Development of PMMA/TiO₂ Nanocomposites and Study Morphology, Optical, Thermal (DSC) and Biological Properties

Although numerous studies have addressed PMMA/TiO₂ nanocomposites and their diverse properties, many have focused on a specific aspect without comprehensively integrating structural, optical, thermal, and biological properties. [1,2] Furthermore, the effect of adding TiO₂ nanoparticles at different concentrations on improving PMMA polymer performance still requires further investigation and the exploration of alternative preparation methods. [3] Therefore, the novelty of this study lies in its preparation of PMMA/TiO₂ nanocomposites and the investigation of the effect of TiO₂ nanoparticles on morphological, optical, and thermal properties using DSC technology, in addition to evaluating biological properties using the casting method. The aim is to develop multifunctional nanomaterials that can be used in advanced medical and optical applications. Polymers such as plastics and rubbers pervade our lives, and we come across them in many dierent forms. Their physical properties are therefore of great importance, and an understanding of them is vital for their use in technology and engineering [4]. Composite materials constitute an advanced engineering system wherein two or more components of differing characteristics are combined to achieve performance attributes that surpass those of the individual pieces [5]. Historically, reinforcement has been supplied by constituents such as fibers, whiskers, or particulates in metals, ceramics, or polymers, improving mechanical, thermal, and chemical properties for diverse applications, including aerospace, structural materials, biomedical devices, and energy technologies [6,7]. Polymethyl methacrylate (PMMA) is produced via the free radical polymerization of methyl methacrylate and is distinguished by its colorless and transparent characteristics. The light transmittance surpasses that of other polymers. Additionally, it is lightweight, durable, demonstrates significant mechanical strength at room temperature, has low water absorption, is resistant to water and specific dilute acids and inorganic salts, endures long-chain alkanes and ethers, is non-toxic, and is flame-retardant upon ignition [8]. The fabrication of composite materials using nanoparticles poses difficulties in attaining a homogeneous particle distribution inside the matrix; nonetheless, it facilitates the creation of composites with varied properties for practical uses. These composites consist of one or more types of nanoparticles of diverse shapes and sizes integrated inside the matrix material. They differ from traditional composites in the distribution of the integrated components, which is partially random yet controlled. The nanocomposites demonstrate isotropy, indicating that their properties are consistent in all directions. This reinforcement approach enables particles to distribute the load with the matrix material, so augmenting its stiffness, creep resistance, impact strength, and resistance to wear and fatigue, while also conferring specialized attributes to achieve the intended goals [9,10]. Metal oxides (including TiO2 [11,12], SiO2, and ZnO [13]) and carbon-based nanofillers have been extensively studied to improve structural, electrical, and optical characteristics [14,15]. The effectiveness of these reinforcements is highly dependent on establishing uniform dispersion throughout the matrix and managing interfacial interactions that control stress transfer and property development [16]. TiO2 nanoparticles have attracted considerable attention owing to their several advantages, such as a high refractive index, photoactivity throughout the UV spectrum, and UV absorption properties, for instance [17]. Their integration into PMMA matrices has been shown to enhance thermal stability, elevate Tg, and augment optical properties as well [18]. The surface characteristics of these oxide nanoparticle species are hydrophilic owing to the presence of hydroxyl groups [19]. To effectively disseminate the nanoparticle within a hydrophobic polymer matrix, enhancement of the interfacial interaction is essential. Modification of the nanoparticle surface via various interfacial techniques has been implemented. These procedures entail the encapsulation of nanoparticles through emulsion polymerization in PMMA [20]. Nonetheless, TiO2 nanoparticles have significant polarity and elevated surface energy; thus, they may agglomerate at increased loading levels, diminishing their reinforcing efficacy. This challenge has prompted endeavors to alter surface particles to reduce particle-particle interactions and enhance uniform distribution inside the organic matrix. Utilizing these approaches, PMMA/TiO2 nanocomposites exhibit enhanced performance in structural and functional applications across optical, electrical, and biological domains [21]. The Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial drug that inhibits observable growth of a microbe following a certain incubation period. The microtiter plate (MTP) broth microdilution method is a prevalent technique for determining minimum inhibitory concentration (MIC) due to its rapidity, cost-effectiveness, reproducibility, and high-throughput capability, enabling the simultaneous testing of numerous isolates and concentrations in a 96-well plate. In MTP MIC testing, bacterial growth is assessed in liquid medium across a serial dilution series of the tested compound(s). Growth can be read by turbidity/optical density, but many laboratories use colorimetric viability indicators to improve clarity—especially when extracts, nanoparticles, or colored compounds may interfere with turbidity assessment. Resazurin is a redox dye used as a cell viability indicator. In its oxidized form, it appears blue/purple; metabolically active (viable) bacteria reduce it to resorufin, which becomes pink (and may further reduce to a colorless form in strongly reducing conditions). Therefore: Blue/purple = no detectable metabolic activity (growth inhibited) and Pink = active metabolism (growth present). Using resazurin provides a clear endpoint for MIC determination, especially when broth becomes slightly cloudy or when test agents affect optical readings [22,23]. In this research, PMMA/TiO2 nanofilms will be fabricated and the effect of titanium on their structural, morphological and biological properties will be studied.

 

 

Poly(methyl methacrylate) (PMMA) [CH₂C(CH₃)(CO₂CH₃)] – ~800,000 g/mol – A vonchem Ltd., UK, Chloroform [CHCl₃]– 119.38 g/mol – ACS Chemicals, India – 99%.

 

 

The solution-casting method was used to make the composite nanofilms. To make a very uniform solution, 1 g of PMMA granules was completely dissolved in 40 mL of chloroform in a glass beaker with magnetic stirring at 25 °C for 1 hour. We took 10 mL of this solution and used it as a base polymer to make more composite structures. The amount of TiO₂ nanoparticles added to the PMMA solution ranged from 5 wt%. Ultrasonic treatment was applied to the suspensions for 8 minutes to ensure equal distribution of nanoparticles and inhibit agglomeration. When you get the nanoparticle suspension.

 

 

Utilizing a ZEISS SIGMA VP field emission scanning electron microscope (FESEM) type. Absorption spectra and Surface Plasmon Resonance (SPR) were obtained for colloids using a Double Beam 1800 UV Spectrometer produced by Shimadzu, Japan, together with Differential Scanning Calorimetry (DSC) analysis. The thermal properties of the fabricated films were analyzed using differential scanning calorimetry (STA. PT-1000 Linseis). The system was calibrated with 9-19 mg of alumina at a temperature ramp rate of 10°C/min in the presence of argon gas.

 

 

The DPPH radical scavenging experiment was used to test how well the nano-compounds worked as antioxidants, as described in [24]. We made a new 0.1 mM DPPH solution in methanol. One milliliter of each nano-compound was added to one milliliter of DPPH solution at concentrations above, at, and below the MIC. The mixtures were kept in the dark at room temperature for 30 minutes, and the absorbance was measured at 517 nm. Ascorbic acid was the positive control. We used the formula [24] to figure out the percentage of radical scavenging activity.

 

 

% Scavenging activity = 100 × [(Acontrol - Asample) / Acontrol]

 

 

Where, Acontrol is the absorbance of DPPH solution without sample, Asample is the absorbance with the test compound.

 

 

Consumables: Sterile 96-well flat-bottom microtiter plates with lid, Sterile microtubes (1.5–2 mL) and Sterile pipette tips (10, 200, 1000 µL). Reagents & Media: Mueller–Hinton Broth (MHB), Solvent for stock preparation (e.g., sterile water / DMSO) and Resazurin solution (commonly prepared as 0.01–0.02% w/v; see below). Microbiology items: Fresh bacterial culture (18–24 h) on suitable agar. Equipment: Micropipettes (10–100 µL, 100–1000 µL) and Incubator at 35–37°C.

 

 

A) Preparation of stock solutions: Prepare a concentrated stock of each tested agent.

B) Preparation of resazurin working solution

A commonly used approach:

Prepare 0.01% w/v resazurin in sterile distilled water (e.g., 10 mg in 100 mL).

Mix until dissolved, filter sterilize, and store protected from light (amber tube) at 4°C.

C) Standardization of bacterial inoculum

Pick 3–5 well-isolated colonies from a fresh agar plate (18–24 h).

Suspend in saline/MHB to match 0.5 McFarland (≈1–2 × 10⁸ CFU/mL for many bacteria).

Dilute this suspension in MHB to obtain the final inoculum used in wells.

Typical final inoculum in each well is around 5 × 10⁵ CFU/mL.

Keep inoculum fresh and use within a short time (avoid long standing).

D) Plate setup (example workflow)

Dispense broth: Add 100 µL MHB to all test wells that will contain dilutions.

Prepare serial two-fold dilutions (2× dilutions)

There are two common ways:

Method 1 (in-plate serial dilution):

Add 100 µL of the test agent at the highest concentration to the first well of the dilution row/column.

Mix well (pipette up/down 5–8 times).

Transfer 100 µL from the first well to the next well (already containing 100 µL broth) to make a two-fold dilution.

Continue across the row/column.

Discard 100 µL from the last well to equalize volumes.

Method 2 (pre-dilution in tubes then dispense):

Prepare the two-fold dilution series in tubes, then add equal volumes to plate wells.

3) Add bacterial inoculum

Add 100 µL of the prepared inoculum to each well containing 100 µL of the diluted agent.

Final volume per well becomes 200 µL.

This ensures the test agent concentration is at the intended final level (because dilutions were made assuming this final volume).

E) Controls (essential)

Include these controls in every plate:

Sterility control (Media only):

MHB (no bacteria, no agent).

Confirms no contamination.

Growth control (Positive control):

MHB + bacteria (no agent).

Confirms organism viability and adequate growth.

F) Incubation

Cover the plate and incubate at 35–37°C for 18–24 hours (typical for many bacteria).

Avoid shaking (shaking may change nanoparticle behavior).

G) Resazurin addition and second incubation

After the main incubation, add 10–20 µL of resazurin working solution to each well (keep it consistent).

Incubate again at 35–37°C for 1–3 hours (time depends on organism metabolic rate and inoculum).

Protect the plate from strong light if possible.

H) Reading results and determining MIC

Visual endpoint:

Pink wells = growth (viable bacteria).

Blue/purple wells = no detectable growth/metabolic activity.

MIC definition:

The MIC is the lowest concentration of the tested agent at which the well remains blue/purple (i.e., no color change to pink) compared with the growth control.

 

 

Field-emission scanning electron microscopy (FESEM) images of PMMA and composites Morphological analysis was performed using FESEM images shown in Figs. 2 and 3. Particle strengthening effects in composites play a crucial role in determining their physical properties [2]. The field emission scanning electron microscopy (FESEM) images in Fig. 2 of the PMMA film show a smooth, spherical surface, consistent with a uniform distribution across the surface. Fig. 3 show the PMMA/TiO2 composition with weight percentages (5 wt%) of TiO2 loaded into the PMMA matrix. It is noted that the 5 wt% PMMA/TiO2 mixture film indicates that the TiO2 nanoparticles are uniformly distributed across the PMMA matrix. They appear as small granules, We observe that the 5% PMMA/TiO2 film has a lumpy structure with small granules on the surface. Adding TiO2 to the PMMA polymer matrix indicates changes in the morphology, which may improve its physical properties [26-28].

 

 

Ultraviolet and visible spectroscopy is used to study the optical properties of (PMMA)/TiO2 composite nanofilms with varying content of TiO2 nanoparticles. Fig. 4 shows the surface plasmon resonance peaks (SPR) obtained for the colloidal solutions of TiO2 nanoparticles as they were nearly constant from (289nm). The UV–Visible absorption spectrum of the PMMA/TiO2 nanocomposite thin films is shown in Fig.4.

The energy gap (Eg) for the allowed indirect transition was calculated using equation [29]:

 

𝛼ℎ𝜐 ≈ 𝛣(ℎ𝜐 − 𝐸𝑔 )1/2                                                      

 

 A tangent is drawn from the best straight line that crosses most points beyond the absorption edge. Table 3 shows the energy gap values for all of the composite films. The table shows that the energy gap value for the pure PMMA film is (4.67) eV. However, when 5wt% TiO2 are added, the energy gap value goes down a little. This result is in line with what was found in earlier studies. Adding small amounts of reinforcement material to the base material (PMMA) makes the energy gap value lower. This is because the reinforcement material makes groups of transmitted charge. These clusters improve electrical conductivity by adding more charges. Moreover, the presence of different reinforcement materials leads the reinforced molecules to close the gap between two local energy levels. This lowers the potential barrier and makes it easier for charge carriers to move between these levels [30]. 

 

 

One of the properties of glass in polymers is that their mechanical and physical properties change at a certain temperature called the resistance to glass transition, which is usually denoted by (Tɡ). It can be defined as the temperature at which a polymer changes from a strong, rigid material to a flexible material above the glass transition, becoming a flexible polymer. [31]

Plastics possess a range of different transformations (transitions) under the influence of high temperature. When purified at a soft temperature below the glass transition point, the plastic material is a rigid solid. Upon transition, it transforms into an elastic plastic material, and then into an elastic state in the case of a non-ductile plastic, and into a highly viscous liquid in the case of a thermoplastic plastic [32]. The glass transition point depends on the number of atoms and molecules in the plastic material. Therefore, the more complex the structure, the greater the capacitance, and the higher the maximum weight possessed by the plastic material, the higher its transition point.

The glass transition temperature of the pure PMMA polymer films reinforced with 5wt % titanium oxide nanoparticles was calculated using a differential calorimeter (DSC). Fig. 6 shows the glass transition temperature values of the prepared films, where we note that the glass transition temperature value of the pure PMMA film is (104.7 ºC) [32]. Studies indicate that adding TiO₂ nanoparticles to polymethyl methacrylate (PMMA) alters the glass transition temperature (Tg), with the value depending on the filler ratio and the preparation method. It has been observed that Tg can fall within a range of 60–70 °C in some modified systems, showing a slight increase or decrease compared to the pure polymer, due to the interaction between the polymer chains and the nanoparticles.[33] Several recent studies have shown that the introduction of TiO₂ improves thermal stability and affects the mobility of polymer chains, leading to a wide range of Tg values ​​that can be either low or high compared to the pure polymer [34].

 

Table 2 presents the statistical analysis of the DPPH radical scavenging activity of TiO₂ nanoparticles at three tested concentrations. The findings reveal a clear concentration-dependent antioxidant effect, with a highly significant overall difference among concentrations (P < 0.001). The highest concentration (1000 µg/ml) showed the greatest scavenging activity, with a mean inhibition percentage of 83.98 ± 0.99%, followed by 500 µg/ml (80.99 ± 0.99%), whereas the lowest concentration (250 µg/ml) exhibited a markedly lower activity (45.93 ± 1.14%). The distinct superscript letters indicate that each concentration differed significantly from the others, confirming that the antioxidant capacity declined significantly as the concentration decreased. This trend is consistent with the principle of the DPPH assay, in which higher scavenging percentages reflect a greater ability of the tested material to donate electrons or hydrogen atoms to neutralize free radicals [35,36]. 

From a functional perspective, the high inhibition values observed at 1000 and 500 µg/ml suggest that the tested TiO₂ nanoparticles possessed substantial in vitro antioxidant potential within the upper concentration range. The relatively small difference between these two concentrations indicates that the nanoparticles retained a high level of radical scavenging efficiency even after a twofold reduction in concentration. In contrast, the marked decline at 250 µg/ml suggests that the antioxidant response became considerably weaker once the nanoparticle concentration dropped below a more effective threshold. Similar concentration-dependent patterns have been described for TiO₂ nanoparticles in previous studies, particularly in green-synthesized systems, where antioxidant activity increased with dose and was influenced by nanoparticle size, surface chemistry, and synthesis-associated bioactive residues [37,38]

The strong activity recorded at 1000 µg/ml is particularly noteworthy because it approaches the level usually associated with potent antioxidant systems in DPPH assays. Although TiO₂ is more commonly discussed in relation to photocatalytic and surface-reactive properties, an increasing body of evidence indicates that TiO₂ nanoparticles can also exhibit appreciable antioxidant behavior under in vitro chemical assay conditions, especially when synthesized through biological or plant-mediated routes. In such systems, the observed scavenging effect may reflect not only the physicochemical characteristics of the TiO₂ core itself but also the contribution of surface-bound organic compounds that enhance redox reactivity [37].

The MIC-based susceptibility ranking demonstrated that the tested microorganisms differed clearly in their response to TiO₂ nanoparticles, indicating variable antimicrobial sensitivity across bacterial and fungal species. Among all tested organisms, Streptococcus pyogenes and Candida albicans showed the highest susceptibility, each with an MIC of 16 µg/ml, whereas Escherichia coli showed an intermediate response with an MIC of 32 µg/ml. In contrast, Staphylococcus aureus and Klebsiella pneumoniae exhibited the lowest susceptibility, both requiring 64 µg/ml for growth inhibition. Since lower MIC values reflect greater antimicrobial potency, these results indicate that TiO₂ nanoparticles were most effective against S. pyogenes and C. albicans, moderately effective against E. coli, and comparatively less effective against S. aureus and K. pneumoniae. This general interpretation is consistent with the established use of MIC values as a quantitative indicator of antimicrobial activity, where lower concentrations indicate stronger inhibitory performance [39,40]. 

The strong susceptibility of S. pyogenes to TiO₂ nanoparticles is biologically plausible, as Gram-positive organisms can be highly affected by nanoparticle-mediated membrane disruption, oxidative stress, and surface interaction, although susceptibility patterns are not always uniform across all Gram-positive bacteria. The lower MIC observed for S. pyogenes compared with S. aureus in the present results suggests that species-specific surface properties, metabolic behavior, or differences in the interaction between nanoparticles and cell envelope structures may have influenced the antimicrobial response. Reviews of TiO₂ nanoparticle antimicrobial activity have similarly noted that susceptibility cannot be predicted solely on the basis of Gram reaction, because microbial sensitivity depends on multiple factors, including nanoparticle size, surface charge, crystallinity, and the physiological properties of the target organism [40,41]. 

The antifungal result against Candida albicans is also noteworthy. The MIC of 16 µg/ml places C. albicans among the most susceptible tested organisms, suggesting that TiO₂ nanoparticles possessed appreciable antifungal activity in addition to their antibacterial effects. This broad-spectrum performance agrees with previous reports showing that TiO₂-based nanomaterials can inhibit fungal cells through oxidative stress generation, membrane-associated damage, and interference with essential cellular processes. Published evidence has likewise highlighted that TiO₂ nanoparticles may act against Candida species and other fungi, especially when the particles possess high surface reactivity or are used in formulations optimized for antimicrobial performance [42,43]. 

The intermediate susceptibility of E. coli, with an MIC of 32 µg/ml, suggests that TiO₂ nanoparticles were also active against this Gram-negative bacterium, although to a lesser extent than against S. pyogenes and C. albicans. This observation is important because Gram-negative bacteria are often considered more difficult to inhibit due to the presence of an outer membrane that can limit the penetration of antimicrobial agents. Nevertheless, TiO₂ nanoparticles may still exert inhibitory effects against such bacteria through direct contact, reactive oxygen species generation, and destabilization of membrane integrity. Previous studies have reported antimicrobial activity of TiO₂ nanoparticles against E. coli, supporting the interpretation that the present result reflects genuine inhibitory potential rather than an isolated finding [44,40]. 

By contrast, S. aureus and K. pneumoniae showed the highest MIC values (64 µg/ml), indicating the lowest susceptibility among the tested isolates. Although these MICs still reflect measurable antimicrobial activity, they suggest that higher nanoparticle concentrations were necessary to achieve inhibition in these organisms. In the case of K. pneumoniae, this may be related to the known structural and protective features of Gram-negative pathogens, including the outer membrane and, in some strains, capsule-associated defense. For S. aureus, the reduced sensitivity relative to S. pyogenes may reflect species-level differences in tolerance to oxidative or membrane-active stress. Similar variability among bacterial species has been reported in previous TiO₂ nanoparticle studies, emphasizing that antimicrobial performance may differ considerably depending on the target organism and the physicochemical characteristics of the nanoparticles used [45,40]. 

Overall, this table demonstrates that the antimicrobial activity of TiO₂ nanoparticles was species-dependent, with the strongest effect observed against S. pyogenes and C. albicans, moderate activity against E. coli, and lower activity against S. aureus and K. pneumoniae. The fold-difference values further emphasize this pattern, showing that the least susceptible organisms required four times the MIC needed for the most susceptible ones. Collectively, these findings support the conclusion that TiO₂ nanoparticles possess broad antimicrobial potential, but that their inhibitory efficiency varies according to the biological characteristics of the target microorganism and the interaction between the nanoparticle surface and microbial cell structures [40,46].

 

 

 This study’s results indicated that the integration of titanium dioxide nanoparticles (at 5 wt%) into a polymethyl methacrylate (PMMA) matrix effectively yielded multifunctional nanocomposites with improved optical and functional characteristics. Field emission scanning electron microscopy (FESEM) coupled with ImageJ analysis shown that augmenting the TiO₂ loading, especially at 0.6 g, promoted uniform dispersion and diminished agglomeration, resulting in a reduction of particle sizes from approximately 52.88 nm to around 40 nm. The composites demonstrated superior thermal stability compared to pure PMMA, while there was no alteration in the glass transition temperature (Tg) between the polymer and the composites. This study established that free radicals can be employed in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to assess the capacity of materials to function as free radical scavengers or hydrogen donors, offering a speedy, straightforward, and cost-effective approach for evaluating antioxidant capabilities. The DPPH analysis approach employs a low concentration of DPPH, a stable free radical. The DPPH free radical interacts with a single electron, resulting in absorption at a peak wavelength of 517 nm (violet hue). Antioxidants interact with DPPH, in the presence of a hydrogen donor (e.g., a free radical scavenger), converting DPPH to DPPHH and diminishing DPPH absorption. In contrast to the DPPH-H form, decolorization (i.e., yellowing) transpires with an increase in electron acquisition. The decolorization capability correlates with the diminishing capacity. The synthesized PMMA/titanium dioxide nanoparticles exhibit antibacterial characteristics.

 

 

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