Concentration-Dependent Modulation of Optical Properties in TiO₂ Nanoparticle-Treated HCT116 Colorectal Cancer Cells

INTRODUCTION
Photon attenuation and redistribution in complicated heterogeneous mediums are controlled by inherent optical characteristics in biological cell systems. Microscale absorption, scattering, and refractive index variability cause diffuse reflectance and transmittance in cellular suspensions. The absorption coefficient measures photon energy dissipation in the medium, while refractive index changes control phase velocity and scattering. These characteristics characterise biological surroundings’ effective optical signature and enable quantitative light–cell interaction analysis [1,2]. Recent biomedical photonics breakthroughs emphasise the role of artificial nanoparticles in optical property modulation. Nanoparticles change local electromagnetic field distributions and refractive index discontinuities in biological systems, affecting scattering cross-sections and attenuation pathways. Since nanoparticle density increases collective scattering and composite medium optical density, these effects are concentration-dependent [3,4]. Titanium dioxide (TiO₂) nanoparticles are important for their optical stability, high refractive index, and well-defined structural phases. The visible spectral area refractive index values for TiO₂ nanostructures vary between 2.4 and 2.7, depending on crystalline phase (anatase or rutile) and wavelength [3,5]. TiO₂ nanoparticles have been extensively studied for their light absorption, band gap, and structural-optical correlations, in addition to their function in photonic and optical materials research [3,6]. TiO₂’s characteristics make it a promising choice for changing light propagation in biological mediums. Few studies have quantified how controlled modification in TiO₂ nanoparticle concentration affects optical parameters in cellular cancer models, despite well-documented intrinsic optical features. A system-level photonic interpretation of nanoparticle–cell interactions requires understanding concentration-dependent absorption coefficient and refractive index changes in nanoparticle-treated cell suspensions. HCT116 colorectal cancer cells are a reproducible and popular in vitro model for biomedical research. Systematic optical characterisation of TiO₂-treated HCT116 cell suspensions, including effective absorption coefficient and refractive index alterations from reflectance and transmittance studies, is still lacking. This study investigates the concentration-dependent adaptation of optical properties in TiO₂ nanoparticle-treated HCT116 cells in the visible spectrum (400-800 nm). Effective absorption coefficient and refractive index changes across nanoparticle concentrations were measured via diffuse reflectance and transmittance. This research uses composite system behaviour rather than inherent nanoparticle constants to establish a quantitative photonic framework for cancer cell nanoparticle-induced light propagation regulation.

MATERIALS AND METHODS
A study compared optical characteristics of HCT116 colorectal cancer cells with and without TiO₂ nanoparticles. Three phases comprised the methodology. TiO₂ nanoparticles were synthesised and characterised using DC sputtering to confirm shape and phase properties. To evaluate nanoparticle uniformity, scanning electron microscopy (SEM) examined surface appearance, particle dispersion, and structural features before biological use. Second, optical measurements were made on both untreated (control) and TiO₂NP-treated cell suspensions. In the visible range (400–800 nm), diffuse reflectance and transmittance spectra were produced, employing 473 nm as a typical excitation wavelength. Third, reflectance and transmittance data from UV–visible spectroscopy determined optical properties such effective absorption coefficient and refractive index changes.

Titanium Oxide (TiO₂NPs) Preparation
TiO₂NPs were made using DC sputtering. During sputtering, nanostructured TiO₂ deposits were formed by ejected material from a titanium target under controlled plasma conditions. Nanoparticulate material was carefully collected and processed into TiO₂ nanopowder for subsequent research. Use 100 mg of TiO₂ nanopowder and 100 mL of distilled water to generate a homogeneous suspension. Stir continuously at 2000 rpm at room temperature for 2 hours. The translucent liquid became milky with evenly scattered nanoparticles. The suspension was centrifuged at 12,000 rpm for 20 min to reduce agglomeration and stabilise dispersion. Extra biological and optical measurements were taken with the supernatant.

Structural Characterization of TiO₂ Nanoparticles
Before biological use, titanium dioxide nanoparticles were characterised for shape, particle size distribution, and crystalline phase. SEM examined surface morphology. Microscopic imaging showed quasi-spherical nanoparticles with homogeneous dispersion. An image-based investigation showed a 60-nm particle diameter for cellular interactions and visible light scattering. The crystal phases were determined by XRD. The anatase phase of TiO₂ was confirmed by diffraction patterns with peaks matching reference data. Anatase TiO₂’s high refractive index (2.4 in visible spectrum) sets it apart from polymorphs like rutile for optical studies [7]. SEM and XRD validated TiO₂ nanoparticle form and phase before dispersion and biological/optical evaluation.

Cell Culture and Nanoparticle Treatment
Human colorectal cancer HCT116 cells were optically evaluated in vitro. Cell culture took place in sterile RPMI growth media at 37 °C in a humidified 5% CO₂ environment. TiO₂ nanoparticle suspensions were freshly produced for each experiment to guarantee consistent dispersion. Separating treated and control cells. Untreated HCT116 cells were the control group, whereas treatment groups received TiO₂ nanoparticles at varied doses (31.25, 62.5, 125, 250, 500, and 1000 µg/mL) over 24 hours. The culture media was carefully collected and cells cleaned to remove loose nanoparticles following incubation. We focused optical measurements on cell-associated TiO₂ nanoparticles, not extracellular ones. Controlled-density cells were collected for optical examination.

Optical Measurements
An absorbance/transmittance double-beam UV-visible spectrophotometer measured 400-800 nm. Standard 1 cm optical path quartz cuvettes held samples. Before measurement, cell suspensions were adjusted to a density of around 1×10⁶cells/mL. Refractive index readings should be interpreted with caution because reflectance values were acquired indirectly from spectrophotometric data rather than an integrating sphere. After 24-hour nanoparticle exposure and washing, treatment and control HCT116-cell suspensions showed identical cell densities before measurement. All groups had diffuse transmittance. Under identical experimental conditions, spectroscopic data showed reflectance-related optical behaviour. Background contributions were eliminated by baseline correction with appropriate reference media. Controlling all measurements decreased instrumental variability and ensured repeatability.

Optical Parameter Estimation
The Beer-Lambert law was utilised to estimate the effective absorption coefficient (μa) from measured transmittance spectra using first-order approximation. The Beer–Lambert law assumes a homogeneous, non-scattering medium, however cell suspensions with distributed nanoparticles may scatter significantly. Quoted μa values approximate apparent absorption coefficients, which may include scattering losses. The absorbance (A) was calculated:

A = – log₁₀ (T)                                                           

The absorption coefficient was estimated using:

μa = 2.303 A / l                                                         

where l represents the optical path length of the cuvette (1 cm).
The refractive index (n) was calculated from reflectance data using Fresnel’s equation under normal incidence. For non-absorbing or slightly absorbing materials at normal incidence, the relationship between reflectance (R) and refractive index (n) is: R = [(n − 1)/(n + 1)]², where n = (1 + √R)/(1 − √R This approach simplifies refractive index values in turbid cell suspensions by assuming specular reflection at a planar contact. R=measured reflectance. To evaluate concentration-dependent modification from TiO₂ nanoparticle treatment, all visible spectrum features (400-800nm) were studied. This study measured single experimental runs without biological duplicates. This study does not provide statistical metrics such as mean ± standard deviation, error bars, or significance tests (e.g., p-values). Since the findings are descriptive, future studies should use triplicate measurements and statistical analysis to draw quantitative conclusions.

Theoretical Calculations of Photonic and Quantum-Optical Parameters
To extend the experimental observations into a quantum-optical framework, a set of theoretical parameters was derived from the measured absorption coefficient (μₐ) and refractive index (n) at the wavelength of maximum optical response (λmax). These parameters characterize the light–matter interaction within the TiO₂-treated HCT116 cellular medium on a photonic and electronic level [15,16].

Photon energy (Eₐₕ)
The energy carried by a single photon at λmax was calculated using the Planck–Einstein relation:

Eph = hc / λ                                                                

where h is Planck’s constant (6.626 × 10⁻³⁴ J·s) and c is the speed of light in vacuum (3 × 10⁸ m/s). The practical form Eₐₕ(eV) = 1240/λ(nm) was used for direct conversion to electron-volt units.

Extinction coefficient (k)
The imaginary part of the complex refractive index, representing the attenuation of the electromagnetic wave within the medium, was estimated from:

k = μaλ / 4π                                                                 

Complex dielectric function (ε = ε₁ + iε₂)
The real (ε₁) and imaginary (ε₂) components describe, respectively, the polarization response and the dissipative behavior of the medium:

ε1 = n2 – k2 ,        ε2 = 2nk                                      

Optical conductivity (σₒₚₜ)
This parameter quantifies the response of bound electronic charges to the oscillating electromagnetic field:

σopt = αnc / 4π                                                          

where α = μₐ is the absorption coefficient in cm⁻¹, and c is the speed of light in cm/s.
Appearing optical transition energy (E𝓕). The wavelength of maximum absorption (E𝓕 ≈ hc/λmax) was used to estimate the dominating electronic transition energy in the spectral window. The composite cellular–nanoparticle system’s photonic transition is taken into account, as the intrinsic band gap of bulk TiO₂ (~3.2 eV for anatase) is in the UV region beyond the measuring range.

RESULTS AND DISCUSSION
Structural Characterization of TiO₂ Nanoparticles
SEM examination showed that synthesised TiO₂ nanoparticles had a quasi-spherical shape and reasonably uniform spatial distribution. Agglomeration was low and particles were well-dispersed. Image-based tests showed an average particle diameter of 60 nm, indicating nanoscale dimensions adequate for optical interaction in cells.
XRD measurement revealed TiO₂ crystalline anatase phase. The diffraction pattern showed anatase structure peaks, indicating phase purity without secondary phases.

Transmittance Spectral Behavior
The transmittance spectra of control and TiO₂-treated HCT116 cell suspensions were measured in the visible range (400-800 nm). The control group had higher spectrum transmittance than treatment groups. Transmittance increased more in treated groups than controls. In the treated groups, transmittance decreased with higher TiO₂ nanoparticle concentration. Higher nanoparticle concentrations (500 and 1000 µg/mL) resulted in a greater fall in transmitted intensity, indicating increased optical attenuation in treated cellular suspensions. Light penetration and optical absorption are key to therapeutic applications. Thus, tissue optical characteristics are crucial to cancer detection and drug delivery. A UV–Vis spectrophotometer measured reflectance and transmittance. In Fig. 3, reflectance (R) and transmittance (T) measurements are given, with findings in Tables 1 and 2.
Results indicate differing reflectance and transmittance levels in untreated and TiO₂NP-treated HCT116 cells. A mathematical model based on the Beer–Lambert law and Fresnel equations estimated the absorption coefficient and refractive index using reflectance and transmittance measurements. Fig. 4 shows measurements, while Tables 3 and 4 provide results. In the treated groups, increasing nanoparticle concentration increases absorption coefficient and refractive index values, although all values remain lower than the untreated control.

Derived Photonic and Quantum-Optical Parameters
Using experimentally obtained absorption coefficient (μₐ) and refractive index (n), theoretical photonic parameters were derived using equations (1)–(4) in Section 2.6. Included parameters: photon energy (Eₐₕ), extinction coefficient (k), complex dielectric function (ε₁, ε₂), and optical conductivity (σₒₚₜ). The calculated values are in Table 5.
The computed photon energy (Eₐₕ) at λmax ranges from 2.376 to 2.408 eV for all samples, corresponding to the visible spectrum’s green area. The observed optical response in anatase TiO₂ is due to sub-gap absorption within the composite cellular–nanoparticle system, not electronic changes in TiO₂. Sub-gap absorption is linked to defect states, surface trap levels, and oxygen-vacancy-related midgap states in nanomaterials [17,18]. The TiO₂ dose increased the extinction coefficient (k) from 1.558 × 10⁻² in control to 1.483 at 1000 μg/mL, after initially decreasing to 0.617. In the cellular medium, nanoparticle concentration considerably affects electromagnetic wave attenuation. This pattern fits the absorption coefficient. Both dielectric function components (ε₁, ε₂) increased concentration-dependently in treated groups, demonstrating higher polarisation response and dissipative behaviour with increasing nanoparticle load [15,19]. The optical conductivity (σₒₚₜ) showed a concentration-dependent increase from 6.446 × 10¹² s⁻¹ at low concentration to 18.32 × 10¹² s⁻¹ at 1000 μg/mL. Rising TiO₂ concentration leads to higher polarisable nanoparticle–cell interface density, resulting in greater bound charge responsiveness to electromagnetic fields. These quantum-optical characteristics describe how TiO₂ nanoparticles affect light-matter interaction in HCT116 cellular suspensions, supporting the dose-dependent modulation found in primary optical studies [16,20].

Observing the Glowing Light of the Synthesized Nanoparticle During the Photomicrographs
Fig. 5 displays HCT116 colon cancer cell optical micrographs taken under an inverted microscope. The glowing effect in cells treated with TiO₂NPs may be due to the optical scattering and reflective properties of the nanoparticles within the cell membrane. No direct cell viability experiment (such as MTT, XTT, or Trypan blue exclusion) was performed in this work, hence microscopic findings cannot be used to quantify cytotoxicity. Previous research suggests that TiO₂NPs may affect cancer cells based on size and concentration [14]. The morphological changes in Fig. 5 suggest dose-dependent effects, but future studies need specialised viability assays to quantify these findings.
This work shows that TiO₂ nanoparticles considerably impact the optical properties of cellular suspensions. These effects can be explained by photons interacting with nanoscale structures in the biological environment. SEM examination showed that the nanoparticles average 60 nm in diameter. Due to their high surface-to-volume ratio, particles in this size range interact well with visible light. This structural feature increases photon–particle interaction, which impacts light transmission across the cellular medium. XRD showed that the nanoparticles were mostly anatase crystalline. The anatase phase is known for its superior optical and photocatalytic properties compared to other TiO₂ phases. This structural characteristic increases photon absorption and scattering, changing biological samples’ optical sensitivity. As nanoparticle concentration grew, transmission intensity decreased in this study’s transmittance spectrum. Two main mechanisms explain this behaviour. Nanoparticles increase photon scattering in suspension. Additionally, nanoparticles can absorb some radiation. Light intensity decreases due to dispersion and absorption. Additionally, nanoparticle concentration clearly affected absorption coefficient values. Note that all treated groups had lower μa values than the untreated control. Higher TiO₂ concentrations in treated groups led to increased μa values over the tested spectral area. This shows that while sample preparation and nanoparticle treatment modify the optical environment, nanoparticles modulate effective attenuation in the treated cellular medium dose-dependently. Previous investigations using nanostructured materials in biological systems showed concentration-dependent variations in optical attenuation. Refractive index estimation showed measured differences between control and treatment samples. The suspension’s effective optical density may explain treated groups’ higher refractive index. Dispersed nanoparticles in biological media create dielectrically distinct areas. Heterogeneity affects the system’s refractive properties and light propagation. The theoretically obtained optical parameters in Section 3.3 illuminate the physical mechanics. The photon energy at λmax (2.38–2.41 eV) is below the inherent band gap of anatase TiO₂ (~3.2 eV), suggesting that the major absorption mechanism in the spectral window does not involve direct band-to-band transitions in crystalline TiO₂. In nanostructured anatase, sub-gap transitions involving oxygen-vacancy midgap states, surface trap levels, and defect-induced energy states likely control the optical response [17,18,21]. Increased TiO₂ concentration leads to higher attenuation coefficient (k) and imaginary dielectric component (ε₂), suggesting greater photon energy dissipation. Enhanced polarisation response of the composite system is indicated by an increase in the real dielectric component (ε₁). The composite’s optical conductivity (σₒₚₜ) increased from 6.4 × 10¹² s⁻¹ at low concentration to 18.3 × 10¹² s⁻¹ at 1000 μg/mL, indicating that higher nanoparticle densities improve the composite’s electromagnetic response. These quantum-optical parameters rationalise experimental results and link concentration-dependent optical modulation to cellular photonic and electrical systems. Other causes for optical changes include nanoparticle-biological interactions. Nanoparticles of TiO₂ can affect the optical environment by interacting with cell membranes or intracellular structures. Cellular components and media affect scattering and refractive index contrast. The optical characteristics of the cellular solution change when TiO₂ nanoparticles are added. Lower transmittance, increased absorption coefficient, and refractive index change. Nanoparticle-assisted optical methods may detect nanoparticle–cell interactions due to optical changes.

Potential Biomedical Applications
This study shows concentration-dependent optical modulation, which has applications in biological photonics and light-based cancer treatment. The difference in absorption coefficient and refractive index between untreated and TiO₂-treated HCT116 cells enables optical biopsy and non-invasive tumour detection. Diffuse reflectance spectroscopy can distinguish cancerous tissue from healthy tissue. Early colorectal cancer diagnosis improves clinical outcomes, making optical contrast useful [22,23,24].
In photodynamic therapy (PDT), TiO₂ nanoparticles can be used as photosensitisers to generate reactive oxygen species. The concentration-dependent increase in absorption coefficient can help determine optimal nanoparticle dosing and excitation wavelength. Optimising photon energy deposition within target cancer cells while minimising off-target exposure to healthy tissue improves treatment selectivity and reduces side effects [25-27].
Laser-assisted tumour ablation and photothermal therapy are also affected by refractive index modulation. To heat tumour areas without overheating neighbouring anatomical structures, the effective optical density of the cellular medium can be controlled to precisely tune therapeutic light penetration depth. Thus, this study’s dose-response pattern can influence colorectal cancer near-infrared laser treatment planning [28,29].
The illuminating effect in inverted microscopy suggests TiO₂ nanoparticles could be used as contrast agents for fluorescence-based and dark-field optical imaging of colorectal cancer cells, facilitating early diagnosis and image-guided surgery. The quantitative framework linking nanoparticle concentration to measurable optical parameters can be used to design nanoparticle-based optical dosimeters which use the local optical response as a real-time feedback signal to monitor drug delivery, tumour response, and treatment progression during clinical intervention [30].

CONCLUSION
The study examined the impact of TiO₂ nanoparticles on the optical properties of cellular suspensions by visible spectrophotometric measurements. SEM and XRD measurements confirmed the anatase crystalline phase and an average particle size of 60 nm for the nanoparticles. Experimental observations show that TiO₂ nanoparticles alter the optical response of the materials. As nanoparticle concentration increased, transmittance dropped, indicating higher light attenuation in the cellular medium. Nanoparticles increase absorption and scattering, causing this behaviour. Nanoparticle concentration clearly affected optical characteristics including absorption coefficient and refractive index. The absorption coefficient and refractive index of all treated groups were lower than the untreated control, but as nanoparticle concentration increased, these metrics increased. Research indicates that TiO₂ nanoparticles alter the optical characteristics of cells and impact light propagation in the biological medium in a concentration-dependent manner. These findings suggest nanostructured materials may affect biological systems’ optical properties. The theoretically determined quantum-optical parameters—photon energy, extinction coefficient, complex dielectric function, and optical conductivity—matched experimental trends and provided a photonic explanation of nanoparticle-induced modulation. The changes in optical properties may help investigate nanoparticle–cell interactions and create nanoparticle-assisted optical diagnostic methods.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.