Cissus quadrangularis mediated biogenic synthesis of silver-nanohydroxyapatite-mesoporous silica nanocomposite, characterization, and cytotoxicity evaluation on dental pulp stem cells

Abstract

This study focuses on tripartite synthesis of Silver (AgNPs), Mesoporous Silica (MSNs), and Hydroxyapatite (n-HAp) nanoparticles with aqueous extract of Cissus quadrangularis (Veldt grape plant; Indian name: Pirandai) as a reducing agent. The dried and powdered form of the plant was subjected to aqueous extraction. The phytochemicals analysis was qualitatively estimated which detected the presence of alkaloid, tannin, phenol, terpenoid, steroid and saponin. The total phenol content of the extract was quantitatively estimated which resulted in Gallic acid equivalent (GAE) of 88.9 mg/g. This aqueous extract was then used in the synthesis of AgNPs, followed by synthesis of MSNs and n-HAp. These three nanoparticles were again combined together and re-synthesized in a tripartite manner to create a novel nanocomposite material. This nanocomposite was characterized by using techniques such as UV–vis spectroscopy, X-ray Diffraction (XRD) and Fourier-Transfer Infrared Spectroscopy (FTIR) to detect the presence, size, stability and functional groups of the synthesized nanoparticles. The bioavailability of synthesized nanoparticles was evaluated by testing their cytotoxicity against dental pulp stem cells (DPSC), which resulted in IC₅₀ values of 49.004, 106.869, 113.711, 131.27 µg/ml for AgNP, n-HAp, MS and tripartite composite respectively. Following this, the proliferation rate of DPSC cells treated culture media diluted composites in the ratio 1:1, 1:2 and 1:4 was evaluated. From the results it was found that cells co-cultured with composite dilution has shown increased proliferation rate. These findings highlight the biocompatibility and the potential of tripartite synthesized nanocomposite, when incorporated in dental restorative materials may give promising results.

Introduction

Herbal therapy, the oldest kind of medicine known to man, uses whole plants or plant parts to treat a variety of life-threatening diseases or promote overall health. The use of herbal remedies to treat a range of medical conditions is still growing quickly worldwide. Both in industrialized and emerging nations, there has been a huge increase in public acceptance and interest in natural cures. Herbal remedies are now sold in both drug and grocery stores. For the vast majority of people in poor nations, herbal medicines serve as their main source of healthcare [1]. The possible molecular targets of herbal medicine extracts have been widely identified in the past few decades, which will make it easier to identify the bioactive substances involved in these plants' pharmacodynamic mechanisms [2]. Various systems, such as traditional Indian medicine, European medicine, Japanese Kampo, traditional Chinese medicine, or traditional Arabic and Islamic medicine and folk medicines, which include not only herbal treatments but also pharmaceuticals made from minerals and metals, have gradually evolved from the accumulated rich knowledge about natural products [3].

India is an herbal centre with a long history of using traditional medicine. There are around 6,000 plant species that are utilized as medicines in it [4]. A member of the Vitaceae family, the genus Cissus comprises 800 species spread across 13 taxa in Africa, Arabia, South Asia, Sri Lanka, India, and other tropical locations. Of these, India is home to 63 distinct species and 8 genera [5]. Cissus quadrangularis belongs to the grape family and is a deciduous, succulent, climbing perennial plant [6]. It is often referred to as adamant creeper, winged treebine, or veldt grape in English and Pirandai in Tamil [7]. The Arabian Peninsula, tropical Asia, and a large portion of Africa are home to the species. It is commonly believed that Cissus quadrangularis originated in Ayurveda, but because it grows in so many places, it is believed to have been utilized medicinally in a variety of locations [8]. The traditional literature on Indian medical systems and literature worldwide indicates that these plants have demonstrated effectiveness in treating a range of conditions, including osteoporosis, fractures of the bones and muscles, injury to ligaments, discomfort, and inflammation [9]. Additionally, studies have demonstrated that the Cissus extracts offer all-natural remedies for a range of oral health issues, which could complement current dental therapies [10].

Nanoscience is a blessing that can lead to unexpected changes in a variety of fields of study and application [11]. The primary focus of nanotechnology is the creation, synthesis, structural analysis, and use of materials smaller than 100 nm. Nanomaterials have been widely discovered in recent years with special qualities and amazing potential, since they open up new avenues for interdisciplinary research and can be used to solve a wide range of real-world issues [12]. Since the early 1900s, it has been recognized that plant extracts can reduce metal ions, but it was unclear what kind of reducing agents were at play. The use of live plants, entire plant extract, and plant tissue to reduce metal salts to nanoparticles has garnered a lot of attention in the past 30 years due to its simplicity [13].

The physical and chemical approaches used in the conventional synthesis of nanoparticles are very laborious, require a lot of energy to maintain high temperatures and pressures, and may be hazardous to humans due to the use of hazardous chemicals [14]. In recent years, biogenic synthesis of metallic nanoparticles has gained popularity as a means of overcoming the drawbacks of conventional methods. Biogenic pathways are thought to be safe, hygienic, and economical. Numerous biological resources, including bacteria, algae, fungi, yeast, and plant extracts, have been used in the biogenic synthesis of metallic nanoparticles [15]. The biomedical industry has long recognized the antibacterial, antifungal, and anti-plasmodial capabilities of silver nanoparticles, which make them an excellent choice for Endodontics, dental prosthesis, Implantology, and restorative dentistry [16].

Mesoporous silica (MS), among the various types of nanoparticles, is unique and can be a promising drug delivery system for dental biomaterials due to their high surface area, large pore volume, ease of synthesis, and amenability to functionalization [17]. Additionally, silica coating of nanoparticles can increase the material's surface area and porosity, making it more viable for use in biological applications [18]. A calcium phosphate molecule called hydroxyapatite (n-HAp) shares the same hexagonal shape as bone apatite. The primary mineral that makes up tooth enamel and dentin is carbonated calcium deficient n-HAp. Since n-HAp has a higher thermodynamic stability under physiological settings than other calcium phosphates, its clinical uses have expanded across a number of medical specialties, particularly due to its osteogenic qualities and biocompatibility [19].

The aim of this study is to synthesize and characterize Silver-Functionalized Mesoporous Silica-Hydroxyapatite Nanocomposite utilizing Cissus quadrangularis extract as a reducing agent, and to evaluate its cytotoxic effects on Dental Pulp Stem Cells (DPSC).

Materials and methods

Identification and sample collection

Cissus quadrangularis was procured from organic farms in and around Chennai and Kancheepuram, India. This sample was authenticated by School of Life Sciences, Sri Ramaswamy Memorial Institute of Science and Technology (SRMIST).

Physicochemical analysis

Soxhlet extraction

Crude plant extract was prepared by Soxhlet extraction method [20]. About 50 gm of powdered plant material was uniformly packed into a muslin cloth and extracted with 150 ml of water. The process of extraction continues for 48 h., or till the solvent in siphon tube of an extractor become color-less. The cooling water supply to the condensers was opened to ensure continuous recycling of the solvent and temperature (90 °C) selected for extraction in the continuous mode. On completion of the extraction process, the samples were left to cool for at least 15 min after which the solvent cups with the extracts were removed.

Concentration of the extract

After the extraction process, the suspension was filtered and concentrated to dryness using a rotary evaporator. Dried extract was kept in refrigerator at 4 °C for their future use in phytochemical analysis.

Extraction yield calculation

The final yield volume of the extracted and dried plant sample was calculated by the following formula [20],

In which Y is Extraction Yield, W_i is initial weight and W_f is final weight.

Qualitative analysis

Qualitative analysis of extract was done for the detection of alkaloids, glycoside, flavonoid, saponin, tannin, steroids, phenol and terpenoids [21].

Quantitative analysis

The phytochemicals present in hydro methanolic extract of Cissus quadrangularis was quantitatively estimated for determination of total phenol content. Total phenolic content was determined using Folin-Ciocalteu method with slight modification [22].

Synthesis of nanoparticles

Silver nanoparticles (AgNPs)

To initiate the synthesis, 80 mL aqueous solution (50 mM) of pure silver nitrate was added drop-wise to 20 mL of the aqueous solution of the plant extract (10 mg/mL). The resulting reaction mixture was maintained at 60 °C while being continuously stirred at 450 rpm using a hot plate magnetic stirrer. Over a duration of 6 h., the reaction mixture underwent precipitation, resulting in the formation of a black color solution composed of AgNPs. After the completion of the reaction, the precipitated AgNPs were separated through centrifugation at 14,000 rpm for 15 min and subsequently washed with 90% alcohol. The washed AgNPs was then dried at 80 °C in a hot air oven for 4 h., resulting in the formation of dried black powder [23].

Mesoporous silica (MSNs)

To 20 ml of 100 mg/ml plant extract, 10 mL of Tetraethyl Ortho-silicate (TEOS) (0.1 mol/L) was drop-wise added. The resulting solution was continuously stirred for 4–6 h., and then 0.1 M Cetyltrimethylammonium bromide (CTAB) buffer prepared in 40% alcohol was added drop-wise to the solution. Finally, the pH of the reaction mixture was maintained at 11 using ammonia and the solution was set at constant agitation overnight. The resulting white mixture was then centrifuged at 12,000 rpm and washed with ethanol for 3 times and dried at 80 °C [24, 25].

Nano-hydroxyapatite (n-HAp)

A total of 80 ml of 0.1 mol/L Calcium chloride was added drop-wise to 20 mL of plant extract(100 mg/ml) under continuous stirring at 2000 rpm for 3 h. The pH of the extract was maintained at 9 by addition of ammonia. 0.1 mol/L of 60 mL disodium hydrogen phosphate dodecahydrate was added drop-wise, then again pH was maintained at 9 by adding ammonia and vigorous shaking for 2 h at 60 °C. The resulting solution was set overnight to remove bubbles trapped in the viscous solution. Finally, the suspension was centrifuged at 3000 rpm for 30 min in order to separate the white precipitate, then dried for 48 h., at − 40 °C in a freeze-dryer [26, 27].

Tripartite synthesis of Ag/n-HAp/Si nanocomposite and characterization

The prepared AgNP and n-HAp was dispersed at a ratio of 1:1 in aqueous solution of 90 ml with constant agitation for 6 h., at 60 °C. This in turn followed by drop-wise addition of TEOS (0.1 mol/L) and (0.1 M) CTAB buffer 5 ml each to the mixture. The pH of the mixture was set at 10 by adding a proper amount of ammonia solution and kept under constant agitation for 4 h. Next, disodium hydrogen phosphate dodecahydrate was added drop wise to the mixture and after overnight of stirring, the precipitate was centrifuged and washed with water and methanol and dried in an oven at 60 °C for 4 h. The synthesized nanoparticles were characterized by techniques such as UV–Vis, FTIR, XRD, SEM-EDAX, DLS and TEM.

In-vitro analysis

Since this study was an in vitro laboratory analysis, clinical trial registration is not applicable for this study.

Cell cytotoxicity

The assay was carried out according to Mosmann, 1983 by using (3‐ (4, 5‐dimethyl thiazol‐2yl) ‐ 2, 5 diphenyl tetrazolium bromide (MTT)[26]. Briefly, trypsinized cells were seeded in 96 well plate and incubated at 37 °C for 48 h. After 48 h incubation, the DPSC cells were treated with nanoparticles dispersed in DMSO at different concentrations for 24–48 h. After treatment, media were removed and wells were added with MTT (5 mg/ml prepared in phosphate-buffered saline) and left for 4 h., at room temperature. The formazan crystals formed were dissolved in 100 µl DMSO, and absorbance was read in a microplate reader at 570 nm [26].

Cell proliferation

DPSC Cells were isolated from freshly extracted tooth. Cultured cells were trypsinized and seeded into 24 well plates at low density (approximately 40% confluence) containing DMEM media with 10% fetal bovine serum and sample extract at dilution 1/10th. Then cells were incubated at 37 °C in 5% CO₂. In the control cultures, cells were placed directly in DMEM medium at the same cell density. The medium was replaced every two days. At predetermined intervals, the culture medium was removed and 100 µL of MTT solution (5 mg/mL) was added to the wells and incubated at 37 °C under 5% CO₂ for 4 h and 500 µL of DMSO was added to each well to solubilize the formazan crystals. The cell number was determined from the absorbance of formazan at 570 nm, read by a microplate reader [28].

Results

Extraction yield

The dried powder of Cissus quadrangularis is extracted in water with a Soxhlet apparatus, the extract is then concentrated with a rotatory evaporator. The result is denoted (Table 1).

Table 1.

Extraction yield percentage of Cissus quadrangularis

Initial weight (g)	Final weight (g)	Extraction yield (%)
51.678	41.33	79.97

Qualitative analysis

The aqueous extract of Cissus quadrangularis was subjected to qualitative analysis for phytochemicals detection, the detected phytochemicals are presented (Table 2).

Table 2.

Phytochemical analysis of the extract

S. No	Phytochemical	Inference
1	Flavonoid	−
2	Alkaloid	+
3	Saponin	++
4	Tannin	+
5	Phenol	+
6	Terpenoid	+
7	Glycoside	−
8	Steroid	+

Presence of phytoconstituents: + (low), ++ (moderate), +++ (high); Absence of phytoconstituent: − 

Quantitative analysis (total phenol content)

The total phenol content detected in the aqueous extract of Cissus quadrangularis was quantitatively estimated by measuring their absorbance at different concentrations are represented (Table 3). The graphical representation of the total phenol content detected in the extract is represented (Fig. 1). The Gallic Acid Equivalent (GAE) of the extract is denoted (Table 4).

Table 3.

Absorbance of the extract for detection of phenol

S. No	Concentration (μg/ml)	Absorbance @650 nm
1	1000	2.258 ± 0.003
2	500	1.321 ± 0.09
3	250	0.945 ± 0.02
4	100	0.661 ± 0.01
5	50	0.437 ± 0.04
6	25	0.285 ± 0.01

Fig. 1.

Graphical representation of total phenol content-gallic acid

Table 4.

Gallic acid equivalent of extract

Sample name	Absorbance @ 650 nm	GAE mg/g of plant extract
Cissus quadrangularis	0.365 ± 0.003	88.90 ± 0.74

Ultraviolet visible spectrophotometry (UV–vis)

The presence and stability of nanoparticles silver, hydroxyapatite, mesoporous silica and their tripartite composite were analyzed by UV–Vis spectroscopy, and the resultant wavelength peaks are shown (Fig. 2).

Fig. 2.

UV–vis spectroscopy of nanoparticles: a AgNPs; b n-HAp; c MSNs; d nanocomposite

Fourier-transfer infrared spectroscopy (FTIR)

The functional groups of nanoparticles were determined using FTIR and the results were given in Table 5. The FTIR peaks of the nanoparticles are characterized (Fig. 3).

Table 5.

FTIR result of nanoparticles

S. No	Sample	Range	Functional groups
1	AgNPs	1747	C=O
		1996	N=C
		3583, 3672	O=H
2	n-HAp	1016	C–O
		2007, 2177	C=N
		3041	OH
3	MSNs	1641	C=O
		2096	C=N
		2173–2243	Si–H
		2852–3302	CH, OH
4	Nanocomposite	1012–1091	C–O, C–N
		1992–2115	C=N
		2333	Si–H

Fig. 3.

FTIR of nanoparticles: a AgNPs; b n-HAp; c MSNs; d nanocomposite

X-ray diffraction (XRD)

The plane values and nanoparticles size for nanoparticles synthesized were analyzed by XRD and results were given in Table 6. Figure 4 shows the graphical representation of XRD results obtained.

Table 6.

XRD results of nanoparticles

S. No	Sample	2θ position	Plane (hkl)	Size(nm)
1	AgNPs	(38.06), (44.2), (64.4), (77.25)	(111), (200), (220), (311)	165.48
2	n-HAp	(25.7), (31.8), (53.1), (46.4), (49.5)	(002), (211), (004), (222), (213)	151.59
3	MSNs	(18.7), (22.9)	(100)	85.55
4	Nanocomposite	(18.9), (31.2), (38.5), (44.8), (77.3)	(100), (211), (111), (200), (311)	169.35

Fig. 4.

Graphical representation of XRD result of nanoparticles: a AgNPs; b n-HAp; c MSNs; d nanocomposite

Scanning electron microscopy (SEM)

SEM images of synthesized nanoparticles AgNP, n-HAp, MS with their tripartite composite was shown (Fig. 5).

Fig. 5.

SEM images of: a AgNPs; b n-HAp; c MSNs; d nanocomposite

Energy-dispersive x-ray spectroscopy (EDX)

EDX results of synthesized nanoparticles AgNPs, n-HAp, MSNs with their tripartite composite are depicted (Fig. 6).

Fig. 6.

EDX analysis: a AgNPs; b n-HAp; c MSNs; d nanocomposite

Dynamic light scattering (DLS)

The zeta potential and size distribution of nanoparticles with their composites were analyzed by DLS. Figure and depicts the pictogram of zeta potential and size distribution respectively. The analyzed data were given in Table 7 (Figs. 7 and 8).

Table 7.

Zeta potential and size distribution of nanoparticles and their composites

Sample	Zeta potential (mV)	Size distribution (d.nm)
AgNP	− 34.7	1113
MSN	− 7.28	1110
n-HAp	10.2	764.1
Nanocomposite	− 9.22	425.2

Fig. 7.

Zeta potential of AgNPs (a), MSN (b), n-HAp (c) and nanocomposite (d)

Fig. 8.

Size distribution of AgNPs (a), MSN (b), n-HAp (c) and nanocomposite (d)

Transmission electron microscopy (TEM)

The TEM images of nanoparticles and their nanocomposite were given in Fig. 9.

Fig. 9.

TEM images of AgNPs (a), MSN (b), n-HAp (c) and nanocomposite (d)

In-vitro study

Cell cytotoxicity

The percentage of cell viability and IC₅₀ value of nanoparticles treated cells are organized (Tables 8 and 9). Figures 10, 11, 12, 13 shows the DPSC cells cultured with nanoparticles at different concentrations.

Table 8.

Cell viability of nanoparticles treated DPSC cells

S.no	Concentrations (µg/ml)	Percentage of cell viability
		AgNPs	n-HAp	MSNs	Nanocomposite
1	512	21.98 ± 0.05	28.67 ± 0.01	29.99 ± 0.02	28.63 ± 0.101
2	256	24.81 ± 0.14	36.64 ± 0.17	39.03 ± 0.20	43.42 ± 0.25
3	128	35.6 ± 0.88	44.59 ± 0.25	49.46 ± 0.01	52.67 ± 0.203
4	64	45.85 ± 0.04	56.47 ± 0.17	55.51 ± 0.26	58.37 ± 0.209
5	32	58.09 ± 0.74	67.13 ± 0.009	64.11 ± 0.202	67.52 ± 0.16
6	16	67.16 ± 0.45	75.4 ± 0.21	75.33 ± 0.202	74.76 ± 0.15
7	8	79.36 ± 0.17	84.61 ± 0.105	87.39 ± 0.02	83.33 ± 0.03
loww8	4	84.65 ± 0.11	95.37 ± 0.03	96.37 ± 0.02	94.59 ± 0.21
9	2	87.45 ± 0.17	96.86 ± 0.005	98.53 ± 0.02	96.66 ± 0.13
10	1	89.54 ± 0.06	97.9 ± 0.03	99.41 ± 0.56	97.63 ± 0.21

Table 9.

IC₅₀ value of nanoparticles treated with DPSC

S. No	Sample	IC50 value (µg/ml)
1	AgNPs	49.004
2	n-HAp	106.869
3	MSNs	113.711
4	Nanocomposite	131.27

Fig. 10.

Cytotoxicity activity of AgNPs against DPSC cell line (a 1 µg/ml; b 2 µg/ml; c 4 µg/ml; d 8 µg/ml; e 16 µg/ml; f 32 µg/ml; g 64 µg/ml; h 128 µg/ml; i 256 µg/ml; j 512 µg/ml)

Fig. 11.

Cytotoxicity activity of MSNs against DPSC cell line (a 1 µg/ml; b 2 µg/ml; c 4 µg/ml; d 8 µg/ml; e 16 µg/ml; f 32 µg/ml; g 64 µg/ml; h 128 µg/ml; i 256 µg/ml; j 512 µg/ml)

Fig. 12.

Cytotoxicity activity of n-HAp against DPSC cell line (a 1 µg/ml; b 2 µg/ml; c 4 µg/ml; d 8 µg/ml; e 16 µg/ml; f 32 µg/ml; g 64 µg/ml; h 128 µg/ml; i 256 µg/ml; j 512 µg/ml)

Fig. 13.

Cytotoxicity activity of nanocomposite against DPSC cell line (a 1 µg/ml; b 2 µg/ml; c 4 µg/ml; d 8 µg/ml; e 16 µg/ml; f 32 µg/ml; g 64 µg/ml; h 128 µg/ml; i 256 µg/ml; j 512 µg/ml)

Cell proliferation

Figure 14 depicts the DPSC cells treated with composite dilutions 1:1, 1:2 and 1:4 at day 1, 3 and 7. The graphical representation of cell proliferation rate are shown in Fig. 15.

Fig. 14.

DPSC cells treated with nanocomposite dilutions 1:1; 1:2 and 1:4 and control (untreated) on day 1, 3 & 7. a Composite dilution 1:1 on day 1; b composite dilution 1:1 on day 3; c composite dilution 1:1 on day 7; d composite dilution 1:2 on day 1; e composite dilution 1:2 on day 3; f composite dilution 1:2 on day 7; g composite dilution 1:4 on day 1; h composite dilution 1:4 on day 3; i composite dilution 1:4 on day 7; j control (untreated) on day 1; k) control (untreated) on day 3; l) control (untreated) on day 7

Fig. 15.

Graphical representation of cell proliferation in dilutions 1:1, 1:2 and 1:4

Discussion

The powdered sample of Cissus quadrangularis is subjected to physio-chemical analysis such as extraction, qualitative and quantitative analysis for the detection of phytocomponents in the extract. The dried sample of Cissus quadrangularis was extracted with water in Soxhlet apparatus, which resulted in an extraction yield of 79.97%. The qualitative analysis of the extract revealed the presence of various phytochemicals, with their levels categorized as high, moderate, low, or absent. The quantitative analysis of the extract revealed the presence of low(+) levels of alkaloid, tannin, phenol, terpenoid and steroid along with moderate(+ +) levels of saponin. Meanwhile, presence of flavonoids and glycoside were not detected in the aqueous extract. These results are consistent with the findings of Rajeshkumar et al., (2021), in which aqueous extract of Cissus quadrangularis was qualitatively estimated [29]. Similarly, the aqueous extract of Cissus quadrangularis was quantitatively analyzed for its total phenol content at varying concentrations which yielded a GAE of 88.9 mg/g. Phenolic compounds have proven to exhibit significant osteoinductive, antioxidant, and anti-inflammatory properties, enhancing the proliferation and osteogenic differentiation of DPSCs while modulating key bone remodeling pathways relevant to osteoporosis and bone regeneration. The important antioxidants that fight free radicals, phenols may shield DPSCs from oxidative stress, encourage osteogenic development, and provide protection [30].

The nanoparticles AgNPs, n-HAp and MSNs were synthesized in a tripartite manner with ratio of 1:1:1. The synthesized nanoparticles with their tripartite composite were characterized using UV, XRD and FTIR analysis. The AgNPs showed a distinct surface plasmon resonance (SPR) peak at 450 nm, confirming successful nanoparticle formation, consistent with reports of SPR peaks in the 420–460 nm range for silver [31]. HAp exhibited a broad absorption near 310 nm, attributed to PO₄³⁻ transitions, aligning with known HAp spectral characteristics [32]. MSNs showed no sharp peaks but a gradual decrease from 200 to 350 nm, typical for non-plasmonic, amorphous silica. The composite spectrum displayed both AgNP and HAp features, along with a broad tail up to 700 nm, suggesting successful integration and interparticle interactions. The UV results of the nanoparticles indicate their presence and stability over time as shown in Fig. 2. The FTIR result of silver nanoparticles denoted the presence of functional groups C=O, N=C, O=H in range of 1747, 1996, 3583 and 3672. These findings were more or less similar to the findings of Duraisamy R et al., 2025, in which AgNPs were synthesized using aqueous extract of Cissus quadrangularis and assessed by FTIR for detection of the functional groups [33]. Functional groups C–O, C=N, OH were detected in hydroxyapatite nanoparticles at range of 1016, 2007, 2177 and 3041 respectively. These results are similar to results obtained by Hooi M et al., 2021, from n-HAp nanoparticles synthesized from fish bone composite [34]. The FTIR of mesoporous silica nanoparticles detected the presence of functional groups C=O, C=N, Si–H, CH, OH at peaks of range 1641, 2096, 2173–2243 and 2852–3302. These were consistent with the findings of Mourhly et al., 2015, in which amorphous silica is analyzed for functional groups detection [35]. The Nanocomposites of AgNPs, n-HAp and MSNs was detected for the presence of functional groups C–O, C–N, C=N, Si–H at ranges of 1012–1091, 1992–2115, 2333 respectively.

The XRD results of AgNPs showed peak values of 2θ = 38.06, 44.2, 64.4 and 77.25 degree corresponding to plane (hkl) values of (111), (200), (220) and (311) respectively. These strong diffraction peaks prove the face-centered cubic (FCC) characteristics of silver, especially the high intensity peak at 38.06 corresponding to plane (111) confirms its predominant crystalline phase. These results are consistent with the findings of Duraisamy et al., (2025) and Santhoshkumar et al., (2012) in which AgNPs synthesized with aqueous extract of Cissus quadrangularis were subjected to XRD [33, 36]. The XRD values of n-HAp denoted peak values of 2θ = 25.7, 31.8, 53.1, 46.4 and 49.5° corresponding to plane (hkl) values of (002), (211), (004), (222) and (213). Multiple characteristics peaks appeared between 20 and 40 degrees as shown in Fig. 4b confirms the presence of crystalline hydroxyapatite, particularly major diffraction peaks at planes (002), (211) and (300) corresponds to standard n-HAp reflections. These results are consistent with the findings of Mondal S et al., (2012), in which n-HAp nanoparticles synthesized from fish scales and bovine bones were assessed by XRD [37]. The mesoporous silica nanoparticles produced peak at angle of 2θ = 18.7 and 22.9 degrees with respective to plane (hkl) value of (100). As shown in Fig. 4c the broad diffraction peak at 22.9 degrees indicates the presence of amorphous silica, and the absence of sharp crystalline peaks confirms its amorphous characteristics. This characteristic is consistent with the reports of Mourhly A et al., 2015, which reported the amorphous characteristics of SiO₂ [35]. Meanwhile, XRD of tripartite composite of all three nanoparticles produced peaks at angle of 2θ = 18.9, 31.2, 38.5, 44.8 and 77.3 corresponding to plane (hkl) values of (100), (211), (111), (200) and (311) respectively. The presence of sharp peaks at 38.5 and 44.8 degrees suggests FCC of silver, the peaks around 18.9and 31.2 confirms n-HAp, and the broad peaks near 20 degrees indicates amorphous silica. These indicates the presence of AgNPs in the composite with confirmed n-HAp phase and MSS contributes to the amorphous background. These phase compositions can be confirmed by comparing with the work conducted by Piecuch A et al., 2023, in which silver-doped silicate-substituted hydroxyapatites were characterized [38].

The peak values obtained from XRD results were used to determine the crystalline size of the synthesized nanoparticles using the Scherrer Equation. The detected crystalline size of the nanoparticles: AgNPs (165.47nm), n-HAp (151.59nm), MSNs (85.55nm) and Nanocomposite (169.35nm).

SEM analysis of revealed that green synthesized AgNPs were predominantly spherical, with individual AgNPs and aggregates being observed. This result indicates the nanoparticles spherical nature and tendency to form larger aggregates influenced by secondary metabolites of the plant. Similar results were observed by Duraisamy et al. 2025, in which AgNPs synthesized with aqueous extract of C. quadrangularis were observed [33]. The SEM analysis of n-HAp revealed porous and granular structures, typical characteristics of n-HAp nanoparticles. The porous structure suggests a high surface area, which is beneficial for dental applications. These results align with the results of Barakat N et al., [39]. The SEM image of mesoporous silica showed irregular and loosely packed particles, with spherical and semi-spherical structures suggesting well-defined morphology. The interconnected porous structure may enhance the drug loading and adsorption properties of the nanoparticles. Similar results can be observed in work done by Sousa et al. [40]. The SEM image of Tripartite composite revealed a highly porous, rough structure with small granular particles present, which are typical characteristics of MS, n-HAp and AgNPs. Agglomeration of particles can also be observed, potentially due to strong interaction between AgNPs, n-HAp and MS. The combination of AgNP, n-HAp and MS with their natures such as antimicrobial, bone integration and high surface area in the tripartite makes it a promising application in dentistry such as implant or coatings. By referring to the Fig. 6a, the presence of Ag is confirmed by the characteristic peaks at around 3 keV, 0.3 keV and 2.9 keV. The most prominent peak at 3 keV, which correspond to Ag Lα emission further supports the result. From EDX report of n-HAp (Fig. 6b), strong P peaks (2 keV) and strong CA Peak (3.7 keV) can be observed indicating the presence of calcium and phosphate, which are main components of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). The oxygen (O) peak (0.5 keV) further supports the phosphate groups. In EDX spectrum (Fig. 6c), Si peaks at 1.8 keV and O peaks at 0.5 keV can be observed indicating the presence of silica (SiO₂). The Ag/MS/n-HAp composite is well-represented in the spectrum (Fig. 6d), with clear signals from Ag (3 keV), silica (1.8 keV) and calcium peaks (2–4 keV) representing n-HAp. This EDX spectrum validates the composition of the Ag/MS/n-HAp tripartite material.

AgNPs exhibited a strongly negative zeta potential (− 34.7 mV), indicating excellent colloidal stability, likely due to effective capping by phytochemicals such as flavonoids and phenolics present in the plant extract. In comparison, MSN (− 7.28 mV) and HAp (+ 10.2 mV) displayed lower absolute values, suggesting limited surface stabilization and a higher propensity for aggregation, particularly in the case of positively charged HAp. The nanocomposite showed an intermediate zeta potential (− 9.22 mV), implying improved stability relative to MSN and n-HAp. This enhancement may be attributed to the presence of AgNPs, which contribute a stabilizing surface charge, thereby reducing aggregation within the composite matrix. Particle size analysis revealed that AgNPs, MSN, and n-HAp exhibited relatively large hydrodynamic diameters (1113 nm, 1110 nm, and 764.1 nm, respectively), likely due to aggregation and the presence of plant-derived capping agents. In contrast, the nanocomposite demonstrated a significantly reduced average size (425.2 nm), indicating improved dispersion and structural compactness resulting from synergistic interactions among the constituent nanoparticles. The crystalline size obtained from XRD for nanoparticles and their nanocomposite confirm their nanostructured nature. However, the corresponding DLS values are significantly larger. This discrepancy can be attributed to the presence of bio-organic capping agents from plant extracts used in green synthesis, as well as particle agglomeration in the aqueous medium. Such trends have been commonly observed in green synthesized nanomaterials, where the hydrodynamic diameter includes the hydration shell and surface-bound biomolecules [31].

TEM analysis further confirmed the structural and morphological features of the synthesized nanoparticles. AgNPs appeared as spherical, polycrystalline particles forming dense aggregates, while MSN exhibited smooth, well-dispersed spherical structures with an amorphous nature. n-HAp showed irregular, flake-like morphology with poor crystallinity, consistent with its tendency to aggregate. Notably, the nanocomposite displayed a heterogeneous structure, with AgNPs and n-HAp fragments embedded within the MSN matrix. The presence of multiple diffraction patterns in SAED validated the successful integration of all components. This structural synergy supports the improved stability and reduced size observed in DLS and zeta potential measurements.

The synthesized nanoparticles were tested for their cytotoxicity against DPSC cells at varying concentrations, in which viability was noted in Table 8. Since according to ISO 10993–5, cell viability greater than 70% was considered non-toxic, the viability of cells treated with AgNP (8 µg/mL), n-HAp (16 µg/mL), MSN (16 µg/mL) and Composite (16 µg/mL) were greater than 70% indicating that nanoparticles were non-toxic at particular concentrations and their subsequent concentrations. IC₅₀ values of nanoparticles were 49.004, 106.869, 113.711, 131.27 µg/ml for AgNP, n-HAp, Silica and Composite respectively. Comparing our IC₅₀ values on DPSCs reveals that AgNPs (49 µg/mL) exhibit moderate cytotoxicity, aligning closely with the threshold concentration (~ 50 µg/mL) reported for CMC-coated silver NPs, which caused significant cell death above a 16% suspension [41]. In contrast, n-HAp and MSNs, with IC₅₀ ~ 107 and ~ 114 µg/mL respectively, demonstrate low toxicity, comparable to reported safe concentration thresholds (~ 50–75 µg/mL) for silica-based NPs in DPSCs [42]. The nanocomposite (IC₅₀ = 131 µg/mL) shows the highest cell tolerance, suggesting enhanced biocompatibility, potentially due to the synergistic effects of component nanoparticles reducing individual toxicity. Cell proliferation is a critical indicator of biocompatibility and regenerative potential, especially in stem cell-based applications such as tissue engineering and dental pulp regeneration. The composites were particularly assessed for its effect on cell proliferation, from which it is found that the proliferation rate of DPSC cells treated with Composite dilutions 1:1 and 1:4 was similar or less than the proliferation rate of control cells that were not treated with composites. Meanwhile, the cells treated with composite dilution 1:2 has shown increased proliferation rate compared to control. The enhanced proliferation of DPSCs at 1:2 dilution indicates that the composite has the potential to promote cellular activities crucial for tissue repair, such as cell division and matrix synthesis, a valuable trait for applications in dental tissue regeneration, scaffold reinforcement and dental pulp capping.

Conclusion

This study successfully tripartite synthesized silver, mesoporous silica and hydroxyapatite nanoparticles using aqueous extract of Cissus quadrangularis as reducing agent. The qualitative analysis of plant extract detected the presence of major phytochemicals alkaloid, tannin, phenol, terpenoid, steroid and saponin. The total phenolic content detected (88.9 mg/g) suggests the extracts potential in shielding DPSCs from oxidative stress, encouraging osteogenic development, and provide protection. The presence and stability of synthesized nanoparticles was confirmed by UV spectroscopy. Meanwhile particles size and functional groups were detected by XRD and FTIR. The cytotoxic assay denotes that tripartite is least lethal against DPSC cell line proving its bioavailability, the proliferation of DPSC cell line treated with composite dilution showed increased proliferation rate which further proves this point. These findings suggest the potential of nanoparticle tripartite synthesized with Ag, MS and n-HAp in its biocompatibility and in dental restorations, coating or therapy.

Author contributions

Shobana K: Conceptualization, nanomaterial synthesis, in vitro analysis and interpretation of the results, drafting of the original manuscript, review & editing. Vidyashree Nandini V: Conceptualization, interpretation of the results, drafting of the original manuscript, review & editing, validation, supervision. Kalaivani Thirunavukarasu: Conceptualization, nanomaterial synthesis, characterization, analysis and interpretation of the results. Udhayakeerthana Chinnathambi : Nanomaterial synthesis, characterization, analysis and interpretation of the results. Vijay Venkatesh K: Interpretation of the results, drafting of the original manuscript, review & editing, validation, supervision. All authors have read and agreed to the publish this version of the manuscript.

Funding

Open access funding provided by SRM Institute of Science and Technology for SRMIST – Medical & Health Sciences. This research received no external funding or grants from any agency or organization.

Data availability

All used data is fully presented in the manuscript.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.