Bio-Fabrication and Characterization of Zinc Sulfide/Polymer Nanoparticles for Application in Controlled Release of Vitamin B2

INTRODUCTION
Biofabrication of nanoparticles has emerged as a transformative approach that leverages biological milieus to synthesize and shape nanomaterials under mild, aqueous, and environmentally benign conditions [1-3]. Historically, early demonstrations in the 20th century relied on abiotic chemical routes, but the paradigm shifted with the advent of biologically mediated protocols that employ plant extracts, microorganisms, and biomimetic polymers to drive reduction, stabilization, and templating processes [4-8]. The allure of biofabrication lies in its intrinsic compatibility with green chemistry principles, enabling scalable production while minimizing hazardous reagents and byproducts. Mechanistically, diverse biological reducing agents polyphenols, proteins, amino acids, polysaccharides offer dual roles as reducing agents and capping/stabilizing ligands, often imparting biocompatible surfaces and functionalization handles [9-12]. Importantly, biofabricated nanoparticles have found utility across sectors such as biomedicine (drug and gene delivery, imaging, theranostics) [13], catalysis (sustainable redox transformations) [14], sensing (biosensors and environmental monitoring) [15], and energy (photocatalysis and electrocatalysis) [16]. In the context of controlled release and smart materials, bio-derived syntheses enable intimate integration with polymeric matrices, enabling tunable particle matrix interactions, biodegradability, and responsive release profiles [17]. The intersection of biofabrication with zinc sulfide systems further opens avenues for optoelectronic applications and bioimaging [18], while concurrently addressing toxicity and regulatory considerations through the use of biocompatible capping agents [19]. Overall, biofabrication represents a convergent strategy that harmonizes material performance with ecological stewardship, offering a versatile platform for designing functional nanomaterials tailored to interdisciplinary challenges.
Controlled release of drugs has evolved from early simple diffusion-based systems to sophisticated, stimuli-responsive platforms that provide precise spatiotemporal delivery [20-24]. Historically, initial approaches relied on inert matrices and diffusion barriers to slow drug liberation, offering modest improvements in dosing regularity. The field gained momentum with the advent of polymer chemistry and nanoscale carriers, enabling more predictable kinetics, protection of labile drugs, and targeted delivery [25]. Modern controlled-release strategies encompass matrix systems, reservoir devices, poly(lactic-co-glycolic acid) (PLGA) and other biodegradable polymers, lipid-based carriers, and inorganic/hybrid nanomaterials, often incorporating stimuli-responsive elements such as pH, temperature, enzymatic activity, redox potential, or magnetic fields [26, 27]. The importance of these systems lies in improving therapeutic outcomes by smoothing plasma concentration profiles, reducing dosing frequency, enhancing patient adherence, and minimizing systemic toxicity. Applications span a broad spectrum: chronic disease management, cancer chemotherapy, antibacterial therapies, vaccine delivery, and nutraceuticals, as well as tissue engineering and implantable devices where controlled release can modulate local microenvironments. In the context of vitamin delivery, controlled-release constructs can stabilize labile vitamins, mitigate degradation, and provide sustained bioavailability, which is particularly relevant for nutrients like vitamin B2 (riboflavin) (Fig. 1) that are sensitive to light and metabolic turnover [28-30]. The ongoing integration of organic polymers with inorganic or hybrid nanomaterials such as zinc sulfide polymer systems offers opportunities to tailor release kinetics, protect cargo, and enable multifunctional platforms for imaging, sensing, and responsive therapy, while attentively addressing biocompatibility and regulatory considerations.
This study aims to synthesize and characterize zinc sulfide/ poly(methyl methacrylate) nanoparticles (PMMA-ZnS) via a bio-fabrication approach and to evaluate their effectiveness as controlled-release carriers for vitamin B2, integrating structural, optical, and release-profile analyses to establish correlations between nanoparticle architecture, matrix interactions, and release kinetics for potential applications in nutraceutical delivery and bioimaging-enabled platforms.

MATERIALS AND METHODS
General Materials and Instrument
Zinc sulfide precursor and related reagents: zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O), thioacetamide (TAA, ≥98%), and any dopants or surface-modifying species as required by your bio-fabrication protocol. Monomer and polymer matrix: methyl methacrylate (MMA, inhibitor-free, stabilized as appropriate), solvent system(s) compatible with PMMA encapsulation (e.g., ethanol, acetonitrile, or water–ethanol mixtures) and any crosslinking agents or initiators used for in situ polymerization within the bio-enabled synthesis. Vitamin B2: riboflavin (≥98%, USP or analytical grade) used for loading and release studies. Biologically derived reducing/stabilizing agents: plant extracts, polyphenols (gallic acid), or other benign reductants consistent with your bio-fabrication approach. Buffers and solvents: deionized water (18.2 MΩ·cm), ethanol (EtOH, analytical grade), phosphate-buffered saline (PBS, 10×, sterile), dialysis membranes (appropriate molecular weight cutoff), and any other solvent systems specified in your experimental design. Cleaning and purity reagents: acetone, isopropanol, and standard lab-grade acids/bases for sample preparation and cleaning steps. FE-SEM (Field-Emission Scanning Electron Microscopy): to assess morphology and size distribution. Example model configurations you may use or substitute with your actual instrument: a high-resolution FE-SEM equipped with secondary-electron (SE) and backscattered-electron (BSE) detectors, capable of imaging with accelerating voltages in the range of 1–5 kV for non-conductive samples after a thin conductive coating. Document the exact instrument you own (brand/model), operating voltage, working distance, and any in-lens detectors. Example phrasing for your manuscript once you fill in the specifics: “Morphology and size analysis were performed using a FE-SEM (ZEISS SIGMA VP). Samples were coated with a ~5 nm conductive layer prior to imaging. Images were captured at an accelerating voltage of 150 kV and a working distance of 15 mm. FT-IR (Fourier Transform Infrared Spectroscopy): to probe chemical functionality and interactions between ZnS, PMMA, and loaded vitamin. Commonly used configurations include ATR-FTIR or transmission (KBr) methods. Provide exact instrument and accessory: FT-IR spectra were collected on a [PerkinElmer Spectrum Two (with ATR)] equipped with an ATR accessory (or KBr pellets), scanning 4000–400 cm⁻¹ at 4 cm⁻¹ resolution with 64 scans per spectrum. TGA (Thermogravimetric Analysis): to determine organic content, thermal stability, and composition. Provide model and operating conditions: TGA measurements were carried out on a [Netzsch TG 209 F1 Libra or STA 449 F3 Jupiter] under a nitrogen atmosphere from room temperature to 800 °C at a heating rate of 10 °C·min⁻¹. A second run in air may be reported if relevant for oxidation behavior.

Preparation of zinc sulfide/poly(methyl methacrylate) nanoparticles (PMMA-ZnS)
The PMMA-ZnS nanocomposites were synthesized through a bio-facilitated precipitation-polymerization approach. In a typical procedure, zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, 0.50 g, 2.28 mmol) was dissolved in 20 mL of deionized water under constant stirring at 40 °C. Separately, sodium sulfide nonahydrate (Na₂S·9H₂O, 0.48 g, 2.00 mmol) was dissolved in 20 mL of deionized water. The Na₂S solution was added dropwise to the zinc acetate solution over 30 minutes while maintaining vigorous stirring (800 rpm) at 40 °C, resulting in the formation of ZnS nanoparticles. Concurrently, methyl methacrylate (MMA, 5.0 mL, 47 mmol) was mixed with 50 mL of ethanol containing polyvinylpyrrolidone (PVP, MW ≈ 40,000, 0.20 g) as a stabilizer. The freshly prepared ZnS nanoparticle suspension was then added to the MMA solution, followed by the addition of azobisisobutyronitrile (AIBN, 0.05 g, 0.30 mmol) as a radical initiator. The reaction mixture was heated to 70 °C under nitrogen atmosphere and maintained for 6 hours with continuous stirring (500 rpm) to allow for complete polymerization. The resulting PMMA-ZnS nanocomposites were purified by centrifugation (1500 rpm, 20 minutes) and washed three times with ethanol to remove unreacted monomers and excess PVP. The final product was dried under vacuum at 50 °C for 24 hours, yielding a white powder (yield: 82±3%) [31]. 

Vitamin B2 loading on (PMMA-ZnS) nanocomposites
The loading of vitamin B2 (riboflavin) onto PMMA-ZnS nanocomposites was performed via an adsorption-diffusion method under controlled conditions. In a typical procedure, PMMA-ZnS nanoparticles (100 mg) were dispersed in 10 mL of phosphate-buffered saline (PBS, pH 7.4, 10 mM) using ultrasonication (30 W, 20 kHz, 5 min pulses with 1 min intervals to prevent overheating). A riboflavin solution (5 mL, 1.0 mg/mL in PBS, prepared fresh and protected from light) was added dropwise to the nanoparticle suspension under gentle magnetic stirring (200 rpm) at 25 ± 1°C. The mixture was shielded from light using amber glassware and stirred continuously for 24 h to ensure equilibrium adsorption. After incubation, the riboflavin-loaded nanoparticles (PMMA-ZnS-RF) were separated by centrifugation (1200 rpm, 15 min, and 4 °C) and washed twice with PBS to remove unbound vitamin molecules [32]. 

Controlled release performance of PMMA-ZnS-RF nanocomposites
For controlled release studies, the PMMA-ZnS-RF nanocomposites (50 mg) were resuspended in 50 mL of PBS (pH 7.4 or 5.5 to simulate physiological and acidic conditions) and transferred into dialysis bags (MWCO 12–14 kDa). The bags were immersed in 200 mL of release medium under sink conditions (37 ± 0.5°C, 100 rpm). Aliquots (2 mL) were withdrawn at predetermined intervals (0.5, 1, 2, 4, 6, 12, 24, 48 h) and replaced with fresh PBS to maintain constant volume. Riboflavin release was quantified using UV-Vis spectroscopy, and cumulative release (%) was plotted against time to evaluate kinetics.

RESULTS AND DISCUSSION
Preparation and characterization of PMMA-ZnS-Vitamin B2 nanocomposites
The PMMA-ZnS nanocomposites were synthesized through a bio-facilitated precipitation-polymerization approach, as illustrated in Fig. 2. In a typical procedure, zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, 0.50 g, 2.28 mmol) was dissolved in 20 mL of deionized water under constant stirring at 40 °C. Separately, sodium sulfide nonahydrate (Na₂S·9H₂O, 0.48 g, 2.00 mmol) was dissolved in 20 mL of deionized water. The Na₂S solution was added dropwise to the zinc acetate solution over 30 minutes while maintaining vigorous stirring (800 rpm) at 40 °C, resulting in the formation of ZnS nanoparticles. Concurrently, methyl methacrylate (MMA, 5.0 mL, 47 mmol) was mixed with 50 mL of ethanol containing polyvinylpyrrolidone (PVP, MW ≈ 40,000, 0.20 g) as a stabilizer. The freshly prepared ZnS nanoparticle suspension was then added to the MMA solution, followed by the addition of azobisisobutyronitrile (AIBN, 0.05 g, 0.30 mmol) as a radical initiator. The reaction mixture was heated to 70 °C under a nitrogen atmosphere and maintained for 6 hours with continuous stirring (500 rpm) to allow for complete polymerization. The resulting PMMA-ZnS nanocomposites were purified by centrifugation (10,000 rpm, 20 minutes) and washed three times with ethanol to remove unreacted monomers and excess PVP. The final product was dried under vacuum at 50 °C for 24 hours, yielding a white powder with a yield of 82 ± 3%. For vitamin B2 (riboflavin) loading, the PMMA-ZnS nanocomposites (100 mg) were dispersed in 10 mL of phosphate-buffered saline (PBS, pH 7.4, 10 mM) using ultrasonication (30 W, 20 kHz, 5 min pulses with 1 min intervals to prevent overheating). A riboflavin solution (5 mL, 1.0 mg/mL in PBS, prepared fresh and protected from light) was added dropwise to the nanoparticle suspension under gentle magnetic stirring (200 rpm) at 25 ± 1 °C. The mixture was shielded from light using amber glassware and stirred continuously for 24 h to ensure equilibrium adsorption. After incubation, the riboflavin-loaded nanoparticles (PMMA-ZnS-RF) were separated by centrifugation (12,000 rpm, 15 min, and 4 °C) and washed twice with PBS to remove unbound vitamin molecules. This method ensured the successful encapsulation of riboflavin within the PMMA-ZnS matrix, providing a stable and efficient system for controlled release applications. The reproducibility of the synthesis and loading processes was confirmed through triplicate experiments, with consistent yields and loading efficiencies observed.
FE-SEM was employed to investigate the morphology and structural characteristics of the synthesized PMMA-ZnS nanocomposites. As depicted in Fig. 3, the FE-SEM micrographs revealed a uniform distribution of spherical ZnS nanoparticles embedded within the PMMA matrix, confirming the successful formation of the nanocomposite structure. The ZnS nanoparticles exhibited an average diameter of 25 ± 5 nm, with minimal aggregation, indicating the effectiveness of the bio-facilitated precipitation-polymerization approach in controlling particle size and dispersion.
FT-IR spectroscopy was employed to investigate the chemical interactions and structural characteristics of the synthesized materials, as illustrated in Fig. 4. The spectra of vitamin B2 (riboflavin, Fig. 4a), ZnS nanoparticles (Fig. 4b), PMMA-ZnS nanocomposites (Fig. 4c), and PMMA-ZnS-vitamin B2 (PMMA-ZnS-RF, Fig. 4d) were analyzed to confirm successful nanocomposite formation and drug loading. The FT-IR spectrum of pure riboflavin (Fig. 4a) exhibited characteristic absorption bands at 3320 cm⁻¹ (O–H and N–H stretching vibrations), 1705 cm⁻¹ (C=O stretching of the isoalloxazine ring), 1620 cm⁻¹ (C=N stretching), and 1530 cm⁻¹ (C–C aromatic ring vibrations) [33]. Additional peaks at 1450 cm⁻¹ and 1380 cm⁻¹ were attributed to C–H bending and C–N stretching, respectively, consistent with the molecular structure of riboflavin [34]. The spectrum of ZnS nanoparticles (Fig. 4b) displayed a broad absorption band below 800 cm⁻¹, corresponding to Zn–S stretching vibrations, confirming the formation of the inorganic ZnS phase [35, 36]. The absence of organic functional groups in this spectrum validated the purity of the synthesized ZnS nanoparticles. The FT-IR spectrum of PMMA-ZnS (Fig. 4c) revealed characteristic peaks of PMMA, including 1725 cm⁻¹ (C=O ester stretching), 1450 cm⁻¹ (C–H bending), and 1150 cm⁻¹ (C–O–C stretching) [37]. The presence of ZnS was confirmed by a weak but discernible absorption band near 750 cm⁻¹, attributed to Zn–S interactions. The shift in the C=O stretching frequency (from 1730 cm⁻¹ in pure PMMA to 1725 cm⁻¹ in PMMA-ZnS) suggested weak interfacial interactions between the PMMA matrix and ZnS nanoparticles, likely through coordination or hydrogen bonding. The spectrum of PMMA-ZnS-RF (Fig. 4d) exhibited a combination of peaks from both PMMA-ZnS and riboflavin. Notably, the O–H/N–H stretching band (3320 cm⁻¹) from riboflavin was preserved, confirming the retention of vitamin B2 after loading. The C=O stretching band of PMMA shifted slightly to 1720 cm⁻¹, suggesting additional interactions between riboflavin’s carbonyl groups and the PMMA-ZnS matrix. The presence of riboflavin’s characteristic peaks (1705 cm⁻¹, 1620 cm⁻¹, 1530 cm⁻¹) in the nanocomposite spectrum confirmed successful drug encapsulation without significant degradation [38].
TGA was conducted to evaluate the thermal stability and composition of the PMMA-ZnS-vitamin B2 (PMMA-ZnS-RF) nanocomposites, as illustrated in Fig. 5. The TGA curve revealed a multi-step degradation profile, reflecting the distinct thermal behaviors of the organic (PMMA and riboflavin) and inorganic (ZnS) components. A minor weight loss (~5%) was observed, attributed to the evaporation of residual moisture and solvent traces from the nanocomposite matrix. This step confirmed the effectiveness of the vacuum-drying process in removing volatile impurities. The most significant weight loss (~65%) occurred in this temperature range, corresponding to the thermal decomposition of the PMMA polymer chains and riboflavin. The onset of degradation at ~280 °C is characteristic of PMMA’s ester bond cleavage, while the broader degradation profile (up to 400 °C) suggests overlapping decomposition of riboflavin, which typically degrades above 300 °C. The absence of a sharp, isolated riboflavin degradation peak indicates its homogeneous dispersion within the PMMA-ZnS matrix. Above 400 °C, the curve stabilized, leaving a residual mass of ~30%, which aligns with the expected ZnS content in the nanocomposite. The high thermal stability of ZnS (decomposition > 1000 °C) ensured its integrity, serving as a thermally resistant framework that enhances the nanocomposite’s structural stability during drug release applications. The TGA results demonstrate that the PMMA-ZnS-RF nanocomposites retain structural integrity up to 250 °C, far exceeding physiological temperatures [39, 40]. This thermal robustness ensures that the material will not degrade prematurely during storage or drug delivery. The homogeneous distribution of riboflavin, as inferred from the degradation profile, supports a diffusion-controlled release mechanism, where the polymer matrix gradually erodes to release the drug.

Controlled Release Performance of PMMA-ZnS-RF Nanocomposites
The controlled release behavior of riboflavin (vitamin B2) from PMMA-ZnS-RF nanocomposites was investigated under simulated physiological (pH 7.4) and acidic (pH 5.5) conditions. For the study, 50 mg of the nanocomposite was resuspended in 50 mL of phosphate-buffered saline (PBS) and loaded into dialysis bags with a molecular weight cutoff (MWCO) of 12–14 kDa. The bags were immersed in 200 mL of release medium (PBS) under sink conditions (37 ± 0.5°C, 100 rpm agitation). Aliquots (2 mL) were withdrawn at predetermined time intervals (0.5, 1, 2, 4, 6, 12, 24, and 48 h) and replaced with fresh PBS to maintain a constant volume. The concentration of released RF was quantified using UV-Vis spectroscopy, and cumulative release percentages were plotted against time to evaluate the release kinetics. The cumulative release profiles (Table 1) demonstrate distinct kinetic behaviors under physiological (pH 7.4) and acidic (pH 5.5) conditions. At pH 7.4, the release exhibited a sustained, diffusion-controlled pattern, with minimal burst release, suggesting stable encapsulation of RF within the PMMA-ZnS matrix. In contrast, the acidic environment (pH 5.5) accelerated RF release, likely due to protonation-induced swelling of the PMMA matrix and enhanced solubility of RF. This pH-responsive behavior is advantageous for targeted drug delivery in acidic microenvironments, such as tumor tissues or inflamed regions.
The release data were fitted to established kinetic models to elucidate the underlying mechanisms: Higuchi Model: A linear relationship between cumulative release and the square root of time (Q=kH​t​) was observed, indicating diffusion-controlled release [41]. The Higuchi rate constant (kH​) was higher at pH 5.5, corroborating the faster release kinetics. Korsmeyer-Peppas Model: The release exponent (n) derived from Mt​​​/M∞=ktn suggested anomalous transport (non-Fickian diffusion) at both pH values, with n values between 0.45 and 0.89. This implies contributions from both diffusion and polymer relaxation [42].
The incorporation of ZnS nanoparticles within the PMMA matrix likely modulated the release kinetics by altering the tortuosity of diffusion pathways [43, 44]. Additionally, electrostatic interactions between ZnS and RF may have influenced the release profile, particularly at acidic pH where surface charges on ZnS could enhance RF dissociation. The sustained release of RF over 48 hours, coupled with pH-responsive behavior, highlights the potential of PMMA-ZnS-RF nanocomposites for applications in nutraceutical delivery or photodynamic therapy. The minimal burst release at physiological pH ensures stable RF delivery, while the accelerated release at acidic pH could be leveraged for site-specific targeting. The release data exhibited low variability (SD < 5%), confirming the reproducibility of the nanocomposite fabrication and release testing protocols. Batch-to-batch consistency was maintained, as evidenced by the narrow confidence intervals in the kinetic parameters [45, 46].
Despite notable advances in the bio-fabrication of zinc sulfide–polymer nanocomposites for the controlled release of riboflavin, several intertwined challenges must be addressed to realize robust, translational impact. Reproducibility remains a critical issue, as subtle variations in ZnS loading, PMMA molecular weight, and nanoparticle dispersion can markedly influence release kinetics and the balance between diffusion-controlled and polymer-relaxation processes; thus, rigorous standardization of synthesis parameters coupled with in-process monitoring is essential to achieve consistent performance across batches. Scaling laboratory procedures to pilot or industrial scales presents additional hurdles, requiring the development of scalable, economically viable dispersion and encapsulation strategies within a robust design-of-experiments framework to maintain release fidelity [47]. Long-term stability under storage and use conditions must be established, including resistance to oxidative degradation, RF leakage, and polymer aging, with stabilizing additives or protective coatings as potential remedies [48]. A deeper mechanistic understanding of release is warranted, integrating kinetic modeling with in situ spectroscopy and high-resolution imaging, complemented by computational modeling, to disentangle diffusion pathways from polymer relaxation and ZnS–RF interactions and to enable predictive design [49, 50]. Biocompatibility and environmental impact considerations should be incorporated early, through comprehensive toxicology and ecotoxicology profiling and by exploring greener polymer matrices and safer nanomaterial constituents, informed by cradle-to-grave analyses. Regulatory readiness and translational potential demand proactive alignment of experimental reporting with journal and regulatory expectations, including comprehensive documentation of calibration procedures, raw data, and statistical analyses to facilitate peer review and eventual clinical or nutraceutical translation. Finally, a forward-looking research trajectory should emphasize the integration of functional performance with translational relevance such as multimodal imaging capability or nutraceutical efficacy while maintaining rigorous data-sharing practices to bolster reproducibility and confidence in the reported findings. 

CONCLUSION
In summary, the present study demonstrates the successful bio-fabrication and comprehensive characterization of zinc sulfide–polymer (PMMA–ZnS) nanocomposites as a versatile platform for the controlled delivery of riboflavin (vitamin B2). The in situ incorporation of ZnS within the PMMA matrix yielded nanocomposites with uniform morphology, enhanced thermal stability, and preserved polymer integrity, as revealed by morphology, spectroscopic, and thermogravimetric analyses. Post-synthesis loading of riboflavin achieved substantial encapsulation efficiency without compromising the structural stability of the host matrix. In vitro release studies conducted under sink conditions in phosphate-buffered saline at physiologic (pH 7.4) and mildly acidic (pH 5.5) environments demonstrated a pronounced pH-responsive release profile: slower, diffusion-controlled release at pH 7.4 and accelerated release at pH 5.5, consistent with a mechanism dominated by Fickian diffusion with contributions from polymer relaxation. Kinetic analyses using Higuchi and Korsmeyer–Peppas models corroborated a predominantly diffusion-driven transport with non-negligible polymer relaxation effects, and highlighted the tunability of release by adjusting ZnS loading, PMMA molecular weight, and RF loading. The incorporation of ZnS not only influences release dynamics but also opens avenues for multimodal applications, including optical imaging and sensing, owing to the intrinsic luminescent properties of ZnS. While the results establish a robust foundational framework for rational design, several challenges were identified that will guide future efforts: ensuring reproducibility across batches through rigorous standardization and in-process monitoring, scaling synthesis and release-testing protocols for pilot-scale production, and evaluating long-term stability and storage under diverse conditions. Furthermore, early integration of biosafety, toxicology, and environmental impact assessments will be essential for translational success. Looking ahead, future work should focus on elucidating detailed structure property relationships via advanced in situ characterization, expanding the repertoire of polymer matrices and ZnS/Cd-free nanostructures, and exploring in vivo models to validate therapeutic or nutraceutical potential. Collectively, the findings provide a coherent strategy for the development of ZnS–polymer nanocomposites as controllable, biocompatible carriers with potential utility in nutraceutical delivery, bioimaging, and beyond. 

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