerium (Ce) NPs with a negative surface charge exhibit preferential internalization in cancer cells compared to normal cells⁶³. This can be due to the altered membrane potential and increased expression of certain anionic transporter proteins in cancer cells, facilitating enhanced uptake of negatively charged NPs. In cancer cells, our KQ-AgNPs may be localized into the lysosomes leading to significant cytotoxicity. Whereas in the case of normal cells they may be accumulated in extracellular space or localizing in the cytoplasm without entering lysosomes thus resulting in lower cytotoxicity. This differential internalization and localization profile can be the reason for observed selective anticancer activity of KQ-AgNPs, making them highly potent against cancer cells while being biocompatible with normal cells. Our results indicate that KQ-AgNPs exhibit enhanced anticancer activity, suggesting their potential for targeted therapy. The lower IC₅₀ value against cancer cells, coupled with a higher value against normal cells, reflects effective treatment with reduced toxicity.

The findings support the increasing recognition of biosynthesized KQ-AgNPs for their eco-friendly synthesis and bioactive properties, which contribute to their enhanced therapeutic efficacy in cancer treatment^64,65,66. However, for large-scale production and biomedical applications, factors such as raw material accessibility, NPs stability, regulatory adherence, and economical manufacturing processes must be considered. Future initiatives will focus on process optimization, industry partnerships, and surface modifications (e.g., PEGylation) to enhance the biocompatibility, circulation time, and therapeutic efficacy of KQ-AgNPs, promoting their commercialization and clinical translation.