Physicochemical characterization of a drug-loaded nanosponge for endodontic regeneration

Abstract

Background:

Regenerative endodontics requires drug delivery systems that are both antimicrobial and bioactive to achieve favorable clinical outcomes.

Aim:

The aim of the study was to develop and characterize a β-cyclodextrin-based nanosponge incorporated with lyophilized platelet concentrate (Ly-PRF) and a modified triple antibiotic paste (m-TAP) to support endodontic regeneration.

Materials and Methods:

β-cyclodextrin-based nanosponge was fabricated and combined with Ly-PRF, ciprofloxacin, metronidazole, and clindamycin (m-TAP) to obtain the drug-loaded nanosponge (DLNS). It was characterized for surface morphology (SEM), crystallinity (XRD), particle size and polydispersity (PDI), functional group interactions (FTIR), drug release (UV), pH analysis, calcium ion release profile, solubility and zeta potential.

Results:

The DLNS exhibited a porous, sponge-like morphology, whereas the XRD analysis confirmed their amorphous state. Drug release profile revealed a moderate, short-term release of antibiotics followed by sustained release over 14 days. Calcium ions released peaked on day 7 and then declined. The formulation demonstrated a zeta potential value of −24.32 mV, indicating moderate stability and a limited aqueous solubility of 4.9%, suitable for sustained drug release.

Conclusion:

The DLNS formulation demonstrated several favorable physicochemical characteristics essential for intra-canal delivery of drugs and growth factors to support regeneration.

INTRODUCTION

Pulpal and periradicular diseases, commonly resulting from caries, trauma, or developmental anomalies, are traditionally treated using root canal therapy, which aims to remove infection while preserving tooth structure. In immature teeth, where root development is incomplete, Mineral Trioxide Aggregate apical barriers halt root development and weaken tooth structure.[1]

Regenerative endodontic procedures have emerged as a biological alternative to repair the pulp–dentin complex, restore immune and sensory function, and promote continued root development.[2] The concept of pulp regeneration was first proposed by Ostby in 1961,[3] whereas the earliest clinical success in revascularization was reported by Iwaya et al.[4] Banchs and Trope[5] utilized a blood clot scaffold and coronal seal to enable tissue regeneration. The current regenerative approaches attempt to induce intracanal bleeding for the release of stem cells and growth factors, the results of which are variable and true regeneration may not always be achieved.[6]

Infection control is important to facilitate regeneration and may be achieved through mechanical debridement combined with irrigants and intracanal medicaments. Calcium Hydroxide (CH) is limited by its prolonged treatment time, potential damage to Hertwig’s epithelial root sheath, and increased risk of root fracture.[7] Triple Antibiotic Paste (TAP), containing ciprofloxacin, metronidazole, and minocycline, has good efficacy but has been shown to discolor the crown, is cytotoxic and weaken the dentin.[8] The modified version (m-TAP), where minocycline is replaced with clindamycin, has been found to reduce staining and improve cell viability.[9]

Scaffolds, natural and synthetic provide a foundation for cell adhesion, proliferation, and differentiation encouraging apical closure, continued root development and lesion healing. They should have satisfactory mechanical properties, favorable degradation profile and the capability to incorporate growth factors or antimicrobials. Platelet-rich fibrin (PRF), a natural scaffold used in endodontic regeneration, is limited by its fast degradation rate and short working time.[10] Lyophilizing PRF (Ly-PRF) enhances its shelf life, preserves growth factor, and reduces immunogenicity due to leukocyte disruption.[11]

Drug delivery systems in the form of microparticles, nanoparticles, liposomes, and micelles, using natural polymers such as PLGA, gelatin, cyclodextrins, alginate, and hydroxyapatite have been investigated for achieving controlled and sustained release of drugs and/or growth factors.[12,13] Among these, β-Cyclodextrin based nanosponges show promise due to their porous, sponge-like structure suitable for drug entrapment and sustained release.[14]

A multifunctional scaffold that integrates antimicrobial agents with bioactive materials can be investigated to control endodontic infection and enhance regenerative outcomes.[15] The integration of β-cyclodextrin nanosponges with Ly-PRF, a source of active growth factors, and a modified triple antibiotic formulation represents a novel platform to simultaneously disinfect the root canal as well as promote regeneration. Our work with this scaffold has found it to be biocompatible with DPSCs and supported its proliferation and demonstrated effective antimicrobial activity against Streptococcus mutans, Enterococcus faecalis and Fusobacterium nucleatum.[16] The drug-loaded nanosponges (DLNS) requires further evaluation to characterize its physicochemical properties and to assess its bioactivity, mineralization, and odontogenic differentiation potential to support regeneration. This study aims to characterize the physicochemical properties of the DLNS with a focus on its potential for application in Regenerative Endodontics.

MATERIALS AND METHODS

The study design received approval from the University Ethics Committee for Human Trials (Reference No: EC-2021/PG/062). All procedures were performed in accordance with the guidelines and recommendations by the Occupational Safety and Health Administration and the Centre for Disease Control and Prevention. Informed consent was obtained from healthy volunteers before withdrawal of blood for PRF preparation.

Preparation of drug-loaded nanosponge

β-cyclodextrin (8.6 g) was dissolved in 50 mL dimethylformamide, followed by the addition of 5.66 mL glutaraldehyde (25%). The mixture was heated until dry, ground, rinsed with 30 mL deionized water, and air-dried for 2 days. It was then re-ground, suspended in water, and lyophilized (Christ Alpha 1-2 Ldplus, Germany) to obtain the nanosponge.[17] Venous blood from healthy volunteers was centrifuged at 3000 rpm for 10 min (Get Eltek Tc 650 D). The PRF layer was isolated, compressed, refrigerated overnight, freeze-dried for 8 h, ground into granules, and stored at 4°C.[11] The nanosponge (100 mg), lyophilized PRF (100 mg), ciprofloxacin, metronidazole, and clindamycin (each 100 mg) were mixed in distilled water (20 mL) and stirred for 24 h. After centrifugation (3000 rpm, 10 min), the supernatant was freeze-dried at −51°C and 13.33 mbar for 8 h to obtain DLNS.[11]

Scanning electron microscope analysis

The morphology and particle size of the plain nanosponge and DLNS were evaluated using high-resolution scanning electron microscopy (ESEM Quanta 200 FEI, USA). Samples were sputter coated with gold (Baltec SCD 500) at 40 mA for 25 s under vacuum and imaged at 15 kV at various magnifications (×100 and ×1000). Images were saved in TIFF format.

X-ray diffraction analysis

This analysis was performed on hydrated and dry DLNS powders using a Rigaku diffractometer (Geiger flex; Tokyo, Japan) with Cu-Kα radiation. Scans were taken from 10° to 60°, and crystallinity was compared using JCPDS database matching.

Particle size, polydispersity index

Particle size and polydispersity index (PDI) were measured using the 90 Plus dynamic light scattering (DLS) system (Brookhaven Instruments Corporation, USA). Samples were diluted to prevent multiple scattering and analyzed in a clean cuvette.[18]

Fourier transform infrared spectroscopy analysis

Fourier transform infrared spectroscopy (FTIR) (Alpha-II, Bruker, UK) was performed on pure antibiotics, unloaded nanosponges, and DLNS using the KBr pellet method.[19] Spectra were recorded from 400 to 4000 cm^⁻1. Samples (0.1–1.0%) were blended with 200–250 mg of KBr and compressed into 13 mm pellets under vacuum pressure (~8 tons). Peak shifts were evaluated to detect drug–polymer interactions.

Ultraviolet spectrophotometry

10 mg of DLNS was suspended in 10 mL Phosphate Buffer Saline (PBS) (pH 7.4) at 37°C. Samples were withdrawn at 2, 4, 6, and 8 h (short-term) and every 48 h for 14 days (long-term), replaced with fresh PBS each time and analysed using ultraviolet (UV) spectrophotometry (UV-1900i UV-Vis Spectrophotometer, Europe) at wavelength specific for each antibiotic, Ciprofloxacin 210 nm, Metronidazole 319 nm, and Clindamycin 210 nm, to evaluate in vitro drug release.[20]

pH measurement

DLNS was diluted in distilled water, and pH was measured using a calibrated digital pH meter (Systronics, Mk-VI). Measurements were taken using a refillable calomel electrode and cleaned between uses.

Calcium ion release

DLNS samples were digested using concentrated nitric acid and Milli-Q water (Merck Millipore, Mumbai), followed by inductively coupled plasma optical emission spectrometry (ICP-OES) analysis (Thermofisher ICAP 7400 ICP-OES, Radial N. America). Calcium concentrations were measured at 4 h, 7^th day, and 14^th day using calibration standards.[21]

Solubility

Pre-weighed DLNS (I = 200 mg) samples were immersed in 30 mL distilled water at 37°C for 24 h. It was suspended, dried, and final weights (D = 190.17 mg) were measured using an analytical balance with an accuracy of 0.001 g to calculate solubility percentage. All values were measured in triplicate.

Solubility (S) = ([I − D]/I) ×100 was calculated as a percentage of the original weight.[22]

Zeta potential evaluation

For measuring Zeta Potential, DLNS was diluted in 0.1 mol/L potassium chloride and loaded into the electrophoretic cell (Sigma-Aldrich Chemicals Pvt. Ltd.). An electric field (15 V/cm) was applied to measure particle mobility, from which Zeta potential was calculated using Zeta Phase Analysis Light Scattering instrument (Brookhaven Instruments Corporation, USA).[23]

Statistical analysis

Statistical analysis was performed using SPSS (version 25, IBM Corp., Armonk, NY: USA). All experiments were conducted in triplicate (n = 3), and the results were expressed as mean ± standard deviation. Normality and homogeneity of variance were assessed before analysis. One-way analysis of variance followed by Tukey’s HSD post hoc test was used to compare drug release and calcium ion release at different time intervals. P < 0.05 was considered statistically significant.

RESULTS

Scanning electron microscope analysis for morphology

It revealed that the nanosponges exhibited a porous, flaky, and irregular morphology characteristic of lyophilized polymeric matrices [Figure 1a and b]. The surface appeared rough with interconnected pores and layered structures, indicating successful formation of a sponge-like network. DLNS showed comparatively denser morphology with irregular crystalline and granular surface deposits, attributed to incorporated antibiotic and PRF residues. Higher magnification images demonstrated coarse surface texture with cavities and crevices, suggesting increased surface area favorable for drug entrapment and sustained release. The absence of major structural collapse indicated the stability of the nanosponge matrix after drug loading.

Figure 1.

(a) Representative scanning electron microscope (SEM) micrographs of Nanosponge surface topography. SEM magnifications: (×1500), (Bar = 10–20 µm), (b) Representative SEM micrographs of drug-loaded nanosponges (DLNS) surface topography. SEM magnifications: (×1000), (Bar = 10–100 µm), (c) X-ray diffraction analysis of DLNS, and (d) Particle size of DLNS

X-ray diffraction analysis

The X-ray diffraction (XRD) pattern of the DLNS demonstrated a broad and diffused halo pattern without distinct sharp crystalline peaks, indicating the predominantly amorphous nature of the formulation [Figure 1c]. The absence of characteristic intense diffraction peaks suggests successful crosslinking and molecular dispersion of the incorporated components within the polymeric matrix during the lyophilization process. This may enhance drug entrapment and dissolution characteristics of the nanosponges.

Particle size and polydispersity index

DLS analysis demonstrated a mean particle size of 458 nm with a log-normal size distribution pattern, indicating successful formation of nanosized particles [Figure 1d]. The particle distribution curve showed a relatively narrow spread with gradual intensity variation, suggesting homogeneous dispersion of the nanosponges in the suspension. The PDI of 0.344 indicated a moderately uniform particle size distribution with acceptable colloidal homogeneity. The absence of multiple distinct peaks further suggests minimal particle aggregation and stable nanosponge formation. Overall, the DLS findings confirm the production of uniformly distributed DLNS within the nanometric range suitable for drug delivery applications.

Drug polymer interaction using Fourier transform infrared spectroscopy

FTIR analysis revealed characteristic functional group peaks of the incorporated drugs and polymeric components [Figure 2a-e]. Broad O–H stretching vibrations were observed at 3261 cm⁻¹ for clindamycin and 3043 cm⁻¹ for metronidazole and ciprofloxacin, confirming the presence of hydroxyl-containing groups. A shift in the C = O stretching vibration from 1658.37 cm⁻¹ in nanosponges to 1690.12 cm⁻¹ in DLNS indicated possible intermolecular interactions and successful drug encapsulation within the polymeric matrix. Minor peak broadening and intensity variations further supported drug–polymer interaction without significant chemical degradation.

Figure 2.

(a) Fourier transform infrared spectroscopy analysis of clindamycin, (b) metronidazole, (c) ciprofloxacin, (d) nanosponge, (e) drug-loaded nanosponges (DLNS), and (f) zeta potential of DLNS

In-vitro drug release using ultraviolet spectrophotometry

In-vitro drug release analysis using UV spectrophotometry demonstrated both short-and long-term sustained release patterns from the DLNS [Table 1]. An initial gradual release was observed within the first 8 h, followed by a sustained increase in drug concentration over 14 days. Ciprofloxacin, metronidazole, and clindamycin exhibited progressive release profiles, reaching peak concentrations on day 10 of 21.53 µg/mL, 25.21 µg/mL, and 29.09 µg/mL respectively. Thereafter, a plateau phase was observed between days 12 and 14, indicating controlled and sustained drug release from the nanosponge matrix. The prolonged release behavior suggests effective drug entrapment and diffusion-mediated release characteristics of the formulation.

Table 1.

Drug release from the drug-loaded nanosponge

Time	Ciprofloxacin (µg/mL), mean±SD	Metronidazole (µg/mL), mean±SD	Clindamycin (µg/mL), mean±SD
0 h	0.00±0.00	0.00±0.00	0.00±0.00
2 h	1.22±0.08ᵃ	0.85±0.05ᵃ	0.56±0.03ᵃ
4 h	1.87±0.11ᵇ	1.23±0.08ᵇ	1.01±0.07ᵇ
6 h	1.88±0.10ᵇ	1.89±0.12ᶜ	1.52±0.09ᶜ
8 h	2.43±0.15ᶜ	2.38±0.14ᵈ	2.01±0.12ᵈ
2 days	15.23±0.84ᵈ	18.56±0.96ᵉ	24.74±1.12ᵉ
4 days	17.98±0.91ᵉ	21.34±1.04ᶠ	28.06±1.23ᶠ
6 days	18.41±0.95ᵉ	22.69±1.08ᶠ	28.33±1.28ᶠ
8 days	20.01±1.02ᶠ	24.07±1.16ᵍ	28.78±1.31ᶠ
10 days	21.53±1.08ᵍ	25.21±1.21ʰ	29.09±1.35ᵍ
12 days	21.67±1.10ᵍ	25.77±1.24ʰ	29.53±1.39ᵍ
14 days	22.02±1.14ᵍ	25.96±1.27ʰ	29.84±1.42ᵍ

Different superscript letters within the same column indicate statistically significant differences (P<0.05). SD: Standard deviation

pH analysis

The pH evaluation demonstrated good stability of both unloaded nanosponges and DLNS over the 7-day observation period. DLNS maintained a stable pH range between 6.80 and 6.84, while unloaded nanosponges exhibited slightly higher values ranging from 6.96 to 7.03. Minimal variation in pH throughout the study period indicates physicochemical stability of the formulations without significant degradation or alteration of the polymeric matrix. The near-neutral pH of both formulations suggests their suitability for biomedical and local drug delivery applications with minimal risk of tissue irritation.

Calcium ion release

Calcium ion release analysis demonstrated an initial release of 1862 mg/L at 4 h, followed by a marked increase reaching a peak value of 3935 mg/L on day 7 [Table 2]. Subsequently, the calcium ion concentration decreased to 1100 mg/L by day 14. The elevated release observed at day 7 suggests active ion diffusion and material interaction during the intermediate phase, whereas the later decline may indicate gradual depletion of available calcium ions from the matrix. Statistical analysis revealed a significant difference (P < 0.05) between the day 7 values and other evaluated time points, indicating a time-dependent calcium ion release pattern.

Table 2.

Mean calcium ion release from the drug-loaded nanosponge

Time interval	Calcium ion release (mg/L), mean±SD
4 h	1862±95ᵃ
7 days	3935±121ᵇ
14 days	1100±72ᶜ

Different superscript letters indicate statistically significant differences (P<0.05). SD: Standard deviation

Solubility

DLNS exhibited limited aqueous solubility of 4.91%, indicating low water uptake and good structural stability of the nanosponge matrix. The reduced solubility suggests effective crosslinking and compact polymeric network formation, which may contribute to controlled swelling and slower drug diffusion. Such limited solubility is advantageous for sustained-release applications, as it helps maintain gradual and prolonged release of the incorporated drugs over an extended period. The findings further support the suitability of DLNS as a stable local drug delivery system.

Zeta potential

Zeta potential analysis revealed a mean surface charge of –24.32 ± 1.8 mV (95% confidence interval: −26.1 to −22.5 mV) indicating moderate colloidal stability of the DLNS formulation [Figure 2f]. The negative surface charge suggests adequate electrostatic repulsion between particles, which may help reduce particle aggregation and maintain dispersion stability. The obtained zeta potential value further indicates stable nanosponge formation with acceptable suspension characteristics suitable for drug delivery applications.

DISCUSSION

In the present study, β-cyclodextrin nanosponge integrated with m-TAP and Ly-PRF was characterized based on various parameters to assess its capacity to support endodontic regeneration. The integration of Ly-PRF within the nanosponge provides a sustained source of growth factors, which are critical for promoting stem cell proliferation, differentiation, and tissue regeneration. Lyophilization preserves the biological activity of PRF while increasing shelf life; however, its inherent porous structure can accelerate degradation. Encapsulation within the nanosponge potentially mitigates this limitation by offering a protective matrix that allows gradual release of growth factors, enhancing their bioavailability in the root canal environment.[24,25,26]

Controlled drug delivery is essential in regenerative Endodontics to eradicate residual microbial infection without compromising the viability of stem cells. Conventional CH and traditional TAP present limitations such as cytotoxicity, prolonged treatment time, crown discoloration, and weakening of dentin. The use of a modified TAP (clindamycin replacing minocycline) combined with the nanosponge addresses these challenges by reducing cytotoxic effects and providing sustained antimicrobial activity. The porous architecture of nanosponges facilitates drug entrapment and controlled release, as reflected in the observed surface morphology.[27,28]

Scanning electron microscope analysis demonstrated a porous, flaky morphology typical of lyophilized polymeric matrices, and particle sizes correlated with DLS analysis, demonstrated a mean particle size of 458 nm which is advantageous for high drug-loading and sustained release. Nanoparticles within the 100–500 nm range are known to penetrate dentinal tubules (mean diameter of 0.6–0.9 micrometers) efficiently. Moreover, consistent particle sizing ensures predictable pharmacokinetics and minimizes aggregation, contributing to formulation stability and therapeutic efficacy. XRD analysis revealed an amorphous nature attributed to crosslinking and the lyophilization process. Amorphous carriers are reported to enhance dissolution behavior and drug dispersion.[29] The PDI value of 0.344 indicates moderate uniformity in particle size distribution, which is acceptable for drug delivery systems, particularly for cyclodextrin-based nanosponges. While values below 0.3 are ideal, a PDI between 0.3 and 0.5 is still considered usable, suggesting the formulation is relatively stable and capable of effective penetration into dentinal tubules for sustained drug action. This range ensures predictable release kinetics and reduces the risk of aggregation during storage or clinical application.[18] In addition, controlled particle size distribution enhances mucoadhesion and interaction with dentin surfaces, critical for prolonged therapeutic efficacy in endodontics.[30]

FTIR spectroscopy confirmed successful fabrication and loading of the nanosponge-based delivery system. The presence of characteristic peaks of clindamycin, metronidazole, and ciprofloxacin validated drug identity, whereas the nanosponge spectrum showed distinct shifts in O–H and C = O bands consistent with β-cyclodextrin crosslinking. The presence of pure drug peaks indicates preserved structural integrity, with no significant changes in drug or polymer functional groups. The observed peak shifts suggest weak intermolecular interactions and successful encapsulation without chemical degradation of the drugs. These findings further support the physicochemical stability of the formulation. The FTIR spectra confirm nanosponge formation, protein presence in PRF, and characteristic drug peaks, with the combined formulation showing compatibility and absence of strong drug–polymer interactions[19] [Figure 2a-e].

Drug release from DLNS was monitored over 14 days. An immediate release was observed, followed by a noticeable burst phase during the first 10 days. Peak release of clindamycin, metronidazole, and ciprofloxacin was observed by day 10, after which the release profile plateaued. The initial burst was attributed to the antibiotics’ water solubility and increased hydrophilicity, followed by a sustained and gradual release. Release rates varied among the drugs, with ciprofloxacin exhibiting comparatively slower release, possibly due to differences in molecular interaction and diffusion within the polymeric matrix.[31] The early burst release is advantageous in rapidly reducing the bacterial load and the controlled release suppresses the residual microorganisms especially those within the dentinal tubules.

In biological systems, pH influences microbial activity, drug stability, molecular function, tissue response, and regeneration. In this study, the pH of plain nanosponge and DLNS ranged from 6.96–7.03 to 6.80–6.84, respectively, with no significant variation over 7 days. These values align with typical peripheral pH levels (6–7.4). Previous research supports enhanced antimicrobial effects through combination therapy.[32] Since E. faecalis can survive highly alkaline conditions (pH ≈ 11), using a combination of antibiotics can enhance antimicrobial efficacy against resistant species.

ICP-OES analysis revealed an initial increase in Ca²⁺ release, reaching a peak followed by a gradual decline, indicating rapid initial dissolution followed by sustained and then diminishing release as calcium sources deplete. Controlled Ca²⁺ release supports tissue regeneration and biocompatibility without inducing toxicity. This release profile is crucial for maintaining ionic balance in the periapical environment, facilitating cellular proliferation and differentiation.[33]

The DLNS exhibited limited aqueous solubility (4.91%) in distilled water, an ideal feature for sustained drug release. Reduced solubility minimizes systemic absorption and supports localized therapeutic action by prolonged drug retention at the target site, enhancing the availability of the drug at the site while reducing the frequency of administration. Low solubility carriers have been shown to maintain therapeutic levels over extended periods, improving clinical efficacy.[34]

Zeta potential is a measure of surface charge on particles and is a critical indicator of colloidal stability. The DLNS presented a zeta potential of −24.32 mV, suggesting moderate colloidal stability, confirming the formulation’s structural integrity and adaptability for the root canal environment. Particles with zeta potential more negative than -20 mV typically exhibit sufficient electrostatic repulsion to resist aggregation, enhancing shelf-life and dispersion. Moreover, optimal zeta potential supports uniform dispersion within the root canal, improving deeper penetration within dentinal tubules to maximize therapeutic efficiency.[35]

The multifunctional scaffold has proven to be compatible with dental pulp stem cells and exhibited antimicrobial efficacy against clinically relevant endodontic pathogens. Such a strategy addresses the limitations of current regenerative approaches, which rely heavily on intracanal bleeding to recruit stem cells and growth factors, a process that often yields variable outcomes. By integrating antimicrobial and regenerative features within a single delivery platform, the present system offers a more predictable environment for pulp-dentin regeneration.

Future investigations should focus on in vitro and in vivo assessment of growth factor release kinetics, its impact on endodontic biofilm, discoloration risk, and compatibility with irrigants and biomaterials to ensure its clinical applicability. In addition, in vivo studies and well-designed clinical trials are essential to confirm the long-term safety and efficacy of DLNS in supporting regeneration.

CONCLUSION

The β-cyclodextrin nanosponge-based scaffold incorporating Ly-PRF and m-TAP represents a promising multifunctional system capable of simultaneously promoting endodontic regeneration and controlling infection. Its physicochemical properties and morphology support its potential as an advanced scaffold for regenerative endodontic therapies.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This research received funding from RUAS Seed Grant under Grant Agreement No ORI-SG/FDS/002/2023.