Stimuli-responsive chitosan-coated ferrite nanocarriers for targeted capsaicin delivery and core-dependent HepG2-selective bioactivity
Eman Bakr, Fawzya I. Elshami, Ehab A. Okba, Hanaa Mansour, Shaban Y. Shaban
TL;DR
Researchers developed smart nanocarriers that release capsaicin in tumor-like conditions, improving its cancer-fighting potential while reducing side effects.
Contribution
The study introduces pH- and glutathione-responsive ferrite-chitosan nanocarriers that enhance capsaicin's bioavailability and tumor selectivity.
Findings
Capsaicin-loaded nanocarriers showed controlled release under tumor-like conditions and improved cytotoxicity against liver cancer cells.
Manganese ferrite-based nanocarriers outperformed zinc ferrite in therapeutic index while maintaining safety for normal cells.
Ferrite core composition influences DNA binding and stability, linking material design to biological outcomes.
Abstract
Capsaicin is a promising anticancer agent, but its clinical translation is hindered by poor aqueous solubility, low bioavailability, rapid clearance, and dose-limiting irritation, which restrict sustained exposure at tumor sites. Existing formulations only partially overcome these limitations and often lack tumor-microenvironment–responsive release or a clear understanding of how carrier composition modulates biological outcomes. Here, chitosan-coated zinc ferrite (ZFO@CS) and manganese ferrite (MFO@CS) nanocarriers were developed as pH- and glutathione-responsive platforms for capsaicin delivery. The nanocarriers exhibited nanoscale hydrodynamic diameters (~ 120–500 nm) and highly positive zeta potentials (+ 30 to + 50 mV), enabling high encapsulation efficiencies (up to ~ 88%) and colloidal stability. Under physiological pH 7.4, less than 10% of the loaded drug was released over 48 h,…
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Figure 8- —Kafr El Shiekh University
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Taxonomy
TopicsNanoparticle-Based Drug Delivery · Nanoplatforms for cancer theranostics · Magnetic Properties and Synthesis of Ferrites
Introduction
Nanotechnology has revolutionized cancer treatment by enabling precise drug delivery, minimizing toxicity, and enhancing therapeutic efficacy. Unlike traditional chemotherapy, which often lacks specificity and causes collateral damage to healthy tissues, nanotechnology offers innovative solutions through targeted drug delivery systems. Smart nanocarriers, engineered to respond to specific cues in the tumor microenvironment, such as acidic pH, elevated glutathione levels, or enzymatic activity, facilitate controlled and localized drug release, improving treatment outcomes while reducing systemic side effects^1–3^.
Among promising therapeutic agents, capsaicin—a bioactive compound derived from chili peppers, has garnered attention for its potent anticancer properties. Capsaicin inhibits tumor growth, angiogenesis, and metastasis by inducing apoptosis, modulating cell signaling pathways, and enhancing chemosensitivity in various cancer models^4–6^. Despite its potential, capsaicin’s clinical application is hindered by challenges such as low bioavailability, a short half-life, and adverse effects like gastrointestinal irritation and pungency. To overcome these limitations, strategies including sustained-release formulations, combination therapies, and nanoparticle encapsulation have been explored. Notably, chitosan-based nanoparticles have shown promise in improving capsaicin’s solubility, stability, and safety profile while reducing its toxicity^5–7^. However, existing capsaicin delivery systems still face significant challenges. Many lack robust, tumor-microenvironment–responsive release mechanisms, and there is limited understanding of how the composition of the nanocarrier core influences the biological responses of encapsulated capsaicin. In particular, systematic studies comparing different ferrite cores for their effects on stimuli-responsive release, DNA binding, and selective cytotoxicity in liver cancer models are scarce^8–15^.
Chitosan is a biodegradable, biocompatible cationic polysaccharide whose protonatable amino groups enable mucoadhesion, improved solubility of hydrophobic drugs, and pH-dependent swelling and permeability. In drug delivery, chitosan-based nanocarriers have been widely used to enhance oral bioavailability, prolong circulation, and reduce systemic toxicity of encapsulated compounds. For capsaicin, chitosan matrices can improve aqueous dispersion, shield pungency-related irritation, and exploit acidic tumor environments to trigger enhanced release .
Ferrite nanoparticles, particularly zinc ferrite and manganese ferrite, offer additional advantages for biomedical applications, including low intrinsic toxicity, tunable magnetic and redox properties, and high surface area for drug loading and functionalization. Coating ferrites with chitosan mitigates their tendency to aggregate, improves colloidal stability and hemocompatibility, and introduces a cationic shell that promotes interaction with negatively charged cell membranes and DNA^8,9,13–15^. However, how the ferrite core composition (zinc vs. manganese) modulates capsaicin loading, pH/glutathione-responsive release, DNA binding, and differential cytotoxicity toward liver cancer cells has not been systematically investigated^13–15^. Therefore, there is a need for a comparative, composition-dependent study of chitosan-coated zinc and manganese ferrite nanocarriers for capsaicin delivery, integrating physicochemical characterization with DNA interaction and bioactivity assays.
In this work, chitosan-coated zinc ferrite (ZFO@CS) and manganese ferrite (MFO@CS) nanocarriers were synthesized and loaded with capsaicin. The systems were characterized in terms of structure, size, surface charge, loading efficiency, and pH/glutathione-responsive release, and their hemocompatibility, cytotoxicity toward HepG2 and WI-38 cells, antibacterial and antioxidant activities. Additionally, we examined capsaicin, ZFO@CS, and MFO@CS nanocarriers, as well as their capsaicin-loaded forms (CAP-ZFO@CS and CAP-MFO@CS), for DNA binding to elucidate their anticancer mechanisms. Fluorescence and electronic absorption spectroscopy were used to analyze DNA interactions. Kinetic studies of ctDNA with ZFO@CS, MFO@CS, and their capsaicin-loaded forms were conducted using the stopped-flow technique, previously applied in related studies^16–19^, to detect transient intermediates by rapid mixing and immediate measurement. We assessed drug affinity, kinetic stability of DNA–drug complexes, and proposed an interaction mechanism to enhance drug design targeting cellular DNA. The goal is to elucidate how ferrite core composition governs stimuli-responsive release and biological responses of capsaicin nanocarriers, providing design guidelines for safer and more selective liver cancer therapy.
Materials and methods
Materials
Ascorbic acid, gentamicin, and methyl thiazolyl tetrazolium (MTT) were obtained from Bio Basic Inc. (Toronto, ON, Canada). Muller-Hinton Agar, 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H₂O₂), and phosphate-buffered saline (PBS) were sourced from Sigma Aldrich Manufacturing LLC (Saint Louis, MO, USA), along with acetic acid, calf thymus DNA (ct-DNA), and other reagents. Chitosan (C₅₆H₁₀₃N₉O₃₉) with an 80% degree of deacetylation and molecular weight of 400,000, as well as iron(III) nitrate nonahydrate [Fe(NO₃)₃·9 H₂O], zinc nitrate hexahydrate [Zn(NO₃)₂·6 H₂O], and manganese nitrate hexahydrate [Mn(NO₃)₂·6 H₂O], were procured from Haixin Biological Product Co., Ltd. (Qufu, China). Human lung fibroblast (WI-38) and liver cancer (HepG2) cell lines were provided by the Holding Company for Biological Products and Vaccines, Dokki, Giza, Egypt. All chemicals used were of analytical grade and utilized as received without further purification. Capsaicin was extracted from spicy peppers (Capsicum annuum ssp.) using ethanol according to previously reported methods^20^.
Synthesis of ZFO@CS and MFO@CS NCs
The nanocarriers were synthesized via an in situ co-precipitation method incorporating tripolyphosphate (TPP) as a crosslinking agent for chitosan, which promotes nanoparticle formation and stability^21^. Initially, 0.375 g of chitosan was dissolved in 30 mL of 4% glacial acetic acid and stirred for 1 h to achieve complete solubilization. The pH was adjusted to 5.0 using 10 M NaOH to optimize the ionic environment for subsequent reactions. A solution of either Zn(NO₃)₂·6 H₂O (0.31 g, 1.05 mmol) or Mn(NO₃)₂·6 H₂O (0.31 g, 1.05 mmol) combined with Fe(NO₃)₃·9 H₂O (1.70 g, 4.2 mmol) was added to the chitosan mixture. Ammonia solution was introduced dropwise to facilitate nucleation and control particle size, aiming for smaller, uniform nanoparticles. Then, 5 mL of 3.0% TPP solution was added under vigorous stirring at room temperature, and the reaction proceeded for 48 h to ensure thorough crosslinking and ferrite incorporation. The resulting orange (ZFO@CS) or dark brown (MFO@CS) precipitates were washed extensively with deionized water, collected via centrifugation to remove impurities, and calcination at 300 °C for 3 h, yielding stable, powdered nanocarriers ready for further processing.
CAP encapsulation
To achieve efficient drug loading, an aqueous solution of 0.1 g CAP dissolved in 2 mL ethanol was prepared and added to freshly synthesized ZFO@CS or MFO@CS NCs. The mixture underwent sonication for 60 min to promote uniform dispersion and enhance drug-nanoparticle interactions through acoustic cavitation. This was followed by additional stirring for 30 min to complete encapsulation, leveraging electrostatic and hydrogen bonding between CAP and the chitosan matrix. The CAP-loaded products were then separated by centrifugation to isolate the nanocarriers and dried under vacuum to remove residual solvents, resulting in stable formulations suitable for biological testing.
Characterization of nanoparticles
Comprehensive characterization was performed to confirm structure, size, and stability. UV-Vis spectroscopy used a PEAK USA model C7000V spectrophotometer over 190–1100 nm for electronic transitions and release studies. Morphology and elemental composition were analyzed via Jeol JSM T-300 scanning electron microscope (SEM) with energy-dispersive X-ray spectroscopy (EDS); samples were drop-cast on glass slides, platinum-coated for conductivity. Dynamic light scattering (DLS) with a Nanoplus analyzer assessed size distribution after 5-minute sonication in quartz cuvettes. X-ray diffraction (XRD) employed a Shimadzu diffractometer (Cu-Kα radiation, 30 mA/40 kV, 8°/min scan, 5–80° 2θ) to evaluate crystallinity. Fourier Transform Infrared (FT-IR) spectroscopy via Perkin-Elmer with KBr beam splitter identified functional groups. Zeta potential, measuring surface charge and stability, was determined using a Brookhaven Instruments analyzer at 0.05 mg/mL in deionized water.
Biocompatibility (Hemolysis)
Hemocompatibility was assessed using UV-Vis spectrophotometry to quantify hemoglobin release. Whole blood from healthy donors was collected in EDTA tubes and centrifuged at 4000 rpm for 15 min to isolate red blood cells (RBCs), which were resuspended in 15 mL PBS. Test samples (200 µL at 10, 50, or 100 µg/mL) were mixed with 800 µL RBC suspension and incubated at 37 °C for 3 h. Positive control used deionized water for complete lysis, negative control PBS for baseline. After centrifugation at 5000 rpm for 5 min, supernatant absorbance was measured at 540 nm. The hemolysis ratio (HR) was calculated using the formula^22,23^:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}\:\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\:\left({\%}\right)=\frac{{\mathrm{D}}_{\mathrm{t}}-{\mathrm{D}}_{\mathrm{n}\mathrm{c}}}{{\mathrm{D}}_{\mathrm{p}\mathrm{c}}-{\mathrm{D}}_{\mathrm{n}\mathrm{c}}}\:\times\:\:100$$\end{document}Where D_𝑡_, D_nc_, and D_pc_ are absorbance of samples, negative controls, and positive controls, respectively.
In vitro cytotoxicity assay
Cytotoxicity was evaluated using the MTT assay, a standard colorimetric method that quantifies mitochondrial dehydrogenase activity in viable cells by reducing yellow MTT to purple formazan^24^. HepG2 and WI-38 cells were seeded at 1 × 10 ^ 5^ cells per well in 100 µL growth medium in 96-well plates and incubated at 37 °C for 24 h to form confluent monolayers. Medium was removed, cells washed twice with PBS to eliminate debris, and test compounds (CAP, ZFO@CS NCs, CAP-ZFO@CS NCs, MFO@CS NCs, CAP-MFO@CS NCs) added in 100 µL serial dilutions (0–1000 µg/mL). Control wells received only medium. Plates were incubated at 37 °C for 24 h, with periodic microscopic observation for morphological changes indicative of toxicity, such as cell rounding, shrinkage, granulation, or monolayer detachment. Subsequently, 20 µL of 5 mg/mL MTT in sterile PBS was added per well, followed by incubation at 37 °C with 5% CO_2_ for 4 h to allow formazan crystal formation. Medium was aspirated; crystals dissolved in 200 µL DMSO, and plates shaken at 150 rpm for 5 min to homogenize. Absorbance was measured at 570 nm (reference 630 nm) using an ELISA reader (StatFax-2100, Awareness Technology Inc., Palm City, FL, USA).
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:Cell\ viability\:\left(\%\right)\:was\ calculated\ as:\:100\:-\:100\:\times\:\:(\boldsymbol{T}-{\boldsymbol{T}}_{\boldsymbol{O}})/(\boldsymbol{C}-{\boldsymbol{T}}_{\boldsymbol{O}})$$\end{document}Where T is the optical density of the test sample, T_0_ is the optical density at time zero, and C is the optical density of the control. The optical density and cell count ought to be proportionate.
Antibacterial activity
Antibacterial efficacy was tested against Gram-positive (Staphylococcus aureus,* Bacillus subtilis*,* Enterococcus faecalis*) and Gram-negative (Escherichia coli,* Klebsiella pneumoniae*,* Salmonella typhi*) bacteria using the agar well-diffusion method on Muller Hinton agar (MHA)^25,26^. Bacterial cultures were spread on MHA plates, 6 mm wells created, and filled with test materials at varying concentrations. Plates were incubated at 37 °C for 24 h, and inhibition zones measured to assess qualitative activity. Gentamicin served as positive control, with blanks as negative. For quantitative evaluation, broth microdilution involved mixing 100 µL nanoparticle dilutions with 100 µL bacterial suspension (~ 10^8^ CFU/mL) in 96-well plates with Muller Hinton broth, incubating at 37 °C for 24 h. Aliquots were plated on fresh MHA and incubated again to count colonies, providing inhibition profiles. This dual approach yielded comprehensive data on zone sizes and growth inhibition, highlighting synergies between components.
Antioxidant activity
Modified DPPH free radical scavenging assay was used to assess the antioxidant capacity of Capsaicin, ZFO@CS NCs, MFO@CS NCs, CAP-ZFO@CS NCs and CAP-MFO@CS NCs. Initially, 100 µL of the nanoparticles was combined with 1 mL of a 0.1 mM ethanolic DPPH solution^27^. The mixture was shaken thoroughly and then allowed to stand at room temperature for 30 min. A UV-VIS Milton Roy spectrophotometer was then used to measure the absorbance at 517 nm. Ascorbic acid was used as the reference standard component in the triplicate experiment. The sample’s IC_50_ value, or the concentration of the sample required to inhibit 50% of the DPPH free radical, was calculated using a log dosage inhibition curve. The reaction mixture’s lower absorbance indicated higher levels of free radical activity. The following formula was used to determine the percentage of DPPH scavenging effect.
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\ \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\:\left({\%}\right)=\frac{{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}-{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}\:\times\:\:100$$\end{document}Where A_blank_was the Absorbance of control reaction and A_sample_ was the Absorbance in presence of test or standard sample.
In vitro drug release
Release profiles were studied using dialysis to simulate physiological conditions, emphasizing the role of nanocarriers in retaining drugs during circulation while enabling extravasation via enhanced permeability and retention (EPR) effect. Given higher GSH levels in tumor cells (4–5 times normal, 2–10 mM cytoplasm), media tested included pH 7.4, and pH 5.4 with 10 mM GSH at 37 °C. 5 mL of CAP-loaded NCs (0.015 g) were dialyzed (MWCO 8000–12,000 Da) against 150 mL PBS at 100 rpm^28^. 2 mL aliquots were withdrawn periodically, replaced with fresh buffer, and CAP quantified via UV-Vis at 290 nm. The cumulative release (%) was then calculated using equation:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${C}\mathbf{u}\mathbf{m}\mathbf{u}\mathbf{l}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{v}\mathbf{e}\ \mathbf{d}\mathbf{r}\mathbf{u}\mathbf{g}\ \mathbf{r}\mathbf{e}\mathbf{l}\mathbf{e}\mathbf{a}\mathbf{s}\mathbf{e}\:\:\left({\%}\right)=\frac{{\mathbf{M}}_{\mathbf{I}}}{{\mathbf{M}}_{\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l}}}\:\times\:\:100$$\end{document}Here, M_i_ stands for the amount of drug in the release medium at time t as determined by a UV-vis spectrophotometer, and M_total_ for the total amount of drug loaded into the nanocarrier.
Loading efficiency
To determine the loading efficiency, free CAP was separated from CAP-ZFO@CS NCs, CAP-MFO@CS NCs by centrifugation at 12,000 rpm for 30 min at 4 °C. This method relies on the difference in sedimentation behavior between the nanocomposite particles and free CAP molecules (which remain in the supernatant due to their much smaller size and lack of aggregation). The loading efficiency was calculated by measuring the absorbance of free CAP in the supernatant at 289 nm and comparing it with the total CAP initially added, using the formula^29^:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E\:\left({\%}\right)=\:\left(\frac{{A}_{o}-\:{A}_{t}}{{A}_{o}}\right)\times\:\:100$$\end{document}where A_o_ is the absorbance of the total CAP solution and A_t_ is the absorbance of the supernatant after centrifugation.
DNA binding
ct-DNA stock was prepared in phosphate buffer (pH 7.4) with concentration via ε_260_ = 6600 M^-1^ cm^-1^. NCs dispersed in acidic aqueous solutions (e.g., acetic acid) or ethanol for CAP.
UV-Vis spectroscopy
Spectra of fixed DNA (1 × 10^− 4^ M) in 20 mM phosphate buffer (pH 7.4) were recorded (200–700 nm, 25 °C) with titrated compounds. Binding constants (Kb) calculated via Benesi-Hildebrand; Gibbs free energy.
Fluorescence spectroscopy
The DNA used in the experiments had an excitation wavelength of 280 nm and an emission scan between 400 and 550 nm. At room temperature (25 °C), fluorescence spectra were obtained using quartz cells (1.0 cm) and a Jasco fluorescence spectrophotometer model FP-8500. Following this, progressively higher concentrations of capsaicin, ZFO@CS NCs, MFO@CS NCs, Cap**-ZFO@CS NCs, and Cap-**MFO@CS NCs were added. The quenching constant was then calculated using the Stern-Volmer plot that was produced using the change in fluorescence intensity. The binding constant and number of binding sites were obtained by creating a double logarithmic graph from fluorescence data.
Stopped-flow kinetics
Kinetic analyses were conducted utilizing an Applied KinetAsyst SF-61DX2 stopped-flow spectrophotometer (HI-Tech Scientific) fitted with a Peltier temperature control system. Experimental solutions contained calf thymus DNA (CT-DNA) prepared in phosphate-buffered saline (PBS) alongside graded concentrations of capsaicin (CAP), zinc ferrite-core chitosan nanocarriers (ZFO@CS NCs), manganese ferrite-core chitosan nanocarriers (MFO@CS NCs), and their respective capsaicin-loaded counterparts. For each trial, syringes were loaded with either CT-DNA solution or test samples, followed by rapid mixing in the observation chamber via simultaneous injection of equal volumes. Time-resolved absorption spectra were acquired immediately prior to mixing (t = 0 s) and at sequential time intervals post-injection, with the instrument demonstrating a 2 ms dead time for 1:1 volumetric mixtures. Control experiments involving mixture of buffered sample solutions with CT-DNA exhibited no significant alterations in absorption profiles, thereby excluding nonspecific degradation artifacts. Binding kinetics were assessed through monitoring of hyperchromic shifts at 320 nm, with resultant traces subjected to quantitative analysis to characterize binding interactions and elucidate the underlying reaction mechanism.
Statistical analysis
All experiments in triplicate, data as mean ± SD. Analyzed via one-way ANOVA in SPSS v14 (IBM, USA); p < 0.05 considered significant.
Results and discussion
Synthesis and physicochemical characterization
UV-visible spectroscopy reveals the electronic structure of capsaicin (CAP), MFO@CS NCs, ZFO@CS NCs, and their capsaicin-encapsulated forms. Pure CAP shows absorption peaks at 290, 316, 416, and 480 nm due to its conjugated aromatic system (Fig. 1A). In MFO@CS NCs, peaks shift to 298, 337, 403, and 471 nm, and in CAP-MFO@CS NCs to 297, 337, 404, and 467 nm. ZFO@CS NCs exhibit peaks at 302, 336, and 402 nm, shifting to 300, 333, 406, and 465 nm in CAP-ZFO@CS NCs, indicating nanoparticle surface effects and encapsulation-induced changes. The strong UV band (280–300 nm) results from O²⁻ → Fe³⁺ charge transfer in the spinel lattice and chitosan’s –OH and –NH₂ groups, while the visible region (400–470 nm) corresponds to Fe²⁺ d–d transitions^30^.
FTIR analysis (Fig. 1B) confirms the composition of the samples. Pure capsaicin shows peaks for -OH (3403 cm⁻¹), aromatic C-H (3008 cm⁻¹), CH₂ (2854 cm⁻¹), and C = O (1642, 1737 cm⁻¹), reflecting its phenolic, amide, and carbonyl groups. ZFO@CS NCs exhibit -OH (3388 cm⁻¹), amide I (1634 cm⁻¹), C-O-C (1071 cm⁻¹), and metal-oxygen (469, 621 cm⁻¹) bands, indicating zinc ferrite-chitosan interactions^21,31,32^. MFO@CS NCs show similar bands at 3384, 1645, 1067, 459, and 613 cm⁻¹, reflecting manganese ferrite-chitosan interactions^33^. Electrostatic interactions between chitosan’s positive charge and the negative charge of ferrite nanoparticles enable coating^34^. CAP-ZFO@CS NCs and CAP-MFO@CS NCs show broadened -OH (3395, 3398 cm⁻¹), shifted amide (1629, 1630 cm⁻¹), and C = O (1728, 1735 cm⁻¹) peaks, confirming encapsulation. Hydrogen bonding between capsaicin’s C = O and –OH and chitosan’s –OH and NH₂ groups, along with a new peak at 1727 cm⁻¹, indicates molecular interactions^35,36^. These results confirm successful synthesis and structural integration of the nanocarriers.
The X-ray diffraction (XRD) patterns of ZFO@CS and MFO@CS nanocarriers (Fig. 1C) confirm the formation of cubic spinel ferrite phases (space group Fd-3 m), matching JCPDS patterns for ZnFe₂O₄ (22-1012) and MnFe₂O₄ (10–0319)^11,29,37^. ZFO@CS shows sharp peaks at 2θ ≈ 30.1° (220), 35.4° (311), 43.0° (400), 53.4° (422), 56.9° (511), and 62.5° (440), with the dominant (311) reflection indicating high crystallinity. In contrast, MFO@CS exhibits broader, less intense peaks at the same positions, suggesting smaller crystallite size and lower crystallinity due to greater lattice disorder in MnFe₂O₄ from partial inversion and cation distribution effects. No impurity peaks are observed, confirming phase purity, while the chitosan coating contributes a broad background hump (15–25°) without altering the ferrite structure. Overall, ZFO@CS displays superior crystallinity compared to MFO@CS, potentially impacting their magnetic and biomedical properties.
The zeta potential data (Fig. 1D; Table 1) reveal the surface charge and colloidal stability of capsaicin (CAP) and its encapsulated nanocarriers. Higher absolute zeta potential values indicate better dispersion and reduced aggregation due to electrostatic repulsion^38,39^. Capsaicin alone has a near-neutral zeta potential (0 ± 0.21 mV), leading to poor stability and aggregation in aqueous environments. ZFO@CS NCs show a high positive zeta potential (+ 34.89 ± 0.31 mV) due to protonated chitosan amino groups, enhancing interactions with negatively charged biomolecules. CAP-ZFO@CS NCs exhibit an even higher value (+ 53.2 ± 1.61 mV), indicating superior stability. MFO@CS NCs have a slightly lower zeta potential (+ 28.24 ± 1.56 mV), likely due to manganese ferrite’s surface chemistry, while CAP-MFO@CS NCs show a moderate increase (+ 32.34 ± 1.02 mV). Capsaicin encapsulation enhances zeta potential and stability, particularly in zinc ferrite systems, which is vital for biomedical applications like drug delivery. The increased zeta potential observed after capsaicin loading suggests enhanced electrostatic repulsion between particles, which may contribute to improved colloidal stability. However, zeta potential alone is not sufficient to confirm long-term dispersion stability. Additional assessments, such as time-dependent dispersion, storage stability, and serum stability, are recommended to provide a more comprehensive evaluation of colloidal behavior. These complementary experiments are recognized as important for future work and will help further validate the stability of the nanocarriers under physiologically relevant conditions.
Dynamic light scattering (DLS) results (Fig. 1E; Table 1) confirm successful drug loading. CAP-ZFO@CS NCs increased in size to 174.2 nm, and CAP-MFO@CS NCs to 490 nm, reflecting polymer-drug shell formation and minor aggregation. All sizes remained < 1000 nm, suitable for nanomaterials in biomedical uses. The polydispersity index (PDI) indicates particle size distribution homogeneity^40^. PDI values below 0.3 suggest monodisperse, stable systems, while 0.3–0.5 indicate acceptable variability, and > 0.5 suggest poor consistency. A slight PDI increase post-capsaicin loading reflects a broader size distribution. The relatively large hydrodynamic diameters and polydispersity indices (PDIs) observed for the chitosan-coated zinc and manganese ferrite nanocarriers are attributed to the formation of small clusters due to magnetic dipole–dipole interactions and the swelling of the chitosan shell in aqueous environments. Additionally, the broad size distribution may result from the synthesis method, which involves in situ co-precipitation and chitosan crosslinking, processes that can lead to moderate aggregation and variability in particle size. While these characteristics are common for magnetic nanocarriers, they may influence pharmacokinetics and clinical translation by affecting biodistribution, tumor accumulation, and clearance rates. To improve the fabrication and achieve smaller, more monodisperse particles in future work, strategies such as optimizing chitosan concentration, adjusting crosslinking agent ratios, reducing reaction time, or employing post-synthesis size-selection techniques (e.g., centrifugation or filtration) could be explored. These approaches may help minimize aggregation and enhance the uniformity of the nanocarriers, potentially improving their in vivo performance and clinical applicability. Overall, DLS and PDI results confirm that these chitosan ferrite nanoparticles are homogeneous, nanoscale, and promising for drug delivery.
Table 1. Mean size, PDI, zeta potential of samples.SamplePDIMean diameter (nm)Zeta potential (mV) ± SDCAP--0.0 ± 0.21MFO@CS NCs0.332348.0+ 28.24 ± 1.56CAP-MFO@CS NCs0.385490.3+ 32.34 ± 1.02ZFO@CS NCs0.305119.3+ 34.89 ± 0.31CAP-ZFO@CS NCs0.343174.2+ 53.2 ± 1.61
Fig. 1(A), (B), (D), (E) are the UV–vis, FTIR spectra, Zeta potentials and DLS distribution of all samples; (C) XRD diffractograms of MFO@CS NCs and ZFO@CS NCs.
SEM and EDS analyses (Fig. 2 and Figure S1) reveal the morphology and elemental composition of the nanocomposite systems. SEM images show pure capsaicin as sheet-like, rough aggregates. SEM images of the nanocarriers revealed the formation of nanoscale particles, though some aggregation was observed, which is common for magnetic ferrite-based systems due to magnetic dipole–dipole interactions and chitosan-mediated bridging. MFO@CS NCs display uneven, agglomerated structures with rough surfaces, likely due to high surface energy and magnetic interactions, forming clusters of smaller nanoparticles stabilized by chitosan^41^. CAP-MFO@CS NCs appear larger and more angular, indicating increased mass and agglomeration post-capsaicin encapsulation. ZFO@CS NCs exhibit granular, moderately aggregated structures, while CAP-ZFO@CS NCs show denser, clustered morphologies, suggesting capsaicin-induced agglomeration. These rough, varied surfaces enhance drug loading and biological interactions, supporting their drug delivery potential. EDS analysis confirms these findings. Pure capsaicin (Figure S1) contains carbon, nitrogen, and oxygen, reflecting its organic composition. MFO@CS NCs and CAP-MFO@CS NCs (Fig. 2 and Figure S1) show iron, carbon, nitrogen, and oxygen, with higher carbon in encapsulated samples, verifying capsaicin incorporation. Similarly, ZFO@CS NCs and CAP-ZFO@CS NCs (Fig. 2 and Figure S1) contain zinc, iron, carbon, nitrogen, and oxygen, with increased carbon and oxygen in encapsulated samples, confirming capsaicin loading. The SEM and EDS results validate the successful synthesis and capsaicin encapsulation of ferrite-chitosan nanocarriers, highlighting their tunable morphology and elemental composition for enhanced drug delivery applications. In summary, the successful green synthesis and thorough physicochemical characterization confirm the formation of stable, nanoscale chitosan-coated zinc and manganese ferrite nanoparticles with distinct structural and surface properties suitable for efficient drug encapsulation and delivery.
Fig. 2SEM of CAP-MFO@CS NCs (A) and CAP-ZFO@CS NCs (C), along with their corresponding EDS graphs (B and D). Scale bar: 100 μm; magnification: ×230.
Loading efficiency
Drug loading profiles in chitosan–ferrite nanocarriers for ZFO@CS NCs and MFO@CS NCs systems exhibited distinct behaviors. Both nanocarriers showed efficient entrapment at higher drug concentrations (100 ppm) (88% for ZFO@CS NCs and 77% for MFO@CS NCs) because of the high affinity of the chitosan–ferrite matrix for CAP. The loading efficiency dropped to 42% for MFO@CS NCs and 50% for ZFO@CS NCs at 50 ppm. To validate the reliability of this approach, control experiments were performed by centrifuging CAP solutions without nanocarriers under identical conditions, which showed negligible loss of CAP signal, confirming that CAP alone does not pellet at 12,000 rpm (Supporting information, Figure S2). Despite having a slightly lower loading efficiency than ZFO@CS NCs, MFO@CS NCs displayed a higher drug release profile. This disparity can be explained by the fact that Mn-based ferrites are more porous, have a larger surface area, and have weaker drug-matrix interactions, all of which facilitate the diffusion of CAP. However, the stronger binding affinity in ZFO@CS NCs results in slower release but more stable entrapment. According to these findings, MFO@CS NCs provide a more favorable platform for controlled drug delivery, even though high initial concentrations aid in effective loading. The ferrite core’s intrinsic physicochemical characteristics are crucial in controlling the release behavior. The nanocarriers demonstrated high capsaicin loading efficiencies, with core composition influencing loading capacity and release behavior, indicating manganese ferrite’s more porous structure facilitates enhanced, controlled drug delivery.
Biocompatibility
The hemolysis rate is a critical indicator of nanomaterial biocompatibility, particularly for blood-contact applications. This study evaluated hemolysis rates of capsaicin (CAP), zinc ferrite-coated chitosan nanocarriers (ZFO@CS NCs), manganese ferrite-coated chitosan nanocarriers (MFO@CS NCs), and their capsaicin-loaded forms across various concentrations. Normal saline (negative control) showed 0% hemolysis, confirming biocompatibility, while distilled water (positive control) caused 100% hemolysis, validating assay sensitivity (Table 2). Capsaicin displayed low hemolytic activity (3.36–4.7% at 10–100 µg/mL), indicating minimal red blood cell damage. ZFO@CS NCs exhibited concentration-dependent hemolysis, ranging from 0.8% at 10 µg/mL to 3% at 100 µg/mL, demonstrating high hemocompatibility, especially at lower doses. Capsaicin-loaded ZFO@CS NCs slightly increased hemolysis but remained below 5%, preserving biocompatibility. MFO@CS NCs showed even lower hemolysis (0–1.9%), with no hemolysis at 10 and 50 µg/mL. Capsaicin-loaded MFO@CS NCs also maintained hemolysis below 5%, confirming retained biocompatibility. Both ZFO@CS and MFO@CS nanocarriers, with or without capsaicin, exhibit excellent hemocompatibility, supporting their safety as drug delivery carriers. Planned in vivo studies will further confirm long-term safety.
Table 2. Hemolysis rate of samples (%).Sample100 µg/mL50 µg/mL10 µg/mLNegative control0Positive control100CAP4.74.23.36ZFO@CS NCs32.80.8CAP-ZFO@CS NCs4.231.6MFO@CS NCs1.90.60.2CAP-MFO@CS NCs1.61.10.9
In vitro drug release and kinetics
The capsaicin release profiles from its encapsulated forms CAP-ZFO@CS, and CAP-MFO@CS nanocarriers demonstrate dual responsiveness to acidic pH and glutathione (GSH), key characteristics of the tumor microenvironment. Under physiological pH (7.4), both nanocarriers exhibited minimal capsaicin release, indicating their stability and potential to minimize premature drug leakage in normal tissues, thereby reducing systemic toxicity. Specifically, CAP-ZFO@CS NCs released only about 3% of the loaded capsaicin, while CAP-MFO@CS NCs released approximately 6% at pH 7.4. In contrast, a marked increase in cumulative release was observed under acidic conditions (pH 5.4) in the presence of 10 mM GSH, simulating tumor intracellular environment, where CAP-ZFO@CS and CAP-MFO@CS nanocarriers released roughly 31% and 37% of capsaicin, respectively (Figs. 3 (A, B)). This significant enhancement confirms the ability of these nanocarriers to provide controlled and stimulus-triggered drug release desirable for cancer-targeted therapy. The enhanced release at acidic pH combined with elevated GSH concentration aligns with the known pH and redox sensitivity of chitosan-based carriers. Protonation of chitosan’s amine groups at lower pH increases polymer solubility and swelling, facilitating drug diffusion^42^. Concurrently, GSH, a reducing agent abundant in cancer cells, can cleave disulfide bonds or interact with metal ions in the ferrite core, further accelerating drug release. This dual stimulus ensures preferential capsaicin release within the tumor microenvironment, potentially enhancing therapeutic efficacy while minimizing off-target effects^43^.
The release kinetics was analyzed using the Korsmeyer-Peppas Eq. (6), plotted in Figs. 3(C, D). and tabulated in Table 3.
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:F=\frac{{M}_{t}}{{M}_{{\infty\:}}}={K}_{KP}{t}^{n}\:\Rightarrow\:\:\mathrm{l}\mathrm{o}\mathrm{g}F=\mathrm{l}\mathrm{o}\mathrm{g}{K}_{KP}+n\mathrm{l}\mathrm{o}\mathrm{g}t$$\end{document}where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{K}_{KP}$$\end{document} is the release rate constant, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:n$$\end{document} is the release exponent related to the drug release mechanism. For CAP-ZFO@CS NCs, the release exponent \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:n$$\end{document} of 0.76 at pH 5.4 + 10 mM GSH indicates anomalous (non-Fickian) transport, suggesting a combination of diffusion and polymer relaxation mechanisms governs capsaicin release. The corresponding kinetic constant \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{K}_{KP}$$\end{document} of 0.35 min^−1^ confirms an accelerated release under tumor-like conditions. At physiological pH, the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:n$$\end{document} value decreases to 0.30, consistent with a Fickian diffusion-controlled mechanism, aligned with the low cumulative release (3% over the studied period). The slightly higher \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{K}_{KP}$$\end{document} of 0.38 min^−1^ at pH 7.4 reflects a sustained, slower drug release. Both conditions exhibit high model fit quality with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{R}^{2}>0.96$$\end{document} . In contrast, CAP-MFO@CS NCs show \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:n$$\end{document} values of 0.43 and 0.40 at acidic GSH and neutral pH, respectively, suggesting predominantly Fickian diffusion-controlled release. The kinetic constants demonstrate a slower release rate at acidic GSH conditions (0.16 min^−1^) compared to neutral pH (0.34 min^−1^, consistent with the observed cumulative release of 37% and 6%, respectively. The good fits ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{R}^{2}>0.95$$\end{document} ) validate the release mechanism interpretation. In conclusion, quantitative release data corroborate the kinetic analyses showing enhanced capsaicin liberation from both nanocomposite types under tumor-mimicking acidic and reductive conditions. CAP-ZFO@CS NCs exhibit release governed by combined diffusion and polymer relaxation, while CAP-MFO@CS NCs show diffusion-dominated release with different sensitivity to microenvironmental stimuli. These findings emphasize the importance of nanocomposite composition and external stimuli in designing efficient, stimulus-responsive drug delivery systems for targeted cancer therapies. In summary, the nanocarriers exhibit minimal capsaicin release under physiological pH but demonstrate significantly enhanced release under tumor-mimicking acidic and reductive conditions, confirming their potential for stimulus-responsive, targeted drug delivery that likely contributes to improved cytotoxicity against HepG2 cells.
Table 3. Korsmeyer-Peppas parameters for CAP release from its encapsulated forms.NanocarrierspH n K_KP_ min^− 1^R²CAP- ZFO@CS NCs5.4 + GSH0.760.350.9877.40.300.380.962CAP- MFO@CS NCs5.4 + GSH0.430.160.9897.40.400.340.951
Fig. 3pH- and GSH-triggered release of capsaicin from its encapsulated nanocarriers: Cumulative capsaicin release (%) from (A) CAP-ZFO@CS NCs, and (B) CAP-MFO@CS NCs at 37 °C under tumor-mimicking conditions (pH 5.4 with 10 mM GSH) and physiological pH (7.4). Panels (C) and (D) show the corresponding Korsmeyer-Peppas kinetic model fits with log–log plots of fractional release versus time for CAP-ZFO@CS and CAP-MFO@CS NCs, respectively, illustrating the release mechanisms under different conditions.
Biological evaluations
This section integrates the cytotoxicity, antibacterial, and antioxidant assessments of capsaicin (CAP), ZFO@CS, MFO@CS, CAP-ZFO@CS, and CAP-MFO@CS nanocarriers, highlighting their multifunctional potential for biomedical applications. By evaluating these activities together, we can better understand the synergistic effects of encapsulation, such as improved selectivity and bioactivity, which are crucial for developing targeted therapies against cancer and related complications like infections or oxidative stress.
Cytotoxicity
The MTT colorimetric assay was employed to assess the cytotoxicity of the tested materials on human liver cancer (HepG2) and normal lung fibroblast (WI-38) cell lines, focusing on metabolic activity via formazan crystal formation (Table 4; Fig. 4(A, B)). Free CAP demonstrated moderate anticancer activity with an IC₅₀ of 78.9 ± 0.2 µg/mL against HepG2 cells and 181.8 ± 1.2 µg/mL against WI-38 cells, resulting in a therapeutic index (TI) of 2.31, which indicates reasonable selectivity but limited potency due to bioavailability issues^42^. The unloaded nanocarriers, ZFO@CS and MFO@CS, exhibited negligible cytotoxicity (IC₅₀ > 1000 µg/mL for both cell lines), underscoring their biocompatibility and suitability as drug delivery platforms. Upon capsaicin encapsulation, efficacy was markedly enhanced: CAP-ZFO@CS showed an IC₅₀ of 102.7 ± 1.3 µg/mL against HepG2 and 167.2 ± 1.3 µg/mL against WI-38 (TI = 1.63), while CAP-MFO@CS displayed superior performance with an IC₅₀ of 109.3 ± 1.3 µg/mL against HepG2 and 352.0 ± 2.6 µg/mL against WI-38 (TI = 3.22). As shown in Table 4; Fig. 4, CAP-MFO@CS showed the lowest IC_50_ against HepG2 cells (109.3 ± 1.3 µg/mL) and the highest therapeutic index among the tested formulations (3.22), with differences that were statistically significant compared with free CAP and the corresponding blank nanocarriers (p < 0.05), indicating enhanced selectivity. This suggests that manganese ferrite nanocarriers offer better biocompatibility and cancer selectivity, likely from differences in surface chemistry, magnetic properties, or cell interactions. While the present study demonstrates the anticancer activity of the nanocarriers using MTT assays, the mechanism of cell death (apoptosis vs. necrosis) was not directly assessed. Future studies will include apoptosis/necrosis assays to further elucidate the mode of cell death and provide a more comprehensive understanding of the anticancer effects. This emphasizes nanoparticle composition’s role in tuning selectivity. Chitosan coating enhances biocompatibility and reduces normal cell toxicity, while ferrite type affects uptake and oxidative stress^44,45^. Most formulations had low TIs, except CAP-MFO@CS NCs, indicating the need for further optimization through surface functionalization, targeting ligands, or size/charge tuning to improve cancer cell targeting and minimize normal cell effects. Representative phase-contrast images of HepG2 and WI-38 cells treated with CAP and CAP-loaded nanocarriers are shown in Figs. 4(D, E), with scale bars and magnifications indicated. Treated HepG2 cells display rounding, shrinkage, and loss of monolayer integrity, particularly with CAP-MFO@CS, consistent with cytotoxic effects. These improvements likely stem from the stimuli-responsive release and stronger binding affinities observed in manganese-based systems, positioning them as more effective for selective cancer cell targeting.
Table 4IC_50_ values obtained for samples against HeptG2 and WI‑38 cells IC_50_ (µg/mL, mean ± SD) and therapeutic index (TI).SamplesWI‑38 CellsHepG2 CellsTICAP181.8 ± 1.278.9 ± 0.22.31MFO@CS NCs162.0 ± 0.7118.9 ± 2.61.36CAP-MFO@CS NCs352.0 ± 2.6109.3 ± 1.33.22ZFO@CS NCs176.4 ± 1.482.2 ± 1.32.15CAP-ZFO@CS NCs167.2 ± 1.3102.7± 1.31.63
Fig. 4. Cell viability from MTT assay for WI-38 normal cells and HepG2 cancer cells treated with nanoparticles (data are presented as mean ± standard deviation (SD) of at least three independent measurements; statistical significance: p < 0.05). (C) IC_50_ values with therapeutic index. Morphological changes in (D) WI-38 normal cells and (E) HepG2 cancer cells following 24 h of treatment with (1) CAP, (2) MFO@CS NCs, (3) CAP-MFO@CS NCs, (4) ZFO@CS NCs, and (5) CAP- FO@CS NCs at 500 μg/mL (n = 3); Scale bar: 25 μm; magnification: ×20.
Antibacterial activity
Figures 5(A-D), and Table S1 illustrate the antibacterial efficacy of CAP, ZFO@CS NCs, MFO@CS NCs, and their encapsulated forms against Gram-positive and Gram-negative bacteria. Zones of inhibition indicate their effectiveness, with higher values reflecting greater antibacterial activity. MFO@CS NCs exhibit strong activity against B. subtilis (29 ± 0.1 mm), E. faecalis (30 ± 0.1 mm), and E. coli (30 ± 0.2 mm), while ZFO@CS NCs show notable activity against B. subtilis (25 ± 0.2 mm) and E. faecalis (27 ± 0.1 mm). The ferrite coating likely enhances chitosan’s antibacterial properties, possibly due to magnetic nanoparticles improving bacterial cell interaction^46,47^. Encapsulated CAP-MFO@CS NCs enhance activity against B. subtilis (30 ± 0.2 mm) compared to MFO@CS NCs alone, and CAP-ZFO@CS NCs are effective against S. Typhi (27 ± 0.1 mm), suggesting synergy between CAP and the nanocomposite structure^48^. CAP alone shows moderate activity, ranging from 19 ± 0.1 mm (S. aureus) to 27 ± 0.2 mm (E. coli). Gentamicin, the control, exhibits consistent activity (21 ± 0.2 mm to 26 ± 0.1 mm). The nanocarriers’ performance, comparable to gentamicin, underscores their potential as broad-spectrum antibacterial agents, supporting their use in nanotechnology-based strategies to combat bacterial infections^49,50^.
Antioxidant activity
The data in Fig. 5(E) illustrate the antioxidant activities of CAP, ZFO@CS NCs, MFO@CS NCs, their encapsulated forms, and ascorbic acid, assessed via the DPPH scavenging assay. Higher DPPH scavenging percentages indicate greater antioxidant activity, while lower IC50 values reflect stronger antioxidant capacity, representing the concentration needed to inhibit 50% of DPPH radicals. DPPH scavenging increases with concentration for all compounds. Ascorbic acid exhibits the highest activity (IC_50_: 4.63 µg/mL), consistent with its known potent antioxidant properties^51–53^. Bare capsaicin shows the lowest activity (IC_50_: 158.74 µg/mL), but its efficacy improves when combined with other materials. ZFO@CS NCs and MFO@CS NCs have IC_50_ values of 47.9 µg/mL and 88.4 µg/mL, respectively, likely due to the synergistic effects of metal ferrites and chitosan, which has inherent antioxidant properties^54,55^. Encapsulated CAP-ZFO@CS NCs (IC_50_: 32.23 µg/mL) and CAP-MFO@CS NCs (IC_50_: 80.7 µg/mL) show enhanced activity compared to their non-encapsulated counterparts, indicating that nanoencapsulation boosts radical scavenging. The improved antioxidant activity of encapsulated capsaicin aligns with studies showing nanoencapsulation enhances bioavailability and bioactivity. Chitosan’s biocompatibility and ability to improve compound stability and delivery further support these findings^55^. Enhanced activity may result from: (i) improved solubility and dispersion of capsaicin, aiding DPPH radical interaction; (ii) chitosan’s protective coating, preventing capsaicin degradation; and (iii) synergistic effects of capsaicin, metal ferrites, and chitosan. Encapsulation of capsaicin in ferrite nanocarriers markedly enhances selective cytotoxicity against HepG2 cells, while also improving antibacterial and antioxidant activities, underscoring the multifunctional therapeutic potential conferred by core-dependent nanocomposite design.
Fig. 5(A and B) Zones of inhibition (mm, mean ± SD); the activity on (C) Gram-negative and (D) Gram-positive of (1) MFO@CS NCs (2) CAP-MFO@CS NCs, (3) ZFO@CS NCs (4) CAP -ZFO@CS NCs and (5) CAP; (E) Antioxidant activities at various concentrations wit antioxidant IC_50_.
DNA binding and kinetics
UV-vis spectroscopy
UV-vis spectroscopy data (Table 5) at 290 nm reveal the binding interactions of ct-DNA with free capsaicin, the blank ferrite-based nanocarriers, and their loaded formulations (Fig. 6). Understanding the strength and thermodynamics of these interactions requires determination of the intrinsic binding constants (K_b_) and Gibbs free energy changes (∆G) by monitoring variations in the π–π* bands’ absorbance with the increase in the compound’s concentration (Eq. (7))^56^
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\:\frac{1}{\:\varDelta\:\mathrm{A}}=\frac{1}{\left({{\upepsilon\:}}_{\mathrm{b}}{-{\upepsilon\:}}_{\mathrm{f}}\right){\mathrm{L}}_{\mathrm{T}}}+\frac{1}{{\left({{\upepsilon\:}}_{\mathrm{b}}{-{\upepsilon\:}}_{\mathrm{f}}\right){\mathrm{L}}_{\mathrm{T}}\:\mathrm{K}}_{\mathrm{a}}\left[\mathrm{M}\right]}$$\end{document}The binding constant (Kb) values indicate that encapsulation of CAP into metal ferrite–chitosan nanocarriers significantly enhances its interaction with DNA compared to free CAP or the unloaded nanocarriers. Free CAP exhibits a relatively weak binding affinity toward DNA (Kb = 0.13 × 10^4^ M^− 1^), accompanied by a small red shift (3 nm), suggesting a weak intercalative or partial groove binding mode driven mainly by hydrophobic interactions. Upon coating ferrite nanoparticles with chitosan, the DNA interaction strength increases moderately to 0.27 × 10^4^ M^− 1^ for ZFO@CS and 0.65 × 10^4^ M^− 1^ for MFO@CS nanocarriers. This enhancement can be attributed to the presence of positively charged chitosan, which promotes electrostatic attraction with the negatively charged phosphate backbone of DNA^57^, as well as to the surface-active properties of the ferrite cores that facilitate adsorption.
The most pronounced binding affinities were observed for the encapsulated systems, particularly CAP-MFO@CS NCs (K_b_ = 1.10 × 10^4^ M^− 1^) and CAP-ZFO@CS NCs (Kb=0.97 × 10^4^ M^− 1^). This marked increase reflects a synergistic effect between the drug and the nanocarrier matrix, likely due to enhanced molecular planarity and the availability of multiple binding domains that favor efficient DNA interaction. The negative values of the Gibbs free energy (ΔG) across all systems, ranging from − 19.90 to − 23.04 kJ mol^− 1^, confirm that the binding processes are spontaneous. Furthermore, the more negative ΔG values for the nanocarrier-loaded complexes indicate thermodynamically more favorable DNA association.
Spectral behavior further supports these trends. The observed red shifts for most systems, especially the 6 nm shift in CAP-ZFO@CS NCs, suggest intercalative binding where the complex inserts between base pairs, resulting in DNA helix stabilization^58,59^. In contrast, the slight blue shift detected for CAP-MFO@CS NCs implies a mixed or electrostatic binding mode, possibly reflecting differences in nanoparticle surface charge density or metal–ligand coordination characteristics of manganese compared to zinc ferrite. Overall, the data denote that encapsulation of CAP within ferrite–chitosan nanocarriers substantially strengthen and modifies its DNA interaction mechanism. This enhancement likely contributes to improved biological reactivity and may underlie the observed increases in cytotoxic and antibacterial activities reported for the loaded formulations.
Table 5. Binding constants of samples interaction to DNA using UV-vis Titration method at 290 nm.DNA–CAPK_b_ [M^− 1^ × 10^4^]UV-vis shift (nm)∆G (kJ mol^− 1^)0.133 (red)-19.90DNA- ZFO@CS NCs0.272 (red)-19.56DNA- MFO@CS NCs0.650 (red)-21.74DNA-CAP-ZFO@CS NCs0.976 (red)-22.73DNA-CAP-MFO@CS NCs1.102 (blue)-23.04
Fig. 6. Changes in UV-vis absorption spectral changes recorded for the reactions between various concentrations of (A) Capsaicin, (B) ZFO@CS NCs, (C) CAP-ZFO@CS NCs. (D) MFO@CS NCs and (E) CAP-MFO NCs. respectively and DNA (0.1 mM).
Fluorescence spectroscopy
The fluorescence enhancement data for Ct-DNA interactions with CAP and nanocarriers reveal strong binding interactions (Figs. 7(A-E)), as evidenced by the Stern-Volmer (SV) equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\left(8\right)$$\end{document} (Fig. 7F)^60^and modified Stern-Volmer Eq. (9) (Fig. 7G)^61^analyses. The linear fits ((R^2^ > 0.95) for SV, (R^2^ > 0.98) for modified SV) indicate robust models for most compounds, with CAP-MFO@CS NCs exhibiting the highest enhancement ((F/F_0_ = 10.6) at 3.44 × 10⁻⁴ M).
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\frac{{F}_{0}}{F}=\left(1+{K}_{SV}\left[Q\right]\right)=\:1+{k}_{q}{{\uptau\:}}_{0}\left[Q\right]$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\frac{{F}_{0}}{{F-F}_{0}}=\frac{1}{{f}_{a}{K}_{SV}\left[Q\right]}+\frac{1}{{f}_{a}}$$\end{document}The observed fluorescence enhancement suggests that these compounds induce conformational changes in Ct-DNA, possibly exposing fluorophores or stabilizing excited states. Unlike typical fluorescence quenching observed in DNA-probe systems (e.g., with ethidium bromide), the intrinsic fluorescence increase here may stem from direct DNA interactions without external probes. The SV constants (K_SV_) range from 0.5772 × 10^4^ M⁻¹ (CAP) to 1.9523 × 10^4^ M⁻¹ (CAP-MFO@CS NCs), indicating stronger interactions in encapsulated forms, particularly with manganese ferrite. The modified SV parameters show (fa > 1) (3.3 to 12.8), which are atypical for quenching models but suggest multiple binding sites or enhanced fluorophore accessibility in enhancement contexts.
CAP exhibits moderate enhancement K_SV_ = 5772 M^− 1^, (Ka = 3529.4 M^− 1^), suggesting weaker DNA binding compared to nanoparticles, likely due to its small molecular size limiting interaction sites. ZFO@CS NCs and MFO@CS NCs both show high KSV (10065.0 and 10804.7 M⁻¹) and similar Ka (~ 2622.6–2783.7 M⁻¹), indicating comparable binding affinities. MFO@CS NCs slightly outperforms ZFO@CS NCs, possibly due to manganese’s redox properties enhancing DNA interactions. Encapsulation significantly boosts enhancement, with CAP-MFO@CS NCs leading (K_SV_ = 1.9523 × 10^4^ M^− 1^, fa = 12.8) and CAP-ZFO@CS NCs (K_SV_ = 1.2444 × 10^4^ M^− 1^, fa = 7.3) (Table 6). The synergy of capsaicin and ferrite-chitosan matrices likely improves solubility, targeting, and electron transfer, amplifying fluorescence. Regarding the fluorescence enhancement mechanism, the linear Stern-Volmer plots and high association constants suggest a predominant static enhancement mechanism, wherein stable complex formation between the nanocarriers and Ct-DNA leads to increased fluorescence intensity. The absence of significant upward curvature rules out major dynamic quenching contributions. Furthermore, the sizeable accessible fluorophore fractions and enhanced binding reflect static interactions that rigidify or expose DNA fluorophores, enhancing emission.
Binding constants and thermodynamics from Double-Logarithmic plots
To further characterize the binding interactions, double-logarithmic plots were analyzed using the Double-Logarithm Eq. (8) (Fig. 7H)^62,63^. This yielded binding constants (K_b_), Hill coefficients (n), and goodness-of-fit (R^2^ > 0.97) for all compounds (Table 6). The thermodynamic free energy change was calculated using Eq. 10. at 298 K. The n values (1.08–1.53) suggest approximately one to two binding sites per DNA molecule, with higher values (n) in nanoparticle systems indicating cooperative binding. The negative ΔG) values confirm spontaneous interactions, with more negative values for encapsulated forms reflecting stronger thermodynamic favorability. Encapsulation increases Kb by orders of magnitude, from ~ 10^3^–10^4^ M⁻¹ for free CAP and uncoated ferrites to ~ 10^5^–10^6^ M⁻¹ for CAP-loaded nanoparticles, suggesting a shift from groove binding to partial intercalation.
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:Log\left\{\right(F-F_0)/F_0\}\:=\:logK_b\:+\:nlog\:\left[Q\right]$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle\:\mathrm{G}\:=\:-\mathrm{R}\mathrm{T}\:\mathrm{l}\mathrm{n}\:{K}_{b}$$\end{document}Table 6. Stern-Volmer, modified Stern-Volmer, and Double-Logarithmic parameters for fluorescence enhancement of Ct-DNA.QuencherK_SV_[10^4^ M] R ^2^ (SV)K_a_(M^-1^)f_a_ R ^2^ (Mod. SV)K_b_(10^4^ M^-1^) n ΔG(kJ/mol) R ^2^ (Double-Log)CAP0.57720.9633529.43.30.9840.821.08-22.330.973ZFO@CS NCs1.00650.9752622.67.10.9916.21.18-27.360.989CAP-ZFO@CS NCs1.24440.9862783.77.30.9961201.53-34.650.981MFO@CS NCs1.08050.9752507.19.50.995791.48-33.640.995CAP-MFO@CS NCs1.95230.9522689.712.80.9891801.51-35.660.994KSV: Stern-Volmer enhancement constant (M^-1^); R^2^(SV): Goodness-of-fit for Stern-Volmer; Ka: Modified Stern-Volmer enhancement constant (M^-1^); fa: Fraction of accessible fluorophores; R^2^ (Mod. SV): Goodness-of-fit for modified Stern-Volmer; Kb: Binding constant (M^-1^); n: Hill coefficient (number of binding sites); ΔG: Free energy change (kJ/mol); R^2^ (Double-Log): Goodness-of-fit for double-logarithmic analysis.
Literature typically reports capsaicin-DNA interactions as quenching processes, with binding constants (K_b_) of 10³–10⁵ M⁻¹ for minor groove binding via hydrogen bonds and van der Waals forces. For instance, studies using EtBr-DNA systems report (K_b_ approx 10^4^ M⁻¹ for capsaicin, lower than intercalators (~ 10⁶–10⁸ M⁻¹), with negative (ΔG) indicating spontaneous binding. Our (K_b_) values for free CAP (8.2 × 10^3^ M⁻¹) align with groove binding, while encapsulated forms (~ 10^6^ M⁻¹) approach intercalator strengths, possibly due to nanoparticle-mediated DNA unwinding or enhanced hydrophobic interactions. Ferrite-chitosan systems are less studied, but capsaicin-encapsulated chitosan nanoparticles enhance anticancer activity by targeting DNA against carcinogens like DMBA. Cobalt ferrite-based capsaicin systems show antimicrobial and photocatalytic properties, suggesting that Mn and Zn ferrites in our study similarly enhance bioactivity. The high (K_b_) and more negative (ΔG) for CAP-MFO@CS NCs parallels findings where magnetic nanoparticles improve drug delivery efficiency, likely due to magnetic field interactions or enhanced cellular uptake. The superior performance of CAP-MFO@CS NCs, with the highest K_b_, n, and most negative ΔG, suggests potential for DNA-targeted therapies, such as cancer treatment or gene delivery, leveraging magnetic properties for guided delivery. However, fa > 1 and n > 1 indicate that models may require adaptation for enhancement scenarios. Missing data at higher concentrations and the unique F_0_ for CAP-MFO@CS NCs suggest experimental variability that warrants further investigation. Future studies should validate these interactions in vivo, measure ΔH and ΔS for full thermodynamics, and explore alternative models for fluorescence enhancement.
Fig. 7. Flourscence of (A) CAP; (B) ZFO@CS NCs; (C) CAP- ZFO@CS NCs; (D) MFO@CS NCs; (E) CAP- MFO@CS NCs; (F) Stern-Volmer, (G) Modified Stern-Volmer, and Double-Logarithmic Parameters for Fluorescence Enhancement of Ct-DNA.
Stopped-flow analysis
The stopped-flow kinetic data at 320 nm reveal distinct interaction dynamics between ct-DNA and various formulations. Capsaicin alone (Fig. 8A) exhibits a biphasic kinetic pattern with a rapid initial absorbance decrease (fast phase) followed by a sustained decline (slow phase), indicating quick binding or intercalation into DNA, followed by prolonged structural changes like unwinding or embedding. In contrast, chitosan-coated zinc and manganese ferrite nanocarriers, with or without capsaicin encapsulation (Fig. 8B), show an initial sharp absorbance increase followed by stabilization, suggesting rapid surface adsorption of DNA with minimal secondary structural changes. These differences highlight the role of material composition. Capsaicin alone induces invasive, prolonged DNA modifications, while nanocarriers promote rapid, stable surface interactions driven by electrostatic forces and the chitosan coating. Encapsulated capsaicin within nanocarriers reduces slow-phase absorbance changes, indicating shielding effects or altered binding site accessibility. Variations in core composition (zinc vs. manganese ferrite) further influence binding dynamics and potential drug delivery efficacy^64^. These kinetic profiles underscore key considerations for DNA-targeted therapeutics. The fast phase ensures immediate interaction, while the slow phase affects long-term DNA structure and drug release. Capsaicin suits therapies needing sustained DNA alteration, whereas nanocarriers offer controlled, surface-mediated delivery with reduced DNA disruption. To study the concentration dependency of the reported reaction rates, 10^− 4^ M solutions of ct-DNA were reacted with different concentrations of CAP, ZFO@CS, CAP- ZFO@CS, MFO@CS and CAP- MFO@CS, while maintaining the pseudo-first-order condition. Table 7 displays the second-order association constant (kon), first-order dissociation constant (koff), equilibrium association constants (Ka [M^− 1^] = (kon/koff), equilibrium dissociation constants (Kd [M] = koff/kon), and Gibbs free energy (ΔG [kJ mol^− 1^]) for these interactions. Table 8 displays these parameters relative to CAP for emphasizing improvements in modified compounds.
Fast phase kinetics
Association rate constants (k₁) increased with encapsulation, peaking at 16.86 ± 1.11 M⁻¹ s⁻¹ for CAP-MFO@CS, followed by CAP-ZFO@CS (12.48 ± 2.59 M⁻¹ s⁻¹), MFO@CS (11.8 ± 1.25 M⁻¹ s⁻¹), ZFO@CS (7.4 ± 1.01 M⁻¹ s⁻¹), and CAP (4.1 ± 0.16 M⁻¹ s⁻¹) (Fig. 8C). Dissociation rates (k₋₁) were minimized in encapsulated systems: 0.13 ± 0.03 s⁻¹ for CAP-MFO@CS and 0.12 ± 0.08 s⁻¹ for CAP-ZFO@CS, versus higher values for unloaded forms and free CAP (0.43 ± 0.0 s⁻¹^65^. Coordination affinities (Kₐ₁) followed suit, with CAP-MFO@CS at 129.69 M⁻¹ (synergistic manganese-capsaicin effects) and CAP-ZFO@CS at 104.0 M⁻¹, exceeding unloaded ZFO@CS (33.63 M⁻¹) and MFO@CS (40.89 M⁻¹). Dissociation constants (Kd₁) were lowest for CAP-MFO@CS (0.008 M) and CAP-ZFO@CS (0.01 M), indicating tight binding. Activation free energies (ΔG₁#) confirmed favorability, with CAP-MFO@CS showing the lowest barrier (-12.05 kJ mol⁻¹) compared to CAP (-5.70 kJ mol⁻¹.
Slow phase kinetics
Slow-phase association rates (k₂) were highest for CAP-MFO@CS (1.08 ± 0.05 M⁻¹ s⁻¹), followed by CAP-ZFO@CS (0.43 ± 0.09 M⁻¹ s⁻¹), MFO@CS (0.42 ± 0.13 M⁻¹ s⁻¹), ZFO@CS (0.27 ± 0.03 M⁻¹ s⁻¹), and CAP (0.20 ± 0.03 M⁻¹ s⁻¹) (Fig. 8D). Dissociation rates (k₋₂) varied, with CAP-MFO@CS at 0.76 ± 0.17 × 10⁻² s⁻¹ reflecting sustained stability, while unloaded forms showed higher values (e.g., 2.7 ± 0.4 × 10⁻² s⁻¹ for MFO@CS. Affinities (Kₐ₂) peaked for CAP-MFO@CS (142.1 M⁻¹) and CAP (62.5 M⁻¹), with low Kd₂ for CAP-MFO@CS (0.007 M). ΔG₂# values were most negative for CAP-MFO@CS (-12.27 kJ mol⁻¹), supporting prolonged interactions^19^.
Overall binding
Using Eq. (12), the overall equilibrium dissociation constant, Kd, can be calculated from the individual equilibrium dissociation constants, Kd1 and Kd2, to obtain the overall association constant, Ka (Fig. 8E). Equation (13) can also be used to calculate the overall binding free energy change, G_bind_.
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\mathrm{K}}_{\mathrm{d}}}={{\mathrm{k}}_{{\mathrm{d1}}}}{{\mathrm{k}}_{{\mathrm{d2}}}}/\left( {{\mathrm{1}}+{{\mathrm{k}}_{{\mathrm{d2}}}}} \right)$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta {{\mathrm{G}}_{{\mathrm{bind}}}}={\mathrm{RT}}\cdot{\mathrm{ln}}\left( {{{\mathrm{K}}_{\mathrm{d}}}} \right)$$\end{document}Overall affinities (Ka) were dramatically enhanced in encapsulated systems: CAP-MFO@CS (1.86 × 10⁴ M⁻¹) far surpassed CAP (687.5 M⁻¹), MFO@CS (640.6 M⁻¹), and ZFO@CS (732.2 M⁻¹), while CAP-ZFO@CS reached 2921 M⁻¹. Dissociation constants (Kd) dropped to 0.05 × 10⁻³ M for CAP-MFO@CS and 0.34 × 10⁻³ M for CAP-ZFO@CS. Overall ΔG# reflected this, with CAP-MFO@CS at -24.34 kJ mol⁻¹ indicating the most spontaneous binding^66^.
DNA binding kinetics: comparison with existing studies
The stopped-flow kinetic data for CAP, ZFO@CS, and MFO@CS nanocarriers reveal a biphasic DNA binding mechanism: rapid initial association followed by slower conformational stabilization, consistent with prior studies on macromolecular DNA interactions^19,64^. Similar biphasic kinetics occurs in ruthenium-based DNA binders, with fast second-order binding followed by slower isomerization. The association (k₁) and dissociation (k₋₁) rate constants of CAP-MFO@CS and CAP-ZFO@CS match those of cationic metal complexes, highlighting the enhanced affinity and stability from ferrite cores and chitosan coatings^19,64^. The higher k₁ and lower k₋₁ for encapsulated CAP-MFO@CS and CAP-ZFO@CS reflect how metal composition and polymer modification improve DNA binding, aligning with protein-DNA studies showing electrostatic-driven binding and structural rearrangements^65^. Thermodynamic analysis shows favorable activation energies (ΔG‡), supporting spontaneous, sustained nucleic acid binding crucial for anticancer and antimicrobial effects, consistent with metal-based complex studies^66^. Manganese ferrite’s redox activity further enhances DNA interaction speed and strength compared to zinc ferrite. This study advances understanding of DNA binding kinetics via stopped-flow methods, emphasizing ferrite core composition and capsaicin encapsulation for optimizing nanocarrier design in biomedicine.
Table 7. Kinetic and thermodynamic parameters for ct-DNA Interactions.ParametersFast PhaseSlow PhaseOver all reactionk_1_[M⁻¹s⁻¹]k_− 1_[s⁻¹]K_a1_ [M⁻¹]K_d1_[M]ΔG_1_[kJ mol⁻¹]k_2_ [M⁻¹s⁻¹]k_− 2_ [10⁻²s⁻¹]K_a2_ [M⁻¹]K_d2_ [M]ΔG_2_[kJ mol⁻¹]K_a_ [M⁻¹]K_d_ [10⁻³M]ΔG[kJ mol⁻¹] CAP 4.1 ± 0.20.43 ± 0.09.50.10-5.700.20 ± 0.030.32 ± 0.062.50.016-10.246881.45-16.18 MFO@CS NCs 11.8 ± 1.20.29±0.0440.90.025-9.130.42 ± 0.132.7 ± 0.415.50.064-6.806401.50-16.10 CAP-MFO@CS NCs
16.86 ± 1.1
0.13 ± 0.0
129.7
0.008
-12.05
1.08 ± 0.05
0.76 ± 0.17
142.1
0.007
-12.27
18,600
0.05
-24.34
ZFO@CS NCs 7.4 ± 1.00.22 ± 0.033.60.02-8.760.27 ± 0.032.41 ± 0.0111.200.089-5.997321.36-16.34 CAP-ZFO@CS NCs 12.48 ± 2.60.12 ± 0.0104.00.01-11.500.43 ± 0.091.55 ± 0.327.740.036-8.2329210.34-19.77
Table 8. Kinetic and thermodynamic parameters for ct-DNA interactions relative to CAP “All values normalized to CAP = 1.0”.Parameters (Fold)Fast PhaseSlow PhaseOver all reactionk_1_k_− 1_K_a1_K_d1_ΔG_1_k_2_k_− 2_K_a2_K_d2_ΔG_2_K_a_K_d_ΔG CAP 1.01.01.01.01.01.01.01.01.01.01.01.01.0 MFO@CS NCs 2.880.674.290.251.602.108.440.254.000.660.931.031.00 CAP-MFO@CS NCs
4.11
0.30
13.61 0.08 2.11
5.40
2.38
2.27
0.44
1.20
27.05
0.03
1.50
ZFO@CS NCs 1.800.513.530.201.541.357.530.185.560.581.060.941.01 CAP-ZFO@CS NCs 3.040.2810.910.102.022.154.840.442.250.804.250.231.22
Fig. 8. Kinetic traces at 320 nm containing two reaction phases for (A) CAP and (B) both ferrites and their CAP encapsulated forms; Plots of k_obs_ versus concentration for the (C) first and (D) second phases recorded during the interaction with DNA in methanol at 296 K along with (E) the overall coordination affinity.
Nanoparticle design: impact of Size, Charge, and composition on cancer targeting and safety
The physicochemical properties of nanoparticles—size, surface charge, and metal composition—critically influence their biological interactions, toxicity, and therapeutic efficacy in cancer treatment. Chitosan-coated zinc ferrite (ZFO@CS) and manganese ferrite (MFO@CS) nanoparticles, used as capsaicin delivery vehicles, showed distinct performance^67^. Dynamic light scattering indicated smaller hydrodynamic diameters for ZFO@CS (~ 119.3 nm) compared to MFO@CS (~ 348.0 nm), increasing to ~ 174.2 nm and ~ 490.3 nm post-capsaicin loading, respectively. Smaller ZFO@CS particles enhanced tumor penetration via the EPR effect, correlating with lower IC₅₀ (197.4 µg/mL against HepG2), while larger MFO@CS particles favored prolonged tumor retention, yielding a higher therapeutic index (TI = 3.22)^68^. Surface charge, assessed via zeta potential, showed CAP-ZFO@CS (+ 53.2 mV) had stronger cancer cell membrane attraction than CAP-MFO@CS (+ 32.3 mV), improving uptake. CAP-MFO@CS’s manganese-driven redox activity may enhance selectivity (TI = 3.22 vs. 1.63) by inducing tumor-specific oxidative stress (IC₅₀ = 45.6 µg/mL)^69^, though the exact mechanisms require further validation. The chitosan coating improved biocompatibility, enabling pH- and glutathione-responsive drug release, minimizing off-target effects. These properties—optimal size (< 200 nm), charge ( > + 30 mV), and metal composition—enable efficient delivery, apoptosis via reactive oxygen species, and controlled drug release, positioning these nanoparticles as promising for precision oncology^70^.
Role of zinc and manganese in binding affinity and biological effects
Zinc ferrite (ZFO@CS) and manganese ferrite (MFO@CS) cores influence DNA binding, cytotoxicity, antibacterial, and antioxidant activities^71^. CAP-MFO@CS exhibited stronger DNA binding (K_b_ = 1.10 × 10⁴ M⁻¹, k₁ = 16.86 s⁻¹, negative ΔG) than CAP-ZFO@CS, indicating stable, spontaneous interactions, potentially due to manganese’s redox activity, versus zinc’s structural stability. CAP-MFO@CS showed blue shifts in DNA spectra (minor groove/external binding), while ZFO@CS showed red shifts (intercalative/groove binding). In cytotoxicity tests, CAP-MFO@CS achieved a higher therapeutic index (TI = 3.22) than CAP-ZFO@CS (TI = 1.63) and free capsaicin (TI = 2.31), possibly driven by manganese’s oxidative and magnetic properties promoting selective cancer cell apoptosis, though further mechanistic studies are needed. Antibacterial tests showed CAP-MFO@CS with broader inhibition zones (29–30 mm) against Gram-positive/negative strains, matching or surpassing gentamicin, due to membrane disruption and oxidative stress. CAP-ZFO@CS excelled in antioxidant activity, likely due to better electron transfer^71^.
Correlation of DNA binding affinity with biological outcomes
The enhanced DNA binding affinities observed in the encapsulated nanocarriers, particularly CAP-MFO@CS with K_b_ = 1.10 × 10⁴ M⁻¹ and overall K_a_ = 1.86 × 10⁴ M⁻¹, correlate with improved cytotoxicity against HepG2 cells, where IC₅₀ values dropped to 109.3 µg/mL compared to free capsaicin’s 224.1 µg/mL, suggesting that stronger intercalative and electrostatic interactions may facilitate apoptosis induction via DNA damage^72,73^. This correlation extends to antioxidant activity, as higher binding constants align with reduced IC₅₀ values (e.g., 32.23 µg/mL for CAP-ZFO@CS), indicating that stable DNA-nanocomposite complexes may mitigate oxidative stress in cancer cells more effectively than unloaded forms^74^. Antibacterial efficacy also shows a positive trend, with larger inhibition zones (up to 30 mm) for CAP-MFO@CS against E. coli, underscoring how kinetic stability (low k_− 1_ = 0.13 s⁻¹) may enhance membrane disruption in pathogens. Enhanced DNA binding affinity in manganese ferrite systems correlates with improved HepG2 cytotoxicity, potent antibacterial effects, and heightened antioxidant capacity, underscoring the therapeutic relevance of core-tuned nanocarrier design. However, these correlations are based on in vitro data and require further validation in cellular and in vivo models.
Mechanistic insights into Nanocomposite-DNA interactions
The biphasic stopped-flow kinetics reveal a two-step equilibrium model for DNA binding: a fast bimolecular association (k₁ up to 16.86 s⁻¹ for CAP-MFO@CS) forming a loosely bound complex via electrostatic attraction between chitosan’s cationic groups and DNA’s phosphate backbone, followed by a slow unimolecular isomerization (k₂ = 1.08 s⁻¹) that stabilizes the tight complex through capsaicin’s hydrophobic intercalation and ferrite-mediated redox modulation. Manganese ferrite’s superior performance over zinc, evidenced by more negative ΔG (− 24.34 kJ/mol vs. −19.77 kJ/mol), may arise from its higher redox potential, which facilitates electron transfer and enhances binding spontaneity in reductive tumor environments. These mechanisms may explain the stimuli-responsive release (37% at pH 5.4 + GSH), as DNA-induced conformational changes trigger capsaicin liberation, linking biophysical interactions to therapeutic selectivity. The biphasic binding kinetics reflect initial rapid electrostatic association followed by slow conformational tightening, with ferrite core composition modulating interaction strength and responsiveness to tumor microenvironment stimuli. Further mechanistic studies are needed to confirm these hypotheses and explore their translation to in vivo systems.
Therapeutic implications and future directions
The multifunctional profile of CAP-MFO@CS nanocarriers, combining pH/GSH-responsive release, selective cytotoxicity (TI = 3.22), and potent DNA binding, positions them as promising platforms for targeted liver cancer therapy, potentially reducing free capsaicin’s limitations like low bioavailability and non-specificity. Comparative advantages over ZFO@CS systems highlight manganese ferrite’s role in optimizing kinetics and efficacy, suggesting hybrid designs for personalized nanomedicine. However, while the present study demonstrates promising in vitro biocompatibility, as evidenced by low hemolysis rates and minimal cytotoxicity toward normal WI-38 fibroblasts, the high positive surface charge (zeta potentials + 30 to + 50 mV) may pose potential risks for off-target membrane interactions, complement activation, and rapid clearance in vivo. Further safety assessments, including in vitro membrane integrity assays and comprehensive in vivo toxicity studies, are warranted to fully evaluate the biosafety profile of these nanocarriers. The current findings should therefore be interpreted within the context of in vitro models, and any clinical translation would require additional safety evaluations and possible surface engineering to moderate surface charge while retaining therapeutic efficacy. Future work should explore in vivo biodistribution, magnetic targeting integration, and clinical translation to validate these correlations in animal models and address scalability for broader antimicrobial and antioxidant applications. These findings position manganese ferrite-chitosan nanocarriers as promising platforms that integrate targeted capsaicin delivery, antibacterial, and antioxidant activities, paving the way for future in vivo evaluation and clinical applications in precision oncology.
Conclusions
This study demonstrates the successful green synthesis and comprehensive evaluation of chitosan-coated zinc ferrite (ZFO@CS) and manganese ferrite (MFO@CS) nanoparticles as multifunctional nanocarriers for capsaicin delivery in liver cancer therapy. The nanocarriers exhibited favorable physicochemical properties, including nanoscale hydrodynamic diameters (119–490 nm), high positive zeta potentials (+ 28 to + 53 mV), and efficient capsaicin loading (up to 88%), enabling stable encapsulation and targeted delivery. Key findings highlight the dual pH- and glutathione-responsive release profiles, with significantly higher capsaicin release (31–37%) in tumor-mimicking environments compared to physiological conditions (< 5%), minimizing off-target effects. Cytotoxicity assays revealed enhanced anticancer efficacy, particularly for CAP-MFO@CS, with a lower IC₅₀ (109.3 µg/mL) and superior therapeutic index (3.22) against HepG2 cells, underscoring improved selectivity over free capsaicin. Additionally, the nanoplatforms showed potent antibacterial activity comparable to gentamicin and boosted antioxidant effects (IC₅₀ reduced to 32.23 µg/mL for CAP-ZFO@CS). Using a stopped-flow technique, a two-step equilibrium model was applied to describe the binding process in which CAP, chitosan-coated ferrite, and the encapsulated forms associate with DNA by a fast bimolecular step to form a loosely bound complex; this was subsequently converted into a tightly bound complex by a slow unimolecular step. Encapsulation markedly enhanced affinity and kinetics, with manganese-based carriers displaying superior binding constants (Kb up to 1.10 × 10⁴ M⁻¹) and association rates, providing mechanistic insights into their anticancer action. Overall, these chitosan-coated ferrite nanocarriers overcome capsaicin’s limitations by improving bioavailability, controlled release, and tumor-specific targeting, positioning them as promising platforms for multifunctional cancer therapy. Future in vivo studies and clinical trials are warranted to validate their translational potential and explore optimizations for broader oncological applications.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1
The reference list from the paper itself. Each links out to its DOI / PubMed record.