Nanoengineered Bezafibrate-Loaded Calcium Nanoparticles for Osteoporosis: A Repurposing Approach for Targeted Bone Therapy

Nanoengineered Bezafibrate-Loaded Calcium Nanoparticles for Osteoporosis: A Repurposing Approach for Targeted Bone Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nanoengineered Bezafibrate-Loaded Calcium Nanoparticles for Osteoporosis: A Repurposing Approach for Targeted Bone Therapy Shikha Yadav, Alka ., Shailendra K Saraf, Neelam Datt This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7197944/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in BioNanoScience → Version 1 posted 10 You are reading this latest preprint version Abstract Osteoporosis is a progressive skeletal disorder characterized by an imbalance between bone resorption and bone formation, leading to reduced bone mineral density and increased fracture risk. Bezafibrate (BZ), a lipid-lowering fibrate, has recently gained attention as a promising repurposed therapeutic agent for osteoporosis due to its potential bone-protective effects. This study aimed to develop and evaluate a novel bezafibrate-loaded calcium nanoparticle (BZ-CNP) system to enhance its therapeutic efficacy against osteoporosis and assess its in vivo performance in an osteoporotic animal model. Calcium nanoparticles (CNPs), known for their biocompatibility and inherent bone-targeting capabilities, were formulated using the chemical precipitation method for efficient drug delivery to bone tissue. BZ-CNPs were optimized using Box-Behnken Design (BBD), and their physicochemical properties were thoroughly characterized. The therapeutic potential of the optimized formulation was evaluated in a dexamethasone-induced osteoporotic rat model. The optimized BZ-CNPs exhibited a particle size of 242.1 nm, a polydispersity index (PDI) of 0.302, and a zeta potential of −32.7 mV, indicating stable nanoscale dispersion. The entrapment efficiency was 87.2%, demonstrating efficient drug loading. In vivo and biochemical parameters results revealed a significant improvement in bone turnover markers, confirming the formulation's efficacy in reversing osteoporosis-induced bone loss. The developed Bezafibrate-loaded calcium nanoparticles represent a promising nanocarrier system for targeted delivery, offering enhanced therapeutic outcomes in osteoporosis management. Bezafibrate calcium nanoparticles osteoporosis repurposing Box-Behnken design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Osteoporosis is the most prevalent metabolic bone disorder globally, posing a significant public health challenge, particularly among the aging population. This condition is characterized by an imbalance between bone resorption and formation, leading to a decrease in bone mineral density (BMD), disruption of bone microarchitecture, and an increased susceptibility to fractures. The impact is most pronounced in postmenopausal women, who experience accelerated bone loss due to estrogen deficiency, with estimates indicating that nearly 70% of individuals over the age of 80 are affected [ 1 ]. Given the rising prevalence and the limitations of existing treatments, there is an urgent need to develop targeted therapeutic strategies, particularly for vulnerable populations, where traditional approaches may be inadequate or accompanied by significant side effects [ 2 ]. Recent advances in translational research have fostered a growing interest in bridging fundamental bone biology with innovative drug-delivery technologies to address these challenges. Nanoparticle-based drug delivery systems, especially those utilizing biocompatible calcium-based carriers, offer promising solutions for enhancing bone regeneration, improving therapeutic targeting, and optimizing clinical outcomes in osteoporotic patients. By leveraging nanotechnology, these systems aim to overcome the limitations of conventional therapies by offering more effective and controlled drug delivery, specifically targeting bone tissue. Osteoporosis results from an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [ 3 ]. Osteoblasts, originating from mesenchymal stem cells (MSCs), play a key role in synthesizing bone matrix and mineralizing it through the deposition of calcium phosphate. Conversely, osteoclasts, derived from hematopoietic stem cells (HSCs), mediate bone resorption through the enzymatic degradation of mineralized tissue [ 4 ]. Bone tissue itself is a composite of collagen, proteins, and mineralized calcium phosphate, which not only provides structural integrity but also supports essential metabolic functions. Calcium, a critical element in maintaining bone homeostasis, is predominantly derived from dietary sources, with its absorption influenced by various physiological factors such as nutritional status and metabolic conditions [ 5 , 6 ]. Bone remodeling is a highly dynamic process involving tightly regulated cycles of bone formation and resorption. Osteoblast function is modulated by various transcriptional, epigenetic, and environmental signals, while osteoclasts contribute to bone degradation within specialized resorption zones known as Howship’s lacunae [ 7 – 9 ]. Advances in nanotechnology have opened new possibilities for enhancing bone-specific drug delivery. Among inorganic carriers, calcium nanoparticles (CNPs) have garnered attention due to their inherent biocompatibility, bone-targeting capabilities, and high drug-loading potential. These nanoparticles not only serve as efficient carriers but also function as therapeutic agents themselves, promoting bone repair by delivering calcium directly to deficient bone sites while maintaining systemic calcium equilibrium [ 10 ]. Compared to organic nanocarriers, CNPs offer superior structural stability and a greater surface area, which can be further optimized for stimuli-responsive drug release. In the context of drug repurposing, fibrates such as fenofibrate and bezafibrate have shown promise in enhancing osteoblast differentiation and improving bone mineral density, suggesting their potential role in osteoporosis management. Bezafibrate, a BCS Class III drug known for its low permeability, may be particularly effective for targeted bone therapy when delivered via an appropriate nanocarrier system. However, despite its therapeutic promise, the clinical translation of bezafibrate for skeletal applications remains underexplored [ 11 ]. This study aligns with the principles of translational research, aiming to translate preclinical discoveries into clinically applicable therapies. The primary objective is to develop and optimize bezafibrate-loaded calcium nanoparticles (BZ-CNPs) for enhanced bone regeneration. The formulation's efficacy will be evaluated in a dexamethasone-induced osteoporotic rat model to assess its potential in improving bone mineral density and restoring bone homeostasis. By bridging the gap between basic research and clinical application, this study seeks to advance targeted and clinically translatable therapeutic options for osteoporosis. 2. Materials and Methods 2.1 Drugs and Reagents Bezafibrate (BZ) was purchased from BLD Pharm, Hyderabad, Telangana, India. Calcium nitrate tetrahydrate was procured from Hi Media Laboratories, Mumbai, Maharashtra, India. Sodium lauryl sulfate (SLS) and sodium hydroxide (NaOH) pellets were obtained from SD Fine Chem, Mumbai, Maharashtra, India. Methanol was sourced from Rankem Chemicals, Pune, Maharashtra, India. All other chemicals and reagents used in preparing and evaluating the formulation were of analytical grade. 2.2 Experimental Design and Selection of Variables To investigate the effect of critical formulation variables on the properties of bezafibrate-loaded calcium nanoparticles, a Box-Behnken Design (BBD) was employed. The selected independent variables were the Ca²⁺: OH⁻ molar ratio (X1), surfactant concentration (X2), and sonication time (X3), each evaluated at three levels (− 1, 0, + 1) as shown in Table I. Design Expert® 12 software generated 15 experimental runs, including 3 center points. The formulations were assessed for particle size, PDI, zeta potential, entrapment efficiency, and cumulative drug release. Table I Formulation matrix of bezafibrate calcium nanoparticles developed using Box-Behnken Design for selection of the optimized batch. Batch Ca2+: OH- (M) (X1) Surfactant (g) (X2) Sonication time(min) (X3) T1 -1 0 1 T2 0 0 0 T3 1 -1 0 T4 1 0 -1 T5 0 -1 1 T6 0 0 0 T7 1 1 0 T8 -1 0 -1 T9 -1 1 0 T10 0 0 0 T11 0 1 -1 T12 1 0 1 T13 -1 -1 0 T14 0 1 1 T15 0 -1 -1 2.3 Method of Preparation of Bezafibrate Calcium Nanoparticles (BZ-CNPs) Bezafibrate-loaded calcium nanoparticles (BZ-CNPs) were synthesized using a chemical precipitation method, as illustrated in Fig. 1 . Initially, a 0.2 M aqueous solution of sodium hydroxide (NaOH) was prepared and maintained under magnetic stirring. A methanolic solution of bezafibrate, along with sodium lauryl sulfate (SLS) as a surfactant, was then added dropwise to the NaOH solution under continuous stirring to facilitate drug solubilization and stabilize nanoparticle formation. Separately, a 0.1 M aqueous solution of calcium nitrate [Ca(NO₃)₂], serving as the calcium ion source, was prepared. The drug-containing mixture was then added dropwise to the calcium nitrate solution under constant stirring, and the entire system was allowed to react for 1 hour to enable nanoparticle formation via controlled precipitation. The resulting colloidal dispersion was subjected to probe sonication (45 seconds ON and 15 seconds OFF cycles) to reduce particle size and ensure uniform dispersion. The nanoparticle suspension was centrifuged at 10,000 rpm for 15 minutes at 4°C to separate the nanoparticles. The formed BZ-CNPs were collected as a pellet and washed, and stored appropriately for further characterization. 2.4 Characterization of the Prepared BZ-CNPs 2.4.1 Particle Size, Polydispersity Index (PDI), and Zeta Potential The average particle size, polydispersity index (PDI), and zeta potential of BZ-CNPs were measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were diluted with Milli-Q water before measurement to ensure appropriate scattering intensity. All measurements were conducted at 25 ± 1°C using disposable polystyrene cuvettes for size and PDI and folded capillary cells for zeta potential. The refractive index of the dispersion medium was set to 1.44. Data were recorded in triplicate and reported as mean ± standard deviation [ 12 ]. 2.4.2 Fourier Transform Infrared Spectroscopy (FTIR) FTIR analysis was performed to identify the characteristic functional groups and assess possible interactions between bezafibrate and the calcium matrix in the prepared nanoparticles. The spectra were recorded using an FTIR spectrophotometer (Alpha II, Bruker, Germany) in the range of 4000–400 cm⁻¹. Samples were prepared by mixing with potassium bromide (KBr) and compressing into a pellet. The obtained spectra were analyzed for shifts or appearance/disappearance of peaks to confirm drug–excipient compatibility and successful nanoparticle formation[ 13 ]. 2.4.3 Scanning Electron Microscopy (SEM) The surface morphology and structural characteristics of the optimized BZ-CNPs were evaluated using SEM (JSM-6490LV, JEOL, Japan). A small aliquot of nanoparticle dispersion was placed onto a clean stub using double-sided carbon adhesive tape and allowed to dry under a vacuum. The dried samples were then sputter-coated with a thin layer of platinum using an auto fine coater (JFC-1600, JEOL, Japan) to ensure conductivity. Imaging was performed at an accelerating voltage of 15 kV to assess particle shape, surface texture, and approximate size[ 14 ]. 2.4.4 Powder X-ray Diffraction (PXRD) PXRD analysis was employed to investigate the crystalline nature and structural transitions of the drug within the calcium nanoparticle matrix. The diffraction patterns were recorded using an X-ray diffractometer (D8 Advance Eco, Bruker, Germany) equipped with a Cu Kα radiation source (λ = 1.5406 Å), operated at 45 kV and 40 mA. Scans were performed over a 2θ range suitable for identifying characteristic diffraction peaks. Changes in peak intensity or position were analyzed to evaluate drug encapsulation and possible alterations in crystallinity due to nanoparticle formulation[ 15 ]. 2.4.5 Differential Scanning Calorimetry (DSC) DSC was employed to determine the melting point and degree of crystallinity of the formulated nanoparticles. This technique is essential for studying thermal transitions of nanoparticles, such as melting points, glass transition temperatures, and crystallization behavior, all of which significantly impact the stability and performance of the drug formulation. The DSC analysis was carried out using a DSC Q2000 (V24.11 Build 124)[ 16 ]. 2.4.6 Entrapment Efficiency (%EE) Entrapment efficiency refers to the ratio of the quantity of drug successfully entrapped within the nanoparticles to the total amount of drug initially added to the dispersion. The EE plays a critical role in determining the release characteristics of the formulation. To assess the entrapment efficiency, the prepared dispersion was centrifuged at 10,000 rpm for 1 hour at 4°C using a SIGMA 3–18 K centrifuge (Sartorius). The supernatant was then diluted with phosphate-buffered saline (PBS, pH 7.4) to a final volume of 10 mL. The drug concentration in the supernatant was determined by UV spectrophotometry at 229.6 nm using a Shimadzu Double Beam Spectrophotometer (UV-1700). The percentage of entrapment efficiency was calculated using the following formula [ 17 ]. 2.4.7 In Vitro Drug Release Using Franz Diffusion Cell The in vitro release behavior of the optimized nanoparticle formulation was assessed using a Franz diffusion cell equipped with an egg membrane as the diffusion barrier. The receptor compartment was filled with phosphate-buffered saline (PBS, pH 7.4) and maintained at 37 ± 0.5°C under constant magnetic stirring to mimic physiological conditions. An aliquot (1 mL) of the formulation was placed in the donor compartment. At predetermined time points (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 24, 25, 26, 27, 28, 29, and 30 hours), 1 mL of the receptor medium was withdrawn and replaced with an equal volume of fresh PBS to maintain sink conditions. The collected samples were appropriately diluted, and drug concentration was determined by measuring absorbance at 229.6 nm using a UV-visible spectrophotometer (UV-1700, Shimadzu, Japan). The cumulative percentage of drugs released was calculated and plotted against time to establish the release profile[ 13 ]. 2.5 Stability Studies The stability of the optimized Bezafibrate-Calcium Nanoparticles was evaluated under different storage conditions per ICH Q1A (R2) guidelines. The formulation was aliquoted into clean, light-protected glass vials and stored at 4°C (refrigerator), 25°C (room temperature), and 40°C (accelerated conditions) for three months. Following the storage period, the samples were analyzed for any changes in physicochemical characteristics, including particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency (%EE) to assess formulation stability. 2.6 In vivo Pharmacodynamic Studies 2.6.1 Experimental Animals Male Wistar rats weighing between 250 and 300 g were used for the in vivo pharmacodynamic evaluation. The animals were kept under standard laboratory conditions: a 12:12 h light-dark cycle, an ambient temperature of 25 ± 2°C, and a relative humidity of 55 ± 5%. They were housed in clean polypropylene cages with stainless steel grid tops and were given free access to standard laboratory chow and water ad libitum. All experimental protocols involving animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Babu Banarasi Das Northern India Institute of Technology (Approval No. BBDNIIT/IAEC/MAY/2024/07) and were conducted in strict adherence to the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). 2.6.2 Experimental Groups The animals were randomly divided into six groups (n = 5 per group). Group I served as the normal control and received no treatment. Group II was assigned as the osteoporosis control group, in which osteoporosis was induced by intraperitoneal administration of dexamethasone (DEX) at a dose of 10 mg/kg, three times a week for 21 days [ 18 ]. This dosing regimen reliably mimics the pathophysiological conditions of glucocorticoid-induced osteoporosis. Groups III to VI were also subjected to the same DEX protocol to induce osteoporosis. Group III received the standard treatment with Zoledronic acid (Zoldonate®) at 0.1 mg/kg, administered subcutaneously once weekly [ 19 ]. Group IV was treated with plain bezafibrate solution at a dose of 10 mg/kg subcutaneously. Groups V and VI received bezafibrate-loaded calcium nanoparticles (BZ-CNPs) at the same dose (10 mg/kg, subcutaneously), differing only in particle size—Group V received the formulation with smaller particle size, while Group VI received the formulation with larger particle size. This study design enabled the evaluation of the therapeutic potential and particle size-dependent efficacy of BZ-CNPs in the management of glucocorticoid-induced osteoporosis. On the 21st day of treatment, the animals were euthanized, and the femur bones were carefully excised and preserved in 10% neutral buffered formalin for subsequent histopathological and morphological analyses. Additionally, the surrounding skeletal muscle tissue was collected and processed for the evaluation of oxidative stress biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase activity, using spectrophotometric methods [ 20 ]. 2.6.3 Collection of Blood Samples and Histopathological Analysis of Bone On the 21st day of treatment, all animals were sacrificed under appropriate anesthesia. Blood samples were collected via cardiac puncture using a 1 mL sterile syringe and transferred into plain collection tubes. The samples were allowed to clot and then centrifuged to separate the serum. Serum was analyzed for biochemical markers associated with bone metabolism, including calcium, phosphorus, osteocalcin, and alkaline phosphatase (ALP), to evaluate the therapeutic efficacy of various treatments in mitigating glucocorticoid-induced osteoporosis [ 21 ]. For histopathological analysis, the left femurs were carefully excised and fixed in 10% neutral buffered formalin. Decalcification was carried out in 20% ethylenediaminetetraacetic acid (EDTA) solution for 20 days to ensure adequate removal of calcium deposits. Following decalcification, the bones were processed through a graded ethanol series for dehydration, cleared in xylene, and embedded in paraffin wax. Thin longitudinal sections of 6 µm thickness were prepared using a microtome and stained with Hematoxylin and Eosin (H&E). The stained sections were examined under a light microscope at magnifications of 10× and 40×. Morphometric parameters such as cortical bone thickness, integrity, and number of osteocyte lacunae, and the structural condition of Haversian canals were analyzed using an image analyzer to assess bone architecture and remodeling. 2.6.4 Effect of Formulation on Body Weight of Rats The body weight of each rat was recorded on Day 0 and weekly for 21 days using a digital balance (± 0.1 g). Measurements were taken in the morning to reduce diurnal variation. Data were expressed as mean ± SD (n = 5) and analyzed to evaluate the systemic effects of treatments and the protective role of BZ-CNPs in dexamethasone-induced osteoporosis. 2.6.5 Bone Mineral Density Bone mineral density (BMD) of the left femur was determined using Archimedes’ principle, which offers a reliable and simple method to assess bone mass by calculating its density. This approach facilitated the evaluation of bone mineralization and treatment efficacy in glucocorticoid-induced osteoporosis. This gravimetric method provided a non-destructive means to quantify changes in bone mass across different treatment groups. The density (d) of each bone sample was calculated using the formula: d = (w 1 /w 1 – w 2 ) × P Equation ……………….2 where d = bone density (in g/cm 3 ), w 1 = weight of bone in air, w 2 = weight of bone in distilled water, and P = density of distilled water (approximately 1 g/cm³ at room temperature) [ 22 ]. 2.6.6 Bone Mineral Content Markers Serum calcium and phosphorus levels were measured to evaluate the bone health of the animals. Serum calcium was quantified using the Arsenazo III colorimetric method, while serum phosphorus was assessed using the Phosphomolybdate UV method [ 23 ]. These biochemical markers provided essential information regarding the mineral content of the bones and served as indicators of overall bone health in the treated rats. 2.6.7. Bone Resorption Biomarker Serum alkaline phosphatase (ALP) levels, a key biomarker for bone resorption, were measured to assess bone degradation associated with osteoclast activity, commonly observed in osteoporosis. The serum ALP levels were quantified using the p-nitrophenol and phosphate (pNPP) kinetic assay, which provides an accurate measurement of ALP activity in serum samples[ 23 ]. 2.6.8. Bone Formation Biomarker Serum osteocalcin, a biomarker indicative of bone formation and osteoblast activity, was also quantified. The osteocalcin levels were assessed using the Chemiluminescent Microparticle Immunoassay (CMIA) method, which provides a sensitive and specific analysis for this biomarker[ 2 ]. 2.6.9. Assessment of Oxidative Stress Biomarkers in Osteoporosis Oxidative stress plays a pivotal role in osteoporosis pathogenesis by disrupting the balance between reactive oxygen species (ROS) and antioxidant defense mechanisms, leading to bone degradation [ 24 ]. In this study, oxidative stress biomarkers such as malondialdehyde (MDA), catalase [ 25 ], and superoxide dismutase (SOD) were assessed to evaluate oxidative damage and the antioxidant response. Muscle tissue (2 g) was homogenized in phosphate-buffered saline (PBS, pH 7.4), centrifuged, and the supernatant was used for biochemical assays. Protein content was quantified using the Bradford method, with bovine serum albumin (BSA) as the standard. MDA levels, an indicator of lipid peroxidation, were measured by reacting the homogenate with thiobarbituric acid (TBA) and trichloroacetic acid (TCA), followed by absorbance measurement at 540 nm. CAT activity was determined by assessing the breakdown of hydrogen peroxide (H 2 O 2 ), with absorbance recorded at 230 nm at two time points[ 26 ], and SOD activity was evaluated by monitoring the reduction of pyragallol at 420 nm[ 27 ]. The respective activities of MDA, CAT, and SOD were calculated using appropriate formulas. These biomarkers provided valuable insights into the oxidative stress status and its association with bone health [ 33 , 34 ]. 2.7 Statistical Analysis The data were expressed as the mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism 9.0.0. To determine the statistical significance between different groups, one-way ANOVA followed by Dunnett’s multiple comparison tests was used. A p-value of < 0.001 was considered statistically significant, indicating a strong effect of the treatments. 3. Results and Discussion 3.1. Challenges in the Fabrication of Bezafibrate Calcium Nanoparticles (BZ-CNPs) The fabrication of Bezafibrate Calcium Nanoparticles (BZ-CNPs) presented significant challenges, particularly in achieving the desired nanoparticle size. A major issue encountered was the rapid aggregation of the calcium nanoparticles upon formulation, leading to the formation of larger particle sizes that were unsuitable for effective bone penetration in osteoporosis treatment. To address this, various fabrication methods were explored, including the reverse microemulsion method and the chemical precipitation method [ 28 ]. The reverse microemulsion method, while widely used in nanoparticle fabrication, posed several challenges. It required large quantities of surfactant and co-surfactant to stabilize the droplets of the microemulsion, which not only increased the complexity of the process but also resulted in a lack of homogeneity within the system. This lack of consistency in the microemulsion system further hindered the formation of nanoparticles with the desired size and uniformity. On the other hand, the chemical precipitation method showed potential for producing nanoparticles of smaller sizes. The solution to these challenges came with the introduction of different stabilizing agents and surfactants. Sodium hydroxide (NaOH) and sodium lauryl sulfate (SLS) were identified as effective agents for stabilizing the nanoparticles and reducing their size to the desired range, thereby enabling their efficient penetration into the bone tissue for the management of osteoporosis. The BZ-CNPs were successfully prepared using the chemical precipitation method (Fig. 1 ). In this approach, SLS acted as the surfactant, adsorbing onto the surface of the nanoparticles. This interaction reduced the surface tension and prevented the aggregation of the nanoparticles, thereby ensuring the production of nanoparticles of a suitable size for their intended therapeutic application. 3.2. Selection of Process Variables For the optimization of bezafibrate-loaded calcium nanoparticles, the influence of various independent variables on critical quality attributes was investigated. The study aimed to establish the relationship between selected process parameters and formulation responses using a design of experiments approach. With an increase in the number of influencing factors, the Box-Behnken Design (BBD) was employed to reduce the number of experimental runs while ensuring systematic evaluation. Three independent variables were selected for the study: the molar ratio of precursors (Ca²⁺: OH⁻) designated as X1, surfactant concentration as X2, and sonication time as X3 (Table II). The formulation’s responses included particle size (Y1), polydispersity index (Y2), zeta potential (Y3), entrapment efficiency (Y4), and cumulative drug release (Y5). Each independent variable was evaluated at three levels: low (-1), medium (0), and high (1). Table II Lists the factor settings for the experimental design. Independent Variables Low (-1) Medium (0) High (1) Ca + 2 : OH − (molar ratio) 1:1 1:2 1:3 SLS concentration (g) 1 2 3 Sonication time (minutes) 30 40 50 The Ca²⁺: OH⁻ molar ratio varied between 1:1, 1:2, and 1:3, with corresponding changes in the surfactant concentration (SLS) and sonication time. The surfactant concentration was adjusted from 1 g to 3 g, and the sonication time was varied between 30 minutes, 40 minutes, and 50 minutes. The optimization process employed the Design Expert 12 software to generate fifteen experimental batches, including three center points, to assess the impact of these variables on the nanoparticle characteristics[ 29 ]. The experimental design allowed for a comprehensive analysis of the formulation's behaviour under varying conditions, and it provided valuable insights into the factors that most significantly affected the quality of the BZ-CNPs. 3.3. Optimization of Bezafibrate Calcium Nanoparticles (BZ-CNPs) The optimization of BZ-CNPs was performed using a Box-Behnken design (BBD), which allowed for the systematic evaluation of different formulation parameters and their effects on the nanoparticle characteristics. The independent variables selected for optimization included the Ca²⁺: OH⁻ molar ratio, surfactant concentration, and sonication time, as outlined in Table III. These variables were carefully chosen to ensure that they could effectively influence the size, zeta potential, polydispersity index (PDI), entrapment efficiency, and drug release profiles of the nanoparticles. Table III Optimization and Physicochemical evaluation of BZ-CNPs formulated via Box-Behnken design. Batch PS (nm) PDI ZP (mV) % Entrapment Efficiency (%EE) % Cumulative Drug Release (% CDR) T1 217.9 0.194 -34.6 92.2 80.1 T2 201.6 0.320 -29.8 85.4 75.2 T3 250.4 0.342 -21.8 86.3 84.4 T4 201.8 0.315 -45.0 86.7 74.1 T5 269.2 0.278 -43.1 87.1 81.3 T6 201.6 0.320 -29.8 85.4 75.2 T7 270.9 0.332 -21.0 88.0 75.3 T8 206.5 0.163 -50.3 90.4 79.1 T9 286.4 0.396 -45.6 91.5 77.5 T10 201.6 0.320 -29.8 85.4 75.2 T11 110.1 0.317 -57.4 91.6 78.1 T12 105.3 0.374 -26.4 86.8 81.0 T13 275.8 0.367 -30.7 85.4 88.3 T14 102.6 0.348 -51.1 92.8 84.9 T15 281.8 0.352 -48.6 90.9 77.7 3.4. Effect of Independent Variables on Dependent Variables 3.4.1. Particle Size, Polydispersity Index (PDI), and Zeta Potential The independent variables outlined in Table III had a significant impact on the dependent variables of the prepared BZ-CNPs. Among these, the particle size emerged as a crucial factor in the fabrication and optimization of BZ-CNPs. A study indicated that nanoparticles with a size of less than 400 nm were effective in significantly enhancing bone density and promoting calcium deposition in bone tissues [ 30 ]. Therefore, achieving a particle size of less than 400 nm was a primary objective of this study. The particle size of the BZ-CNPs was primarily influenced by the sonication time, with a noticeable reduction in particle size as sonication time increased. This observation aligns with previous reports where increased sonication duration led to finer particles, presumably due to better dispersion and energy imparted to the system [ 31 ]. Furthermore, the molar ratio of Ca²⁺: OH⁻ was also found to significantly impact the particle size. In this study, the intermediate molar ratio (Ca²⁺: OH⁻ = 1:2) resulted in smaller nanoparticles. The results from the Design Expert supported these observations (Fig. 2 a). The reduced quadratic model developed for particle size was statistically significant (p = 0.0080), with a Model F-value of 7.43. Among the factors, surfactant concentration (B) and sonication time squared (C²) significantly affected the particle size (p = 0.0332 and p = 0.0108, respectively). The model exhibited an R² of 0.5531 and an adjusted R² of 0.4787, indicating moderate predictive power. The lack of fit was not significant (p = 0.4410), suggesting good model adequacy. The adequate precision ratio was 7.29, which is greater than the desired value of 4, confirming an adequate signal (Table IV). The optimized BZ-CNPs had a particle size of 242.1 nm and a Polydispersity Index (PDI) of 0.314 (Fig. 3 a). The PDI value indicated a narrow size distribution, which is essential for the uniformity and reproducibility of the nanoparticle formulation. The particle size and PDI results were in line with the desired characteristics for effective bone penetration and therapeutic efficacy. Regarding PDI (Fig. 2 b), a reduced cubic model was found to be significant (p = 0.0009), with sonication time squared (C², p = 0.0054) and the interaction between Ca²⁺: OH⁻ ratio and sonication (AC², p = 0.0011) playing key roles. The model had a high R² value of 0.7650 and an adjusted R² of 0.7009, with a non-significant lack of fit (p = 0.8198). The low standard deviation (0.0377) and coefficient of variation (CV = 11.58%) reflected the model’s precision and reliability (Table IV). The zeta potential, another critical parameter for the stability of nanoparticles, was also influenced by the independent variables. It was observed that increasing the molar ratio of the precursors led to a higher zeta potential (Fig. 2 c). Additionally, lower concentrations of surfactants resulted in improved zeta potential values[ 32 ]. An increase in sonication time further optimized the zeta potential, resulting in a more stable dispersion. The zeta potential of the optimized BZ-CNPs was measured at -32.7 mV (Fig. 3 ), indicating good stability and reduced chances of nanoparticle aggregation over time [ 33 ]. These results were validated by the reduced quadratic model developed for zeta potential (Table IV), which was also statistically significant (p = 0.0067) with a Model F-value of 6.74. Significant factors included sonication time (C) and its quadratic term C² (p = 0.0397 and p = 0.0046, respectively). The model had an R² value of 0.7295 and an adjusted R² of 0.6213. The lack of fit was not significant (p = 0.7851), confirming the model’s reliability. Table IV ANOVA summary of model statistics for Particle Size, PDI, and Zeta Potential of BZ-CNPs Response Particle Size PDI Zeta Potential Model Type Reduced Quadratic Reduced Cubic Reduced Quadratic Model F-value 7.43 11.94 6.74 p-value 0.0080 0.0009 0.0067 Significant Terms B, C² C², AC² C, C² R² 0.5531 0.7650 0.7295 Adjusted R² 0.4787 0.7009 0.6213 Predicted R² 0.2824 0.5531 0.4028 Lack of Fit (p) 0.4410 0.8198 0.7851 Adequate Precision 7.2883 9.6105 6.6296 3.4.2. Entrapment Efficiency (EE) and Cumulative Drug Release (CDR) The entrapment efficiency (EE) and cumulative drug release (CDR) were also influenced by the independent variables. As the sonication time increased, the EE decreased, likely due to heat generation during prolonged sonication, which could cause the drug to leach out from the nanocarriers [ 34 ]. On the other hand, a shorter sonication time resulted in lower EE, suggesting that a moderate sonication time is optimal for achieving a higher EE. Surfactant concentration played a pivotal role in facilitating the solubilization of the drug, leading to an increase in drug release from the nanoparticles. This is due to the surfactant’s ability to lower the interfacial tension between the nanoparticles and the surrounding medium, promoting the dissolution and release of the encapsulated drug [ 35 ]. However, changes in the Ca²⁺: OH⁻ molar ratio showed less significant influence on cumulative drug release, indicating that surfactant behavior and sonication are more dominant factors in controlling drug release kinetics. These trends were statistically confirmed by the ANOVA results obtained from Design Expert® software (Fig. 2 d and Fig. 2 e). The EE data followed a reduced quadratic model that was statistically significant (p = 0.0026), with a Model F-value of 9.04. Surfactant concentration (B), along with its squared term (B²), and sonication time squared (C²), were found to be significant factors (p < 0.05). The model showed good reliability with an R² of 0.8340, an adjusted R² of 0.7417, and a predicted R² of 0.5382. The lack of fit was not significant (p = 0.8138), indicating that the model was suitable for prediction. Adequate precision was 8.20, which is well above the minimum threshold of 4, suggesting a strong signal-to-noise ratio (Table V). Similarly, the cumulative drug release (%CDR) was best described by a reduced cubic model, which was also statistically significant (p = 0.0011) with a Model F-value of 11.42. The drug release was significantly affected by sonication time (C), the square of surfactant concentration (B²), and the interaction term A²B (p < 0.05). The model had a high R² value of 0.7570 and an adjusted R² of 0.6907, with a predicted R² of 0.5649, indicating good agreement among the three. The non-significant lack of fit (p = 0.8939) and a high adequate precision of 8.73 supported the model’s predictive validity (Table V). The optimized formulation exhibited an EE of 87.2% and a cumulative drug release of 82.7%, as shown in Fig. 8 B, closely matching the predicted values from the desirability function. These findings affirm that the Box-Behnken Design successfully guided the formulation process toward achieving an ideal balance of high drug loading and sustained release, suitable for potential use in osteoporosis treatment. Table V ANOVA summary of model statistics for Entrapment Efficiency (%EE) and Cumulative Drug Release (%CDR) of BZ-CNPs Response Entrapment Efficiency Cumulative Drug Release Model Type Reduced Quadratic Reduced Cubic Model F-value 9.04 11.42 p-value 0.0026 0.0011 Significant Terms B, B², C² B², A²B R² 0.8340 0.7570 Adjusted R² 0.7417 0.6907 Predicted R² 0.5382 0.5649 Lack of Fit (p) 0.8138 0.8939 Adequate Precision 8.1967 8.7252 3.5. Scanning Electron Microscopy (SEM) Scanning Electron Microscopy (SEM) analysis was performed on the prepared Bezafibrate Calcium Nanoparticles (BZ-CNPs) to observe their surface morphology. As shown in Fig. 4 A, the SEM images revealed that the bezafibrate is irregular in shape and size, while drug-incorporated calcium nanoparticles exhibited a mix of spherical and smooth surface morphology as depicted in Fig. 4 B. Similar results were observed earlier by researchers [ 36 ]. Some nanoparticles were well-defined and spherical, while others showed an irregular morphology. Additionally, it was observed that the particles had agglomerated to some extent, which is a common occurrence during nanoparticle formulation due to the inherent tendency of nanoparticles to aggregate in the absence of proper stabilizing agents. This agglomeration could potentially impact the uniformity and performance of the nanoparticles in drug delivery systems [ 37 ]. 3.6. Fourier Transform Infrared Spectroscopy (FTIR) FTIR analysis was conducted to assess the chemical structure of the Bezafibrate Calcium Nanoparticles and to detect any possible interactions between the drug and the excipients used in the formulation. The FTIR spectrum (Fig. 5 ) of the BZ-CNPs revealed sharp, characteristic peaks that correspond to specific functional groups present in Bezafibrate. Notably, the peak at 2916.13 cm-¹ corresponded to the stretching vibration of hydroxide (-OH), a key functional group in Bezafibrate. The spectrum also exhibited peaks for the ether group at 1081.06 cm⁻¹, the bending vibration of the -NH group at 1450.48 cm⁻¹, and the C-Cl stretch at 832.55 cm⁻¹. Additionally, the N = O peak from calcium nitrate appeared at 1412.41 cm⁻¹, and the S = O stretch from sodium lauryl sulfate (SLS) was detected at 1213.13 cm⁻¹ [ 25 , 36 ]. The FTIR results indicated that the functional groups of the drug and excipients remained intact, suggesting that there were no significant chemical interactions between them. This stability of the functional groups is indicative of the chemical stability of the BZ-CNPs formulation. The unaltered structure of both the drug and excipient implies that the encapsulation process did not cause any detrimental changes to their chemical composition or integrity. These findings align with previous reports where bioactive compounds, even when encapsulated into nano-formulations, retained their original functional groups and structural configurations, ensuring their stability and potential efficacy in drug delivery systems[ 38 ]. These results from the FTIR analysis support the successful preparation of BZ-CNPs with preserved chemical stability, which is crucial for ensuring the therapeutic effectiveness and safety of the formulation in clinical applications. 3.7. X-ray diffraction (XRD) X-ray diffraction (XRD) analysis was conducted to investigate the crystallinity of bezafibrate and its distribution within the calcium nanoparticles. The XRD pattern of the pure bezafibrate (Fig. 6 a) showed distinct peaks at 2θ angles of 10.1°, 15.5°, 16.6°, 18.5°, 19.9°, 20.4°, 24.8°, 25.3°, and 30.4°, which are indicative of the crystalline nature of bezafibrate[ 25 ]. These findings were consistent with previous reports on bezafibrate's crystallinity [ 39 ]. However, in the XRD pattern of the bezafibrate calcium nanoparticles (Fig. 6 b), the peaks corresponding to bezafibrate were completely absent, suggesting that the drug was fully entrapped within the nanoparticles. The absence of the characteristic crystalline peaks indicates that the drug had been converted into an amorphous state within the nanoparticle matrix. This shift to an amorphous form is beneficial for enhancing the solubility and bioavailability of bezafibrate. 3.8. Differential Scanning Calorimetry (DSC) Differential Scanning Calorimetry (DSC) was performed to further evaluate the thermal properties and confirm the amorphous state of bezafibrate within the calcium nanoparticles. The DSC thermogram of pure bezafibrate (Fig. 7 a) exhibited a sharp endothermic peak at 188.57°C, corresponding to the melting point of the crystalline drug. This peak is characteristic of the crystalline nature of the drug, and the melting point was observed between 181°C and 185°C, consistent with previous findings [ 40 ]. In contrast, the DSC thermogram of the bezafibrate calcium nanoparticles (Fig. 7 b) displayed an endothermic peak at 100.97°C, which is significantly lower than the melting point of pure bezafibrate. The lack of any sharp peak near the melting point of bezafibrate indicates that the drug had been completely entrapped and no longer existed in its crystalline form. The shift in the melting point and the absence of characteristic peaks further confirm the transformation of the drug into an amorphous state within the nanoparticle formulation. This amorphous form is expected to enhance the dissolution rate and improve the therapeutic efficacy of the drug. 3.9. In-vitro cumulative Drug Release of Bezafibrate-Calcium Nanoparticles The in vitro cumulative drug release of the optimized Bezafibrate-Calcium Nanoparticles (BZ-CNPs) was evaluated and compared to that of a bezafibrate solution. Over 28 hours, approximately 86.42% of the drug was released from the optimized BZ-CNPs formulation, demonstrating a controlled and sustained release profile as depicted in Fig. 8 B. In contrast, the bezafibrate solution exhibited a rapid burst release, with 82.68% of the drug being released within the first 5 hours. This significant difference in release patterns indicates that the nanoparticle formulation can provide prolonged drug release, which is essential for maintaining therapeutic concentrations over an extended period. 3.9.1 Release Kinetics of BZ-CNPs The in vitro drug release data of BZ-CNPs were analyzed using various kinetic models, including Zero-order, First-order, Higuchi, and Korsmeyer–Peppas, to determine the release mechanism. The Korsmeyer–Peppas model showed the best fit (R² = 0.9771), indicating its suitability in describing the release pattern. The release exponent (n = 0.6139) suggested a non-Fickian (anomalous) diffusion mechanism, implying a combination of drug diffusion and matrix erosion. This sustained release behavior supports the potential of BZ-CNPs for prolonged therapeutic action in osteoporosis management[ 41 ]. 3.10 Stability Studies The stability of bezafibrate calcium nanoparticles (BZ-CNPs) was assessed under different storage conditions, and the results indicated that the nanoparticles maintained their stability over three months. As shown in Table VI, the nanoparticles demonstrated minimal changes in particle size, PDI, and zeta potential at various temperatures (4˚C, 25˚C, and 40˚C). The particle size increased slightly at higher temperatures, with the most significant change observed at 40˚C, where the size reached 302.6 nm, compared to the initial size of 242.1 nm at 4˚C.[ 42 ]. The PDI also showed a slight increase at elevated temperatures, indicating a minor enhancement in particle distribution, although the changes were not significant enough to impact the formulation's overall stability. The zeta potential remained relatively stable, with a slight decrease in values across all temperatures. Initially, the zeta potential at 4˚C was − 32.7 mV, decreasing to -36.1 mV at 40˚C; however, these values still suggest that the nanoparticles maintained sufficient stability due to the repulsive forces between particles, which prevent aggregation. Regarding the entrapment efficiency, there was only a slight reduction across all storage conditions, with the highest retention observed at 4˚C. The entrapment efficiency was 87.2% at the beginning and dropped minimally to 85.9% at 4˚C, indicating that the nanoparticles maintained a high degree of drug retention. In contrast, at higher temperatures (25˚C and 40˚C), the entrapment efficiency decreased more noticeably, but it remained within an acceptable range (80.6% at 40˚C). These findings suggest that the BZ-CNPs exhibit good stability at lower temperatures, with only slight changes observed in critical parameters like particle size, zeta potential, and entrapment efficiency. The results support the potential for long-term storage and application of BZ-CNPs, particularly when stored at 4˚C, where stability was most effectively preserved. Table VI Stability studies of nanoparticles under various storage conditions over time. Parameter Storage Conditions Initial Value After Three Months 4 ± 2˚C 25 ± 2˚C 40 ± 2˚C Particle size (nm) 242.1 ± 3.45 254.8 ± 2.85 287.3 ± 4.25 302.6 ± 2.15 PDI 0.302 ± 0.023 0.387 ± 0.008 0.449 ± 0.029 0.486 ± 0.011 Zeta Potential (mV) -32.7 ± 0.23 -36.1 ± 0.96 -38.4 ± 0.66 -39.2 ± 0.67 %Entrapment Efficiency 87.2 ± 2.55 85.9 ± 2.85 82.3 ± 3.35 80.6 ± 4.11 3.11. In vivo Studies 3.11.1. Effect of Formulation on Body Weight of Rats The body weight changes of rats over three weeks were monitored to evaluate the impact of the inducer and different treatments. Significant weight loss was observed in the inducer group (Group II), with a marked decrease in body weight from Week 1 to Week 3. The body weight of animals in Group II decreased from 302.14 g to 261.42 g, indicating that the dexamethasone-induced osteoporosis protocol caused substantial weight loss (Fig. 10 A). In comparison, Group I (normal control) exhibited gradual weight gain throughout the study period, with body weight increasing from 254.28 g to 267.85 g. Groups III (Zoledronic acid treatment) and IV (bezafibrate solution treatment) also displayed minor reductions in body weight; however, these changes were not statistically significant. Group III’s body weight remained relatively stable, ranging from 259.28 g to 260.00 g, while Group IV’s body weight experienced slight fluctuations, decreasing from 268.57 g to 260.00 g. Groups V and VI, treated with bezafibrate-loaded calcium nanoparticles (BZ-CNPs) of different particle sizes, showed no significant changes in body weight, suggesting that the formulation did not have a detrimental impact on the animals’ overall health. Specifically, Group V maintained consistent body weight, starting at 267.85 g and ending at 266.42 g, while Group VI showed a slight decline (257.85 g to 248.57 g), but this reduction was not significant when compared to the control group. [ 42 ] Overall, these findings suggest that while the inducer and the bezafibrate solution formulation (Group IV) led to a noticeable reduction in body weight, the bezafibrate-loaded calcium nanoparticles in Groups V and VI did not significantly affect body weight, indicating their safety in terms of systemic weight changes (Table VII). Table VII Changes in body weight of experimental subjects throughout the in vivo study. Group Week 1 (g) Week 2 (g) Week 3 (g) Control 254.28 ± 1.88 258.57 ± 57.00 267.85 ± 3.93 Inducer 302.14 ± 2.67 285.71 ± 4.49 261.42 ± 12.14 Standard 259.28 ± 6.72 265.00 ± 4.08 260.00 ± 4.08 Test 1 268.57 ± 5.56 272.14 ± 4.87 260.00 ± 4.08 Test 2 267.85 ± 4.87 266.42 ± 5.56 266.42 ± 5.56 Test 3 257.85 ± 3.93 260.71 ± 4.49 248.57 ± 2.43 3.11.2. Bone Mineral Density (BMD) Group V, which was treated with bezafibrate-loaded calcium nanoparticles (BZ-CNPs), showed a BMD of 3.838 ± 0.008 g/cm³, which was comparable to the normal control group (Group I, 3.968 ± 0.013 g/cm³). This suggests that BZ-CNPs effectively improved bone density in osteoporotic rats. This was further supported by the data presented in Fig. 9 , which visually demonstrated the positive impact of BZ-CNPs on bone health. In contrast, the inducer group (Group II) exhibited the lowest BMD of 2.168 ± 0.016 g/cm³, indicating significant bone loss induced by dexamethasone. This result (Table VII) underscores the detrimental effects of osteoporosis and highlights the efficacy of BZ-CNPs in counteracting these effects [ 23 ]. Other treatment groups also showed improvements in BMD, with Group III (Zoledronic acid treatment) and Group VI (bezafibrate-loaded calcium nanoparticles with different formulations) exhibiting BMD values of 3.602 ± 0.017 g/cm³ and 3.642 ± 0.013 g/cm³, respectively. While these values were lower than those observed in Group I and Group V, they still indicated a therapeutic effect compared to the inducer group. 3.11.3. Bone Mineral Content Markers Serum calcium (Fig. 10 B) and phosphorus (Fig. 10 C) levels are important indicators of bone mineral content. The control group had significantly higher serum calcium levels (p < 0.001), while in the Inducer group, the calcium levels were notably lower (7.90 ± 0.56 mg/dL, p < 0.001 vs Control), suggesting a condition like osteoporosis. The Standard treatment group showed moderate recovery (p < 0.001 vs Inducer), and the Drug solution group also displayed some improvement. However, in the NP-1 and NP-2 treatment groups, calcium levels showed significant improvement (p < 0.001 vs Inducer), with values approaching those of the Control group (NP-1: 11.50 ± 0.39, NP-2: 11.14 ± 0.41 mg/dL), indicating that nanoparticle-based treatments effectively restored calcium homeostasis. Similarly, phosphorus levels in the Inducer group were significantly low (2.61 ± 0.01 mg/dL; p < 0.001). Significant recovery in phosphorus levels was observed in the NP-1 (3.80 ± 0.02) and NP-2 (3.69 ± 0.01) groups (p < 0.001 vs Inducer), while no significant difference was seen in the Drug Solution and Standard groups when compared to the Inducer group [ 20 ]. 3.11.4. Bone Resorption Biomarker Serum alkaline phosphatase (ALP), as shown in Fig. 10 D is a key biomarker of bone resorption. In the control group, the ALP level was 292.10 ± 0.02 U/L, while in the Inducer group, the value was significantly elevated (725.05 ± 0.11 U/L; p < 0.001), indicating severe bone resorption. Both the Standard treatment group (407.19 ± 0.04 U/L) and the Drug solution group (567.43 ± 0.02 U/L) showed a reduction in ALP levels, although these values remained considerably higher compared to the Control group [ 23 ]. The NP-1 group (291.99 ± 0.44 U/L) significantly reduced ALP levels (p < 0.001 vs Inducer; ns vs Control), while the NP-2 group (378.34 ± 0.03 U/L) also demonstrated a statistically significant improvement (p < 0.001 vs Inducer). These results suggest that the anti-resorptive effect of NP-1 was slightly more effective than that of NP-2. 3.11.5. Bone Formation Biomarker Osteocalcin, a marker of bone formation, is depicted in Fig. 10 E. In the control group, the osteocalcin level was 17.33 ± 0.03 ng/mL, while in the Inducer group, it drastically decreased to 1.78 ± 0.05 ng/mL (p < 0.001). The Standard treatment group showed significant improvement (14.27 ± 0.04 ng/mL; p < 0.001 vs Ind), whereas the Drug solution group (7.71 ± 0.02 ng/mL) exhibited less pronounced improvement. Both NP-1 (17.27 ± 0.02 ng/mL) and NP-2 (17.22 ± 0.02 ng/mL) groups restored osteocalcin levels to those similar to the Control group (p < 0.001 vs Inducer; ns vs Control), indicating that both nanoparticle formulations substantially supported bone formation (Table VIII). Table VIII Biochemical parameters measured across experimental groups. Group Calcium Level (mg/dL) Phosphorous Level (mg/dL) Alkaline Phosphatase Level (mg/dL) Osteocalcin Level (mg/dL) Group I 12.18 ± 0.55 3.84 ± 0.03 292.10 ± 0.02 17.33 ± 0.03 Group II 7.90 ± 0.56 2.61 ± 0.01 725.05 ± 0.11 1.78 ± 0.05 Group III 9.68 ± 0.41 3.07 ± 0.03 407.19 ± 0.04 14.27 ± 0.04 Group IV 8.48 ± 0.50 2.64 ± 0.03 567.43 ± 0.02 7.71 ± 0.02 Group V 11.50 ± 0.39 3.80 ± 0.02 291.99 ± 0.44 17.27 ± 0.02 Group VI 11.14 ± 0.41 3.69 ± 0.01 378.34 ± 0.03 17.22 ± 0.02 3.11.6. Assessment of Oxidative Stress Biomarkers in Osteoporosis The assessment of oxidative stress biomarkers in osteoporosis revealed notable variations (Fig. 10 ) across the experimental groups. Group II exhibited the highest protein content (0.591 ± 0.055 mg/mL), suggesting a greater concentration of proteins compared to other groups. However, this group also showed elevated levels of malondialdehyde (MDA) (0.460 ± 0.012 nM/mg of protein content), indicating increased oxidative stress and lipid peroxidation. In contrast, Group I, which served as the control, had lower MDA levels (0.163 ± 0.016 nM/mg of protein content), suggesting minimal oxidative damage. Catalase [ 25 ] activity, which plays a crucial role in mitigating oxidative stress, was highest in Group I (0.204 ± 0.005 nM H 2 O 2 /min/mg of protein content), reflecting a more efficient antioxidant defense. Group II, however, demonstrated significantly lower CAT activity (0.040 ± 0.006 nM H 2 O 2 /min/mg of protein content), indicating a compromised ability to neutralize oxidative damage.[ 26 ] Superoxide dismutase (SOD) activity, another important antioxidant enzyme, was highest in Group III (4.045 ± 0.055 U/mg of protein content), suggesting a strong defense against oxidative stress in this group. On the other hand, Group IV showed a reduced SOD activity (3.811 ± 0.052 U/mg of protein content), which may indicate a less effective antioxidant response [ 43 ]. Overall, the findings highlight (Fig. 10 ) the intricate relationship between oxidative stress and antioxidant defense in osteoporosis, suggesting that antioxidant enzyme activities such as CAT and SOD are crucial in modulating the extent of oxidative damage. These results also point to the potential benefits of antioxidant-based therapies to counteract the oxidative stress associated with osteoporosis. [ 24 ] 3.11.7. Histopathological Analysis The histopathological examination of femur bones in rats revealed distinct variations among the experimental groups (Fig. 11 ). In the control group, the bone structure was normal, with a cortical bone thickness of 150 µm, well-preserved periosteum and endosteum, and healthy osteocyte lacunae containing nuclei. On the other hand, the osteoporosis-induced group showed a significant reduction in cortical bone thickness (119 µm), along with irregularities in the periosteum and endosteum, and a reduced number of osteocytes, many of which lacked nuclei. The Haversian canal in this group appeared enlarged and irregular [ 44 ]. The standard group displayed a cortical bone thickness of 132 µm, with slight improvements in bone architecture and a smaller Haversian canal compared to the osteoporosis-induced group. The group treated with smaller-sized BZ-CNPs (Group IV) exhibited a cortical bone thickness of 130 µm, slight irregularities in the periosteum and endosteum, and several lacunae devoid of osteocyte nuclei [ 45 ]. In contrast, the group treated with smaller-sized BZ-CNPs (Group V) showed a normal bone structure, with a cortical bone thickness of 156 µm and numerous osteocytes with well-defined nuclei, suggesting active bone regeneration [ 46 ]. Lastly, the group treated with larger-sized BZ-CNPs (Group VI) demonstrated moderate improvement, with a cortical bone thickness of 148 µm and a regular periosteum and endosteum, although the osteocyte count was lower than in Group V[ 47 ]. 4. Conclusion In this study, bezafibrate-loaded calcium nanoparticles were created utilizing the co-precipitation approach to treat osteoporosis. It was found that bezafibrate was a white, crystalline, odorless powder with melting points of 182 ± 0.56˚C and 183.66 ± 0.34˚C. 99.92% pure. Functional groups were determined using Fourier Transform Infrared Spectroscopy (FTIR), showing characteristic peaks at reported frequencies. Compatibility studies confirmed no interaction between the drug and excipients. Box Behnken Design was used for optimization, and fifteen batches were developed. One optimized batch was selected based on desired responses, and further optimized batches were evaluated for various parameters. The optimized bezafibrate calcium nanoparticles showed a particle size of 242.1 nm, PDI of 0.302, -32.7 mV of zeta potential, and 87.2% drug entrapment efficiency with 82.7% drug release in 29 hours. Effects of temperature and heat on the lattice structure of the crystal structure were verified by DSC and XRD. The drug release kinetics were plotted, and the calcium nanoparticles followed the Korsmeyer Peppas model, indicating non-Fickian diffusion. Stability studies demonstrated that the calcium nanoparticles were more stable at 4˚C storage conditions. Biochemical tests showed the successful creation of calcium in bones and elevated levels of osteocalcin, indicating bone formation. Serum ALP levels were higher in the inducer groups compared to the control and test groups. Oxidative stress biomarkers show the effectiveness of BZ-CNP for anti-osteoporotic therapy. The current study's findings indicate that these generated BZ-CNPs effectively promote bone creation in the rat model of osteoporotic DEX-induced osteoporosis. Additionally, BZ-CNPs, a novel pharmaceutical treatment that has demonstrated exceptional therapeutic benefits for the effective management of osteoporosis with excellent selectivity for bone tissues, are anticipated to help cure senile osteoporosis. Declarations Ethics approval and consent to participate : All the procedures related to the animal study were approved by the Institutional Animal Ethics Committee (IAEC) with approval number BBDNIIT/IAEC/MAY/2024/07 Consent for publication : Not applicable Competing interests : The authors declare that they have no competing interests. Consent to Participate : Not applicable Funding: This research did not receive a specific grant from funding agencies in the public, commercial, or not-for-profit sector Author Contribution Conceptualization: S KS, N D, S Y; Data curation: N D; Formal analysis: S KS, Alka; Investigation: S Y, Alka; Methodology: SY, ND; Roles/Writing - original draft: SY, Alka; and Writing – review and editing: N D, SKS Acknowledgement Acknowledgments: The authors want to acknowledge the USIC facility at BBAU, CSIR-IITR Availability of data and material: All data generated or analysed during this study are included in this published article. Data Availability: Not applicable References Prince, R. L., et al. (2019). Adding Lateral Spine Imaging for Vertebral Fractures to Densitometric Screening: Improving Ascertainment of Patients at High Risk of Incident Osteoporotic Fractures. Journal Of Bone And Mineral Research , 34 (2), 282–289. Gera, S., & Sampathi, S. (2022). Therapeutic Potential of Naringenin Nanosuspension: In Vitro and In Vivo Anti-Osteoporotic Studies. Pharmaceutics, 14: p. 1449. Marcucci, G. (2023). Oxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches . Antioxidants (Basel) , 12(2). Florencio-Silva, R. (2015). Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells. Biomed Res Int, 2015: p. 421746. Clarke, B. (2008). Normal bone anatomy and physiology. Clinical Journal Of The American Society Of Nephrology , 3 (Suppl 3), S131–S139. Ji, W., Chen, M., & Ji, H. (2022). The calcium supplementation effect of calcium-binding oligopeptides from bonito (Auxis thazard) hydrolysate in rats (p. 42). Food Science and Technology. Kim, J. M. (2020). Osteoblast-Osteoclast Communication and Bone Homeostasis . Cells , 9(9). Tresguerres, F. G. F. The osteocyte: A multifunctional cell within the bone. 2024(1618 – 0402 (Electronic)). Ponzetti, M., & Rucci, N. (2021). Osteoblast Differentiation and Signaling: Established Concepts and Emerging Topics . International Journal Of Molecular Sciences , 22(13). Park, E. (2020). Osteoprotective Effects of Loganic Acid on Osteoblastic and Osteoclastic Cells and Osteoporosis-Induced Mice . International Journal Of Molecular Sciences , 22(1). Bonewald, L. F. (2021). Chap. 7 - Osteocytes . In D. W. Dempster, et al. (Eds.), Marcus and Feldman's Osteoporosis (Fifth Edition) (pp. 135–163). Academic. Datt, N., Poonuru, R. R., & Yadav, P. K. (2022). Development and characterization of griseofulvin loaded nanostructured lipid carrier gel for treating dermatophytosis. Food Hydrocolloids for Health , 2 , 100074. Yadav, M., et al. (2025). Colloidal Copper Nanoparticles Loaded with Vanillic Acid as Liquid Dressings: Development, Characterisation and Evaluation. Pharmaceutical Innovation , 20 (2), 40. de Bôlla, L., et al. (2024). Calcium oxide nanoparticles: Biosynthesis, characterization and photocatalytic activity for application in yellow tartrazine dye removal. Journal of Photochemistry and Photobiology A: Chemistry , 447 , 115182. Rani, M., et al. (2024). Biowaste-derived nanocomposite of calcium oxide incorporated in nickel oxide for efficient removal of organic pollutants. Biomass Conversion and Biorefinery , 15 (4), 6495–6510. Nuruzzaman, M., et al. (2025). Effect of Heat Treatment on Polymorphism and Particle Size Distribution of Calcium Carbonate Nanoparticle Synthesized via Mechanochemical Process. Applied Nano , 6 (2), 8. Elmsmari, F., et al. (2021). Calcium hydroxide-loaded PLGA biodegradable nanoparticles as an intracanal medicament. International Endodontic Journal , 54 (11), 2086–2098. Mada, S. B., et al. (2020). Milk-derived bioactive peptides with antiosteoporotic effect: A mini review. Fudma Journal of Sciences , 4 (3), 351–357. Pizzino, G., et al. (2017). Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model. Frontiers In Pharmacology , 8 , 558. Lucinda, L. M. F., et al. (2017). Evaluation of the anti-osteoporotic effect of Ginkgo biloba L. in Wistar rats with glucocorticoid-induced-osteoporosis by bone densitometry using dual-energy x-ray absorptiometry (DEXA) and mechanical testing. Anais Da Academia Brasileira De Ciências , 89 (4), 2833–2841. Honary, S., & Zahir, F. (2013). Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems - A Review (Part 1) . Tropical Journal of Pharmaceutical Research , 12(2). Muhammad, S. I. (2013). Bone mass density estimation: Archimede’s principle versus automatic X-ray histogram and edge detection technique in ovariectomized rats treated with germinated brown rice bioactives . Clinical Interventions in Aging , : pp. 1421–1431. Banji, D., et al. (2014). Role of Triticum aestivum aqueous extract in glucocorticoid induced osteoporosis in rats. Indian Journal Of Experimental Biology , 52 (2), 153–158. Iantomasi, T., et al. (2023). Oxidative Stress and Inflammation in Osteoporosis: Molecular Mechanisms Involved and the Relationship with microRNAs. International Journal Of Molecular Sciences , 24 (4), 3772. Lemmerer, A., et al. (2009). Concomitant polymorphs of the antihyperlipoproteinemic bezafibrate. Crystal Growth , 9 (6), 2646–2655. Tang, S., et al. (2019). Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris. Journal of Central South University , 26 (1), 184–195. Prajapati, P., et al. (2023). Azilsartan Ameliorates Skeletal Muscle Wasting in High Fat Diet (HFD)-induced Sarcopenic Obesity in Rats via Activating Akt Signalling Pathway. Archives Of Gerontology And Geriatrics , 112 , 105025. Khine, E. E., et al. (2022). Synthesis and characterization of calcium oxide nanoparticles for CO2 capture. Journal of Nanoparticle Research , 24 (7), 139. Rakić, T., et al. (2014). Comparison of full factorial design, central composite design, and box-behnken design in chromatographic method development for the determination of fluconazole and its impurities. Analytical Letters , 47 (8), 1334–1347. Dashnyam, K., et al. (2020). Nanoscale Calcium Salt-Based Formulations As Potential Therapeutics for Osteoporosis. ACS Biomater Sci Eng , 6 (8), 4604–4613. Maleki Dizaj, S., et al. (2016). Application of Box–Behnken design to prepare gentamicin-loaded calcium carbonate nanoparticles. Artificial cells nanomedicine biotechnology , 44 (6), 1475–1481. Ahmad, S., et al. (2021). Experimental investigation of microalgal harvesting with low cost bottom ash: Influence of temperature and pH with zeta potential and thermodynamic function. Environmental Technology Innovation , 22 , 101376. Cacua, K., et al. (2019). Surfactant concentration and pH effects on the zeta potential values of alumina nanofluids to inspect stability. Colloids Surfaces A: Physicochemical Engineering Aspects , 583 , 123960. Abeywardena, M. R., et al. (2020). Surfactant assisted synthesis of precipitated calcium carbonate nanoparticles using dolomite: Effect of pH on morphology and particle size. Advanced Powder Technology , 31 (1), 269–278. Emami, J., Yousefian, H., & Sadeghi, H. (2018). Targeted Nanostructured Lipid Carrier for Brain Delivery of Artemisinin: Design, Preparation, Characterization, Optimization and Cell Toxicity. Journal Of Pharmacy & Pharmaceutical Sciences : A Publication Of The Canadian Society For Pharmaceutical Sciences, Société Canadienne Des Sciences Pharmaceutiques , 21 (1s), 225s–241s. Sun, R., et al. (2020). Electrosprayed Polymeric Nanospheres for Enhanced Solubility, Dissolution Rate, Oral Bioavailability and Antihyperlipidemic Activity of Bezafibrate. Int J Nanomedicine , 15 , 705–715. Habte, L. (2019). Synthesis of Nano-Calcium Oxide from Waste Eggshell by Sol-Gel Method . Sustainability , 11(11). Kamaraj, S., et al. (2018). Curcumin drug delivery by vanillin-chitosan coated with calcium ferrite hybrid nanoparticles as carrier. European journal of pharmaceutical sciences , 116 , 48–60. Sun, R., et al. (2020). The effects of additives on the porosity and stability of amorphous calcium carbonate. Microporous mesoporous materials , 292 , 109736. Afzal, A., et al. (2020). Molecular dynamics in the supercooled liquid and glassy states of bezafibrate and binary mixture of fenofibrate. of Non-Crystalline Solids , 550 , 120407. Muhammad Mailafiya, M., et al. (2019). Cockle Shell-Derived Calcium Carbonate (Aragonite) Nanoparticles: A Dynamite to Nanomedicine. Applied Sciences , 9 (14), 1–25. Huang, S., et al. (2009). Effects of nano calcium carbonate and nano calcium citrate on toxicity in ICR mice and onbone mineral density in an ovariectomized mice model. Nanotechnology , 20 (37), 375102. Shen, X., et al. (2024). Associations of global biomarkers of oxidative stress with osteoporosis, bone microstructure and bone turnover: Evidence from human and animal studies. Bone , 183 , 117077. Rahmani, D., et al. (2023). The beneficial effects of simultaneous supplementation of Lactobacillus reuteri and calcium fluoride nanoparticles on ovariectomy-induced osteoporosis. BMC Complement Med Ther , 23 (1), 340. Khajuria, D. K., Razdan, R., & Mahapatra, D. R. (2015). Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model. European Journal Of Pharmaceutical Sciences , 66 , 173–183. Guo, Z., et al. (2023). Investigation of the treatment potential of Raloxifene-loaded polymeric nanoparticles in osteoporosis: In-vitro and in-vivo analyses. Heliyon , 9 (9), e20107. Elghareeb, M. M., et al. (2022). Estradiol and zinc-doped nano hydroxyapatite as therapeutic agents in the prevention of osteoporosis; oxidative stress status, inflammation, bone turnover, bone mineral density, and histological alterations in ovariectomized rats. Frontiers in Physiology , 13 , 989487. Additional Declarations No competing interests reported. Supplementary Files GA.jpg Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2025 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 08 Sep, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviewers agreed at journal 09 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Editor assigned by journal 04 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 23 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7197944","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496691996,"identity":"2425eca3-3480-4f59-ae92-9b2d41731320","order_by":0,"name":"Shikha Yadav","email":"","orcid":"","institution":"Babu Banarasi Das Northern India Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shikha","middleName":"","lastName":"Yadav","suffix":""},{"id":496691998,"identity":"b138d1f1-2512-4ebe-ac0a-ea0e2891b27e","order_by":1,"name":"Alka .","email":"","orcid":"","institution":"Babu Banarasi Das Northern India Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Alka","middleName":"","lastName":".","suffix":""},{"id":496691999,"identity":"69dccb42-74f2-496c-aa0f-aba39c70a5c7","order_by":2,"name":"Shailendra K Saraf","email":"","orcid":"","institution":"Babu Banarasi Das Northern India Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shailendra","middleName":"K","lastName":"Saraf","suffix":""},{"id":496692000,"identity":"1f44bd5d-e1b6-43ee-8d29-c0a64a44ea91","order_by":3,"name":"Neelam Datt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIie3PsUrEMBjA8ZQDXT5wTeEeonAQOSjtg7ikBOoSH0A8sMHB5e4NfAXXzJHA3VKva6WD5gk8cLrNpILnEuN4YP5Lafh+/VKEYrHjLFEUIThD4F5ydyBUyDgyTZuR1I40YeK+nqmR6O8Tb+end0oZXgAaVk8f17wrHu+13bLIL3xkvlxTVUkGycOWpc9yYLKtLFnXV42HZD3PLJnABPMsFZYQZUnSaD95fd9ZcgsnmM/2Qm4Z6UyA9IAs0QCYE7tFFaQPbWlrd7EN4GlL5kIySnq7hf72LxttzF7elOWwnL0IWZSkuzRvu0XuJYfw16MaJ2lw/Acp/zQci8Vi/6pP6eZqIxFWRvAAAAAASUVORK5CYII=","orcid":"","institution":"Babu Banarasi Das Northern India Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Neelam","middleName":"","lastName":"Datt","suffix":""}],"badges":[],"createdAt":"2025-07-23 15:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7197944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7197944/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02305-7","type":"published","date":"2025-12-17T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88519471,"identity":"6a1c0c5f-8320-4527-bf5f-492eeebb5302","added_by":"auto","created_at":"2025-08-07 09:33:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96952,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the synthesis of bezafibrate-loaded calcium nanoparticles (BZ-CNPs) using the chemical precipitation method. The process involves the co-precipitation of calcium ions and bezafibrate under controlled conditions to form biocompatible nanoparticles suitable for targeted bone therapy.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/e38f95b3454fcfd3c49fe9d0.jpg"},{"id":88520929,"identity":"8662f434-b2e1-4234-b241-3939eaab9d2e","added_by":"auto","created_at":"2025-08-07 09:41:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312864,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional response surface plots illustrating the effect of independent variables on (a) particle size, (b) polydispersity index (PDI), (c) zeta potential, (d) entrapment efficiency (%EE), and (e) cumulative drug release (%CDR).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/124ef3c6233485c353029fc2.png"},{"id":88520930,"identity":"6a15f334-a129-4cd8-915d-e0b520865c11","added_by":"auto","created_at":"2025-08-07 09:41:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208485,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic light scattering (DLS) analysis showing (a) Particle size distribution and Polydispersity index (PDI), and (b) Zeta potential of BZ-CNPs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/d27fe53e86c008f573198bc7.png"},{"id":88519474,"identity":"47a09cc5-7093-4150-bff2-e7692039213b","added_by":"auto","created_at":"2025-08-07 09:33:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":274447,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Microscopy (SEM) images (A) Calcium Nanoparticles (B) bezafibrate-loaded calcium nanoparticles (BZ-CNPs), depicting surface morphology, particle shape, and structural characteristics.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/43d21d15f523b433038bcb8b.png"},{"id":88521185,"identity":"448304c3-d184-45bb-9dd6-bcf2c9f2fcd0","added_by":"auto","created_at":"2025-08-07 09:49:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20965,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra representing (a) pure Bezafibrate, (b) Sodium Lauryl Sulfate (SLS), (c) Calcium Nitrate, (d) physical mixture of all components, and (e) formulated BZ-CNPs, highlighting characteristic peaks and possible interactions among drug and excipients.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/adad46fe2aef02528e257273.png"},{"id":88522778,"identity":"1b8dc349-5a4f-49ff-9681-7b53c37d3825","added_by":"auto","created_at":"2025-08-07 09:57:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77878,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of (a) pure Bezafibrate and (b) BZ-CNPs, illustrating changes in crystallinity and confirming the encapsulation of the drug within the nanoparticulate matrix.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/d397ca218f51d42f653b6b17.png"},{"id":88521184,"identity":"5d08706b-c9f1-48de-ad05-a1e6a5494d2e","added_by":"auto","created_at":"2025-08-07 09:49:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57078,"visible":true,"origin":"","legend":"\u003cp\u003eDSC thermograms of (a) pure Bezafibrate and (b) BZ-CNPs, depicting thermal transitions and indicating possible changes in crystallinity and drug-excipient interactions upon nanoparticle formation.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/a871488b8ff22eb84777d353.png"},{"id":88519480,"identity":"40ba5414-c9b6-4aad-a8dc-1a93b479ab05","added_by":"auto","created_at":"2025-08-07 09:33:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":55079,"visible":true,"origin":"","legend":"\u003cp\u003eIn- vitro\u003cem\u003e \u003c/em\u003eDrug Release of (A) All Batches, (B) Drug solution, and BZ-CNPs\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/681d0b97cbabccd19a30d35c.png"},{"id":88520935,"identity":"029fcb9e-6aa7-4abb-b8f1-7399e910d4d7","added_by":"auto","created_at":"2025-08-07 09:41:43","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":43274,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of bone mineral density (BMD) in rats across all experimental groups, demonstrating the effect of various interventions\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/212c025d0dd669ba23511900.jpg"},{"id":88520933,"identity":"89de19f2-7328-4e0f-9469-c3c056cd5f1d","added_by":"auto","created_at":"2025-08-07 09:41:43","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":190586,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of physiological and biochemical parameters across experimental groups, including (A) body weight, (B) serum calcium, (C) serum phosphorus, (D) serum alkaline phosphatase, and (E) serum osteocalcin levels. The graph illustrates the impact of disease induction and treatment interventions on bone health and metabolic activity.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/0e8d28b7c8f139a63cb449fc.jpg"},{"id":88522779,"identity":"9efca125-685b-4189-9310-8f02c8e7fd9f","added_by":"auto","created_at":"2025-08-07 09:57:43","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":107455,"visible":true,"origin":"","legend":"\u003cp\u003eLevels of oxidative stress biomarkers of (A) Protein content, (B) MDA level, (C) Catalase level, and (D) SOD level in different experimental groups, highlighting oxidative damage and antioxidant defence in osteoporotic conditions.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/5b99243fad4261e829d8a91e.jpg"},{"id":88519488,"identity":"22e08a7a-286e-4deb-82f8-8ee35aaacb98","added_by":"auto","created_at":"2025-08-07 09:33:44","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1179784,"visible":true,"origin":"","legend":"\u003cp\u003eHistological images illustrating cortical bone thickness, osteocyte lacunae, and Haversian canals. Panels (a-b) depict the control group, (c-d) the inducer group, (e-f) the standard group, (g-h) test group 1, (i-j) test group 2, and (k-l) test group 3, respectively.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/e563a57dc4b65e72ca8559d7.png"},{"id":98814052,"identity":"e694a448-1610-4829-84b9-06f80cd8c593","added_by":"auto","created_at":"2025-12-22 16:10:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4878813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/1d10e907-3d37-44f7-8b64-eb690fa15fd4.pdf"},{"id":88519469,"identity":"8a658ec7-0484-4a8f-90f2-a38555fc9e07","added_by":"auto","created_at":"2025-08-07 09:33:43","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":97173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7197944/v1/5f19e06b685e92cbb9515da4.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanoengineered Bezafibrate-Loaded Calcium Nanoparticles for Osteoporosis: A Repurposing Approach for Targeted Bone Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteoporosis is the most prevalent metabolic bone disorder globally, posing a significant public health challenge, particularly among the aging population. This condition is characterized by an imbalance between bone resorption and formation, leading to a decrease in bone mineral density (BMD), disruption of bone microarchitecture, and an increased susceptibility to fractures. The impact is most pronounced in postmenopausal women, who experience accelerated bone loss due to estrogen deficiency, with estimates indicating that nearly 70% of individuals over the age of 80 are affected [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Given the rising prevalence and the limitations of existing treatments, there is an urgent need to develop targeted therapeutic strategies, particularly for vulnerable populations, where traditional approaches may be inadequate or accompanied by significant side effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent advances in translational research have fostered a growing interest in bridging fundamental bone biology with innovative drug-delivery technologies to address these challenges. Nanoparticle-based drug delivery systems, especially those utilizing biocompatible calcium-based carriers, offer promising solutions for enhancing bone regeneration, improving therapeutic targeting, and optimizing clinical outcomes in osteoporotic patients. By leveraging nanotechnology, these systems aim to overcome the limitations of conventional therapies by offering more effective and controlled drug delivery, specifically targeting bone tissue.\u003c/p\u003e\u003cp\u003eOsteoporosis results from an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Osteoblasts, originating from mesenchymal stem cells (MSCs), play a key role in synthesizing bone matrix and mineralizing it through the deposition of calcium phosphate. Conversely, osteoclasts, derived from hematopoietic stem cells (HSCs), mediate bone resorption through the enzymatic degradation of mineralized tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBone tissue itself is a composite of collagen, proteins, and mineralized calcium phosphate, which not only provides structural integrity but also supports essential metabolic functions. Calcium, a critical element in maintaining bone homeostasis, is predominantly derived from dietary sources, with its absorption influenced by various physiological factors such as nutritional status and metabolic conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBone remodeling is a highly dynamic process involving tightly regulated cycles of bone formation and resorption. Osteoblast function is modulated by various transcriptional, epigenetic, and environmental signals, while osteoclasts contribute to bone degradation within specialized resorption zones known as Howship\u0026rsquo;s lacunae [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Advances in nanotechnology have opened new possibilities for enhancing bone-specific drug delivery. Among inorganic carriers, calcium nanoparticles (CNPs) have garnered attention due to their inherent biocompatibility, bone-targeting capabilities, and high drug-loading potential. These nanoparticles not only serve as efficient carriers but also function as therapeutic agents themselves, promoting bone repair by delivering calcium directly to deficient bone sites while maintaining systemic calcium equilibrium [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Compared to organic nanocarriers, CNPs offer superior structural stability and a greater surface area, which can be further optimized for stimuli-responsive drug release.\u003c/p\u003e\u003cp\u003eIn the context of drug repurposing, fibrates such as fenofibrate and bezafibrate have shown promise in enhancing osteoblast differentiation and improving bone mineral density, suggesting their potential role in osteoporosis management. Bezafibrate, a BCS Class III drug known for its low permeability, may be particularly effective for targeted bone therapy when delivered via an appropriate nanocarrier system. However, despite its therapeutic promise, the clinical translation of bezafibrate for skeletal applications remains underexplored [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aligns with the principles of translational research, aiming to translate preclinical discoveries into clinically applicable therapies. The primary objective is to develop and optimize bezafibrate-loaded calcium nanoparticles (BZ-CNPs) for enhanced bone regeneration. The formulation's efficacy will be evaluated in a dexamethasone-induced osteoporotic rat model to assess its potential in improving bone mineral density and restoring bone homeostasis. By bridging the gap between basic research and clinical application, this study seeks to advance targeted and clinically translatable therapeutic options for osteoporosis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Drugs and Reagents\u003c/h2\u003e\n \u003cp\u003eBezafibrate (BZ) was purchased from BLD Pharm, Hyderabad, Telangana, India. Calcium nitrate tetrahydrate was procured from Hi Media Laboratories, Mumbai, Maharashtra, India. Sodium lauryl sulfate (SLS) and sodium hydroxide (NaOH) pellets were obtained from SD Fine Chem, Mumbai, Maharashtra, India. Methanol was sourced from Rankem Chemicals, Pune, Maharashtra, India. All other chemicals and reagents used in preparing and evaluating the formulation were of analytical grade.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Experimental Design and Selection of Variables\u003c/h2\u003e\n \u003cp\u003eTo investigate the effect of critical formulation variables on the properties of bezafibrate-loaded calcium nanoparticles, a Box-Behnken Design (BBD) was employed. The selected independent variables were the Ca\u0026sup2;⁺: OH⁻ molar ratio (X1), surfactant concentration (X2), and sonication time (X3), each evaluated at three levels (\u0026minus;\u0026thinsp;1, 0, +\u0026thinsp;1) as shown in Table I. Design Expert\u0026reg; 12 software generated 15 experimental runs, including 3 center points. The formulations were assessed for particle size, PDI, zeta potential, entrapment efficiency, and cumulative drug release.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable I\u003c/strong\u003e Formulation matrix of bezafibrate calcium nanoparticles developed using Box-Behnken Design for selection of the optimized batch.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCa2+: OH- (M)\u003c/p\u003e\n \u003cp\u003e(X1)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSurfactant (g)\u003c/p\u003e\n \u003cp\u003e(X2)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSonication time(min)\u003c/p\u003e\n \u003cp\u003e(X3)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Method of Preparation of Bezafibrate Calcium Nanoparticles (BZ-CNPs)\u003c/h2\u003e\n \u003cp\u003eBezafibrate-loaded calcium nanoparticles (BZ-CNPs) were synthesized using a chemical precipitation method, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Initially, a 0.2 M aqueous solution of sodium hydroxide (NaOH) was prepared and maintained under magnetic stirring. A methanolic solution of bezafibrate, along with sodium lauryl sulfate (SLS) as a surfactant, was then added dropwise to the NaOH solution under continuous stirring to facilitate drug solubilization and stabilize nanoparticle formation. Separately, a 0.1 M aqueous solution of calcium nitrate [Ca(NO₃)₂], serving as the calcium ion source, was prepared. The drug-containing mixture was then added dropwise to the calcium nitrate solution under constant stirring, and the entire system was allowed to react for 1 hour to enable nanoparticle formation via controlled precipitation. The resulting colloidal dispersion was subjected to probe sonication (45 seconds ON and 15 seconds OFF cycles) to reduce particle size and ensure uniform dispersion. The nanoparticle suspension was centrifuged at 10,000 rpm for 15 minutes at 4\u0026deg;C to separate the nanoparticles. The formed BZ-CNPs were collected as a pellet and washed, and stored appropriately for further characterization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Characterization of the Prepared BZ-CNPs\u003c/h2\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.1 Particle Size, Polydispersity Index (PDI), and Zeta Potential\u003c/h2\u003e\n \u003cp\u003eThe average particle size, polydispersity index (PDI), and zeta potential of BZ-CNPs were measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were diluted with Milli-Q water before measurement to ensure appropriate scattering intensity. All measurements were conducted at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C using disposable polystyrene cuvettes for size and PDI and folded capillary cells for zeta potential. The refractive index of the dispersion medium was set to 1.44. Data were recorded in triplicate and reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.2 Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\n \u003cp\u003eFTIR analysis was performed to identify the characteristic functional groups and assess possible interactions between bezafibrate and the calcium matrix in the prepared nanoparticles. The spectra were recorded using an FTIR spectrophotometer (Alpha II, Bruker, Germany) in the range of 4000\u0026ndash;400 cm⁻\u0026sup1;. Samples were prepared by mixing with potassium bromide (KBr) and compressing into a pellet. The obtained spectra were analyzed for shifts or appearance/disappearance of peaks to confirm drug\u0026ndash;excipient compatibility and successful nanoparticle formation[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.3 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\n \u003cp\u003eThe surface morphology and structural characteristics of the optimized BZ-CNPs were evaluated using SEM (JSM-6490LV, JEOL, Japan). A small aliquot of nanoparticle dispersion was placed onto a clean stub using double-sided carbon adhesive tape and allowed to dry under a vacuum. The dried samples were then sputter-coated with a thin layer of platinum using an auto fine coater (JFC-1600, JEOL, Japan) to ensure conductivity. Imaging was performed at an accelerating voltage of 15 kV to assess particle shape, surface texture, and approximate size[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.4 Powder X-ray Diffraction (PXRD)\u003c/h2\u003e\n \u003cp\u003ePXRD analysis was employed to investigate the crystalline nature and structural transitions of the drug within the calcium nanoparticle matrix. The diffraction patterns were recorded using an X-ray diffractometer (D8 Advance Eco, Bruker, Germany) equipped with a Cu K\u0026alpha; radiation source (\u0026lambda;\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;), operated at 45 kV and 40 mA. Scans were performed over a 2\u0026theta; range suitable for identifying characteristic diffraction peaks. Changes in peak intensity or position were analyzed to evaluate drug encapsulation and possible alterations in crystallinity due to nanoparticle formulation[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.5 Differential Scanning Calorimetry (DSC)\u003c/h2\u003e\n \u003cp\u003eDSC was employed to determine the melting point and degree of crystallinity of the formulated nanoparticles. This technique is essential for studying thermal transitions of nanoparticles, such as melting points, glass transition temperatures, and crystallization behavior, all of which significantly impact the stability and performance of the drug formulation. The DSC analysis was carried out using a DSC Q2000 (V24.11 Build 124)[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.6 Entrapment Efficiency (%EE)\u003c/h2\u003e\n \u003cp\u003eEntrapment efficiency refers to the ratio of the quantity of drug successfully entrapped within the nanoparticles to the total amount of drug initially added to the dispersion. The EE plays a critical role in determining the release characteristics of the formulation. To assess the entrapment efficiency, the prepared dispersion was centrifuged at 10,000 rpm for 1 hour at 4\u0026deg;C using a SIGMA 3\u0026ndash;18 K centrifuge (Sartorius). The supernatant was then diluted with phosphate-buffered saline (PBS, pH 7.4) to a final volume of 10 mL. The drug concentration in the supernatant was determined by UV spectrophotometry at 229.6 nm using a Shimadzu Double Beam Spectrophotometer (UV-1700). The percentage of entrapment efficiency was calculated using the following formula [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 593px;\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.7 \u003cem\u003eIn Vitro\u003c/em\u003e Drug Release Using Franz Diffusion Cell\u003c/h2\u003e\n \u003cp\u003eThe in vitro release behavior of the optimized nanoparticle formulation was assessed using a Franz diffusion cell equipped with an egg membrane as the diffusion barrier. The receptor compartment was filled with phosphate-buffered saline (PBS, pH 7.4) and maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C under constant magnetic stirring to mimic physiological conditions. An aliquot (1 mL) of the formulation was placed in the donor compartment. At predetermined time points (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 24, 25, 26, 27, 28, 29, and 30 hours), 1 mL of the receptor medium was withdrawn and replaced with an equal volume of fresh PBS to maintain sink conditions. The collected samples were appropriately diluted, and drug concentration was determined by measuring absorbance at 229.6 nm using a UV-visible spectrophotometer (UV-1700, Shimadzu, Japan). The cumulative percentage of drugs released was calculated and plotted against time to establish the release profile[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Stability Studies\u003c/h2\u003e\n \u003cp\u003eThe stability of the optimized Bezafibrate-Calcium Nanoparticles was evaluated under different storage conditions per ICH Q1A (R2) guidelines. The formulation was aliquoted into clean, light-protected glass vials and stored at 4\u0026deg;C (refrigerator), 25\u0026deg;C (room temperature), and 40\u0026deg;C (accelerated conditions) for three months. Following the storage period, the samples were analyzed for any changes in physicochemical characteristics, including particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency (%EE) to assess formulation stability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 In vivo Pharmacodynamic Studies\u003c/h2\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.1 Experimental Animals\u003c/h2\u003e\n \u003cp\u003eMale Wistar rats weighing between 250 and 300 g were used for the in vivo pharmacodynamic evaluation. The animals were kept under standard laboratory conditions: a 12:12 h light-dark cycle, an ambient temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and a relative humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. They were housed in clean polypropylene cages with stainless steel grid tops and were given free access to standard laboratory chow and water ad libitum. All experimental protocols involving animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Babu Banarasi Das Northern India Institute of Technology (Approval No. BBDNIIT/IAEC/MAY/2024/07) and were conducted in strict adherence to the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.2 Experimental Groups\u003c/h2\u003e\n \u003cp\u003eThe animals were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;5 per group). Group I served as the normal control and received no treatment. Group II was assigned as the osteoporosis control group, in which osteoporosis was induced by intraperitoneal administration of dexamethasone (DEX) at a dose of 10 mg/kg, three times a week for 21 days [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. This dosing regimen reliably mimics the pathophysiological conditions of glucocorticoid-induced osteoporosis. Groups III to VI were also subjected to the same DEX protocol to induce osteoporosis. Group III received the standard treatment with Zoledronic acid (Zoldonate\u0026reg;) at 0.1 mg/kg, administered subcutaneously once weekly [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Group IV was treated with plain bezafibrate solution at a dose of 10 mg/kg subcutaneously. Groups V and VI received bezafibrate-loaded calcium nanoparticles (BZ-CNPs) at the same dose (10 mg/kg, subcutaneously), differing only in particle size\u0026mdash;Group V received the formulation with smaller particle size, while Group VI received the formulation with larger particle size. This study design enabled the evaluation of the therapeutic potential and particle size-dependent efficacy of BZ-CNPs in the management of glucocorticoid-induced osteoporosis. On the 21st day of treatment, the animals were euthanized, and the femur bones were carefully excised and preserved in 10% neutral buffered formalin for subsequent histopathological and morphological analyses. Additionally, the surrounding skeletal muscle tissue was collected and processed for the evaluation of oxidative stress biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase activity, using spectrophotometric methods [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.3 Collection of Blood Samples and Histopathological Analysis of Bone\u003c/h2\u003e\n \u003cp\u003eOn the 21st day of treatment, all animals were sacrificed under appropriate anesthesia. Blood samples were collected via cardiac puncture using a 1 mL sterile syringe and transferred into plain collection tubes. The samples were allowed to clot and then centrifuged to separate the serum. Serum was analyzed for biochemical markers associated with bone metabolism, including calcium, phosphorus, osteocalcin, and alkaline phosphatase (ALP), to evaluate the therapeutic efficacy of various treatments in mitigating glucocorticoid-induced osteoporosis [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. For histopathological analysis, the left femurs were carefully excised and fixed in 10% neutral buffered formalin. Decalcification was carried out in 20% ethylenediaminetetraacetic acid (EDTA) solution for 20 days to ensure adequate removal of calcium deposits. Following decalcification, the bones were processed through a graded ethanol series for dehydration, cleared in xylene, and embedded in paraffin wax. Thin longitudinal sections of 6 \u0026micro;m thickness were prepared using a microtome and stained with Hematoxylin and Eosin (H\u0026amp;E). The stained sections were examined under a light microscope at magnifications of 10\u0026times; and 40\u0026times;. Morphometric parameters such as cortical bone thickness, integrity, and number of osteocyte lacunae, and the structural condition of Haversian canals were analyzed using an image analyzer to assess bone architecture and remodeling.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.4 Effect of Formulation on Body Weight of Rats\u003c/h2\u003e\n \u003cp\u003eThe body weight of each rat was recorded on Day 0 and weekly for 21 days using a digital balance (\u0026plusmn;\u0026thinsp;0.1 g). Measurements were taken in the morning to reduce diurnal variation. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5) and analyzed to evaluate the systemic effects of treatments and the protective role of BZ-CNPs in dexamethasone-induced osteoporosis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.5 Bone Mineral Density\u003c/h2\u003e\n \u003cp\u003eBone mineral density (BMD) of the left femur was determined using Archimedes\u0026rsquo; principle, which offers a reliable and simple method to assess bone mass by calculating its density. This approach facilitated the evaluation of bone mineralization and treatment efficacy in glucocorticoid-induced osteoporosis. This gravimetric method provided a non-destructive means to quantify changes in bone mass across different treatment groups. The density (d) of each bone sample was calculated using the formula:\u003c/p\u003e\n \u003cp\u003ed = (w\u003csub\u003e1\u003c/sub\u003e/w\u003csub\u003e1 \u0026ndash;\u003c/sub\u003e w\u003csub\u003e2\u003c/sub\u003e) \u0026times; P Equation \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.2\u003c/p\u003e\n \u003cp\u003ewhere d\u0026thinsp;=\u0026thinsp;bone density (in g/cm\u003csup\u003e3\u003c/sup\u003e), w\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;weight of bone in air, w\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;weight of bone in distilled water, and P\u0026thinsp;=\u0026thinsp;density of distilled water (approximately 1 g/cm\u0026sup3; at room temperature) [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.6 Bone Mineral Content Markers\u003c/h2\u003e\n \u003cp\u003eSerum calcium and phosphorus levels were measured to evaluate the bone health of the animals. Serum calcium was quantified using the Arsenazo III colorimetric method, while serum phosphorus was assessed using the Phosphomolybdate UV method [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. These biochemical markers provided essential information regarding the mineral content of the bones and served as indicators of overall bone health in the treated rats.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.7. Bone Resorption Biomarker\u003c/h2\u003e\n \u003cp\u003eSerum alkaline phosphatase (ALP) levels, a key biomarker for bone resorption, were measured to assess bone degradation associated with osteoclast activity, commonly observed in osteoporosis. The serum ALP levels were quantified using the p-nitrophenol and phosphate (pNPP) kinetic assay, which provides an accurate measurement of ALP activity in serum samples[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.8. Bone Formation Biomarker\u003c/h2\u003e\n \u003cp\u003eSerum osteocalcin, a biomarker indicative of bone formation and osteoblast activity, was also quantified. The osteocalcin levels were assessed using the Chemiluminescent Microparticle Immunoassay (CMIA) method, which provides a sensitive and specific analysis for this biomarker[\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.9. Assessment of Oxidative Stress Biomarkers in Osteoporosis\u003c/h2\u003e\n \u003cp\u003eOxidative stress plays a pivotal role in osteoporosis pathogenesis by disrupting the balance between reactive oxygen species (ROS) and antioxidant defense mechanisms, leading to bone degradation [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, oxidative stress biomarkers such as malondialdehyde (MDA), catalase [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], and superoxide dismutase (SOD) were assessed to evaluate oxidative damage and the antioxidant response. Muscle tissue (2 g) was homogenized in phosphate-buffered saline (PBS, pH 7.4), centrifuged, and the supernatant was used for biochemical assays. Protein content was quantified using the Bradford method, with bovine serum albumin (BSA) as the standard. MDA levels, an indicator of lipid peroxidation, were measured by reacting the homogenate with thiobarbituric acid (TBA) and trichloroacetic acid (TCA), followed by absorbance measurement at 540 nm. CAT activity was determined by assessing the breakdown of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), with absorbance recorded at 230 nm at two time points[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], and SOD activity was evaluated by monitoring the reduction of pyragallol at 420 nm[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The respective activities of MDA, CAT, and SOD were calculated using appropriate formulas. These biomarkers provided valuable insights into the oxidative stress status and its association with bone health [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eThe data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM). Statistical analysis was performed using GraphPad Prism 9.0.0. To determine the statistical significance between different groups, one-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparison tests was used. A p-value of \u0026lt;\u0026thinsp;0.001 was considered statistically significant, indicating a strong effect of the treatments.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Challenges in the Fabrication of Bezafibrate Calcium Nanoparticles (BZ-CNPs)\u003c/h2\u003e\u003cp\u003eThe fabrication of Bezafibrate Calcium Nanoparticles (BZ-CNPs) presented significant challenges, particularly in achieving the desired nanoparticle size. A major issue encountered was the rapid aggregation of the calcium nanoparticles upon formulation, leading to the formation of larger particle sizes that were unsuitable for effective bone penetration in osteoporosis treatment. To address this, various fabrication methods were explored, including the reverse microemulsion method and the chemical precipitation method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The reverse microemulsion method, while widely used in nanoparticle fabrication, posed several challenges. It required large quantities of surfactant and co-surfactant to stabilize the droplets of the microemulsion, which not only increased the complexity of the process but also resulted in a lack of homogeneity within the system. This lack of consistency in the microemulsion system further hindered the formation of nanoparticles with the desired size and uniformity. On the other hand, the chemical precipitation method showed potential for producing nanoparticles of smaller sizes. The solution to these challenges came with the introduction of different stabilizing agents and surfactants. Sodium hydroxide (NaOH) and sodium lauryl sulfate (SLS) were identified as effective agents for stabilizing the nanoparticles and reducing their size to the desired range, thereby enabling their efficient penetration into the bone tissue for the management of osteoporosis. The BZ-CNPs were successfully prepared using the chemical precipitation method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this approach, SLS acted as the surfactant, adsorbing onto the surface of the nanoparticles. This interaction reduced the surface tension and prevented the aggregation of the nanoparticles, thereby ensuring the production of nanoparticles of a suitable size for their intended therapeutic application.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Selection of Process Variables\u003c/h2\u003e\u003cp\u003eFor the optimization of bezafibrate-loaded calcium nanoparticles, the influence of various independent variables on critical quality attributes was investigated. The study aimed to establish the relationship between selected process parameters and formulation responses using a design of experiments approach. With an increase in the number of influencing factors, the Box-Behnken Design (BBD) was employed to reduce the number of experimental runs while ensuring systematic evaluation.\u003c/p\u003e\u003cp\u003eThree independent variables were selected for the study: the molar ratio of precursors (Ca\u0026sup2;⁺: OH⁻) designated as X1, surfactant concentration as X2, and sonication time as X3 (Table II). The formulation\u0026rsquo;s responses included particle size (Y1), polydispersity index (Y2), zeta potential (Y3), entrapment efficiency (Y4), and cumulative drug release (Y5). Each independent variable was evaluated at three levels: low (-1), medium (0), and high (1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable II\u003c/b\u003e Lists the factor settings for the experimental design.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIndependent Variables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow (-1)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedium (0)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh (1)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e: OH\u003csup\u003e\u0026minus;\u003c/sup\u003e (molar ratio)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1:2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1:3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSLS concentration (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSonication time (minutes)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe Ca\u0026sup2;⁺: OH⁻ molar ratio varied between 1:1, 1:2, and 1:3, with corresponding changes in the surfactant concentration (SLS) and sonication time. The surfactant concentration was adjusted from 1 g to 3 g, and the sonication time was varied between 30 minutes, 40 minutes, and 50 minutes. The optimization process employed the Design Expert 12 software to generate fifteen experimental batches, including three center points, to assess the impact of these variables on the nanoparticle characteristics[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The experimental design allowed for a comprehensive analysis of the formulation's behaviour under varying conditions, and it provided valuable insights into the factors that most significantly affected the quality of the BZ-CNPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Optimization of Bezafibrate Calcium Nanoparticles (BZ-CNPs)\u003c/h2\u003e\u003cp\u003eThe optimization of BZ-CNPs was performed using a Box-Behnken design (BBD), which allowed for the systematic evaluation of different formulation parameters and their effects on the nanoparticle characteristics. The independent variables selected for optimization included the Ca\u0026sup2;⁺: OH⁻ molar ratio, surfactant concentration, and sonication time, as outlined in Table III. These variables were carefully chosen to ensure that they could effectively influence the size, zeta potential, polydispersity index (PDI), entrapment efficiency, and drug release profiles of the nanoparticles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable III\u003c/b\u003e Optimization and Physicochemical evaluation of BZ-CNPs formulated via Box-Behnken design.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePS\u003c/p\u003e\u003cp\u003e(nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZP\u003c/p\u003e\u003cp\u003e(mV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% Entrapment Efficiency\u003c/p\u003e\u003cp\u003e(%EE)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e% Cumulative Drug Release\u003c/p\u003e\u003cp\u003e(% CDR)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e217.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.194\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-34.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e80.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e201.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-29.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e75.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e250.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-21.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e86.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e84.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e201.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-45.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e86.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e74.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e269.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.278\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-43.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e87.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e81.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e201.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-29.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e75.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e270.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.332\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-21.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e88.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e75.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e206.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-50.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e79.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e286.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-45.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e91.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e77.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e201.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-29.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e85.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e75.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e110.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.317\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-57.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e91.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e78.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e105.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-26.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e86.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e81.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eT13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e275.8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.367\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-30.7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e85.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e88.3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e102.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-51.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e84.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e281.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.352\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-48.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e77.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of Independent Variables on Dependent Variables\u003c/h2\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. Particle Size, Polydispersity Index (PDI), and Zeta Potential\u003c/h2\u003e\u003cp\u003eThe independent variables outlined in Table III had a significant impact on the dependent variables of the prepared BZ-CNPs. Among these, the particle size emerged as a crucial factor in the fabrication and optimization of BZ-CNPs. A study indicated that nanoparticles with a size of less than 400 nm were effective in significantly enhancing bone density and promoting calcium deposition in bone tissues [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, achieving a particle size of less than 400 nm was a primary objective of this study.\u003c/p\u003e\u003cp\u003eThe particle size of the BZ-CNPs was primarily influenced by the sonication time, with a noticeable reduction in particle size as sonication time increased. This observation aligns with previous reports where increased sonication duration led to finer particles, presumably due to better dispersion and energy imparted to the system [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, the molar ratio of Ca\u0026sup2;⁺: OH⁻ was also found to significantly impact the particle size. In this study, the intermediate molar ratio (Ca\u0026sup2;⁺: OH⁻ = 1:2) resulted in smaller nanoparticles.\u003c/p\u003e\u003cp\u003eThe results from the Design Expert supported these observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The reduced quadratic model developed for particle size was statistically significant (p\u0026thinsp;=\u0026thinsp;0.0080), with a Model F-value of 7.43. Among the factors, surfactant concentration (B) and sonication time squared (C\u0026sup2;) significantly affected the particle size (p\u0026thinsp;=\u0026thinsp;0.0332 and p\u0026thinsp;=\u0026thinsp;0.0108, respectively). The model exhibited an R\u0026sup2; of 0.5531 and an adjusted R\u0026sup2; of 0.4787, indicating moderate predictive power. The lack of fit was not significant (p\u0026thinsp;=\u0026thinsp;0.4410), suggesting good model adequacy. The adequate precision ratio was 7.29, which is greater than the desired value of 4, confirming an adequate signal (Table IV).\u003c/p\u003e\u003cp\u003eThe optimized BZ-CNPs had a particle size of 242.1 nm and a Polydispersity Index (PDI) of 0.314 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The PDI value indicated a narrow size distribution, which is essential for the uniformity and reproducibility of the nanoparticle formulation. The particle size and PDI results were in line with the desired characteristics for effective bone penetration and therapeutic efficacy.\u003c/p\u003e\u003cp\u003eRegarding PDI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), a reduced cubic model was found to be significant (p\u0026thinsp;=\u0026thinsp;0.0009), with sonication time squared (C\u0026sup2;, p\u0026thinsp;=\u0026thinsp;0.0054) and the interaction between Ca\u0026sup2;⁺: OH⁻ ratio and sonication (AC\u0026sup2;, p\u0026thinsp;=\u0026thinsp;0.0011) playing key roles. The model had a high R\u0026sup2; value of 0.7650 and an adjusted R\u0026sup2; of 0.7009, with a non-significant lack of fit (p\u0026thinsp;=\u0026thinsp;0.8198). The low standard deviation (0.0377) and coefficient of variation (CV\u0026thinsp;=\u0026thinsp;11.58%) reflected the model\u0026rsquo;s precision and reliability (Table IV).\u003c/p\u003e\u003cp\u003eThe zeta potential, another critical parameter for the stability of nanoparticles, was also influenced by the independent variables. It was observed that increasing the molar ratio of the precursors led to a higher zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Additionally, lower concentrations of surfactants resulted in improved zeta potential values[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. An increase in sonication time further optimized the zeta potential, resulting in a more stable dispersion. The zeta potential of the optimized BZ-CNPs was measured at -32.7 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating good stability and reduced chances of nanoparticle aggregation over time [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese results were validated by the reduced quadratic model developed for zeta potential (Table IV), which was also statistically significant (p\u0026thinsp;=\u0026thinsp;0.0067) with a Model F-value of 6.74. Significant factors included sonication time (C) and its quadratic term C\u0026sup2; (p\u0026thinsp;=\u0026thinsp;0.0397 and p\u0026thinsp;=\u0026thinsp;0.0046, respectively). The model had an R\u0026sup2; value of 0.7295 and an adjusted R\u0026sup2; of 0.6213. The lack of fit was not significant (p\u0026thinsp;=\u0026thinsp;0.7851), confirming the model\u0026rsquo;s reliability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable IV\u003c/b\u003e ANOVA summary of model statistics for Particle Size, PDI, and Zeta Potential of BZ-CNPs\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResponse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParticle Size\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZeta Potential\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eModel Type\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReduced Quadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReduced Cubic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReduced Quadratic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eModel F-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0080\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0067\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSignificant Terms\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB, C\u0026sup2;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC\u0026sup2;, AC\u0026sup2;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC, C\u0026sup2;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eR\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5531\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7650\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7295\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAdjusted R\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.6213\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePredicted R\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.2824\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5531\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.4028\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLack of Fit (p)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4410\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.8198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.7851\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAdequate Precision\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.2883\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.6105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.6296\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2. Entrapment Efficiency (EE) and Cumulative Drug Release (CDR)\u003c/h2\u003e\u003cp\u003eThe entrapment efficiency (EE) and cumulative drug release (CDR) were also influenced by the independent variables. As the sonication time increased, the EE decreased, likely due to heat generation during prolonged sonication, which could cause the drug to leach out from the nanocarriers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. On the other hand, a shorter sonication time resulted in lower EE, suggesting that a moderate sonication time is optimal for achieving a higher EE. Surfactant concentration played a pivotal role in facilitating the solubilization of the drug, leading to an increase in drug release from the nanoparticles. This is due to the surfactant\u0026rsquo;s ability to lower the interfacial tension between the nanoparticles and the surrounding medium, promoting the dissolution and release of the encapsulated drug [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, changes in the Ca\u0026sup2;⁺: OH⁻ molar ratio showed less significant influence on cumulative drug release, indicating that surfactant behavior and sonication are more dominant factors in controlling drug release kinetics.\u003c/p\u003e\u003cp\u003eThese trends were statistically confirmed by the ANOVA results obtained from Design Expert\u0026reg; software (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The EE data followed a reduced quadratic model that was statistically significant (p\u0026thinsp;=\u0026thinsp;0.0026), with a Model F-value of 9.04. Surfactant concentration (B), along with its squared term (B\u0026sup2;), and sonication time squared (C\u0026sup2;), were found to be significant factors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The model showed good reliability with an R\u0026sup2; of 0.8340, an adjusted R\u0026sup2; of 0.7417, and a predicted R\u0026sup2; of 0.5382. The lack of fit was not significant (p\u0026thinsp;=\u0026thinsp;0.8138), indicating that the model was suitable for prediction. Adequate precision was 8.20, which is well above the minimum threshold of 4, suggesting a strong signal-to-noise ratio (Table V). Similarly, the cumulative drug release (%CDR) was best described by a reduced cubic model, which was also statistically significant (p\u0026thinsp;=\u0026thinsp;0.0011) with a Model F-value of 11.42. The drug release was significantly affected by sonication time (C), the square of surfactant concentration (B\u0026sup2;), and the interaction term A\u0026sup2;B (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The model had a high R\u0026sup2; value of 0.7570 and an adjusted R\u0026sup2; of 0.6907, with a predicted R\u0026sup2; of 0.5649, indicating good agreement among the three. The non-significant lack of fit (p\u0026thinsp;=\u0026thinsp;0.8939) and a high adequate precision of 8.73 supported the model\u0026rsquo;s predictive validity (Table V).\u003c/p\u003e\u003cp\u003eThe optimized formulation exhibited an EE of 87.2% and a cumulative drug release of 82.7%, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, closely matching the predicted values from the desirability function. These findings affirm that the Box-Behnken Design successfully guided the formulation process toward achieving an ideal balance of high drug loading and sustained release, suitable for potential use in osteoporosis treatment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable V\u003c/b\u003e ANOVA summary of model statistics for Entrapment Efficiency (%EE) and Cumulative Drug Release (%CDR) of BZ-CNPs\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResponse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEntrapment Efficiency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCumulative Drug Release\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eModel Type\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReduced Quadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReduced Cubic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eModel F-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0011\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSignificant Terms\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB, B\u0026sup2;, C\u0026sup2;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u0026sup2;, A\u0026sup2;B\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eR\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.8340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7570\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAdjusted R\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.7417\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.6907\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePredicted R\u0026sup2;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5382\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5649\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLack of Fit (p)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.8138\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.8939\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAdequate Precision\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.1967\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.7252\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eScanning Electron Microscopy (SEM) analysis was performed on the prepared Bezafibrate Calcium Nanoparticles (BZ-CNPs) to observe their surface morphology. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the SEM images revealed that the bezafibrate is irregular in shape and size, while drug-incorporated calcium nanoparticles exhibited a mix of spherical and smooth surface morphology as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Similar results were observed earlier by researchers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Some nanoparticles were well-defined and spherical, while others showed an irregular morphology. Additionally, it was observed that the particles had agglomerated to some extent, which is a common occurrence during nanoparticle formulation due to the inherent tendency of nanoparticles to aggregate in the absence of proper stabilizing agents. This agglomeration could potentially impact the uniformity and performance of the nanoparticles in drug delivery systems [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\u003cp\u003eFTIR analysis was conducted to assess the chemical structure of the Bezafibrate Calcium Nanoparticles and to detect any possible interactions between the drug and the excipients used in the formulation. The FTIR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) of the BZ-CNPs revealed sharp, characteristic peaks that correspond to specific functional groups present in Bezafibrate. Notably, the peak at 2916.13 cm-\u0026sup1; corresponded to the stretching vibration of hydroxide (-OH), a key functional group in Bezafibrate. The spectrum also exhibited peaks for the ether group at 1081.06 cm⁻\u0026sup1;, the bending vibration of the -NH group at 1450.48 cm⁻\u0026sup1;, and the C-Cl stretch at 832.55 cm⁻\u0026sup1;. Additionally, the N\u0026thinsp;=\u0026thinsp;O peak from calcium nitrate appeared at 1412.41 cm⁻\u0026sup1;, and the S\u0026thinsp;=\u0026thinsp;O stretch from sodium lauryl sulfate (SLS) was detected at 1213.13 cm⁻\u0026sup1; [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The FTIR results indicated that the functional groups of the drug and excipients remained intact, suggesting that there were no significant chemical interactions between them. This stability of the functional groups is indicative of the chemical stability of the BZ-CNPs formulation. The unaltered structure of both the drug and excipient implies that the encapsulation process did not cause any detrimental changes to their chemical composition or integrity. These findings align with previous reports where bioactive compounds, even when encapsulated into nano-formulations, retained their original functional groups and structural configurations, ensuring their stability and potential efficacy in drug delivery systems[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese results from the FTIR analysis support the successful preparation of BZ-CNPs with preserved chemical stability, which is crucial for ensuring the therapeutic effectiveness and safety of the formulation in clinical applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.7. X-ray diffraction (XRD)\u003c/h2\u003e\u003cp\u003eX-ray diffraction (XRD) analysis was conducted to investigate the crystallinity of bezafibrate and its distribution within the calcium nanoparticles. The XRD pattern of the pure bezafibrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) showed distinct peaks at 2θ angles of 10.1\u0026deg;, 15.5\u0026deg;, 16.6\u0026deg;, 18.5\u0026deg;, 19.9\u0026deg;, 20.4\u0026deg;, 24.8\u0026deg;, 25.3\u0026deg;, and 30.4\u0026deg;, which are indicative of the crystalline nature of bezafibrate[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These findings were consistent with previous reports on bezafibrate's crystallinity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, in the XRD pattern of the bezafibrate calcium nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), the peaks corresponding to bezafibrate were completely absent, suggesting that the drug was fully entrapped within the nanoparticles. The absence of the characteristic crystalline peaks indicates that the drug had been converted into an amorphous state within the nanoparticle matrix. This shift to an amorphous form is beneficial for enhancing the solubility and bioavailability of bezafibrate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Differential Scanning Calorimetry (DSC)\u003c/h2\u003e\u003cp\u003eDifferential Scanning Calorimetry (DSC) was performed to further evaluate the thermal properties and confirm the amorphous state of bezafibrate within the calcium nanoparticles. The DSC thermogram of pure bezafibrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) exhibited a sharp endothermic peak at 188.57\u0026deg;C, corresponding to the melting point of the crystalline drug. This peak is characteristic of the crystalline nature of the drug, and the melting point was observed between 181\u0026deg;C and 185\u0026deg;C, consistent with previous findings [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, the DSC thermogram of the bezafibrate calcium nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) displayed an endothermic peak at 100.97\u0026deg;C, which is significantly lower than the melting point of pure bezafibrate. The lack of any sharp peak near the melting point of bezafibrate indicates that the drug had been completely entrapped and no longer existed in its crystalline form. The shift in the melting point and the absence of characteristic peaks further confirm the transformation of the drug into an amorphous state within the nanoparticle formulation. This amorphous form is expected to enhance the dissolution rate and improve the therapeutic efficacy of the drug.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e3.9. \u003cem\u003eIn-vitro cumulative\u003c/em\u003e Drug Release of Bezafibrate-Calcium Nanoparticles\u003c/h2\u003e\u003cp\u003eThe in vitro cumulative drug release of the optimized Bezafibrate-Calcium Nanoparticles (BZ-CNPs) was evaluated and compared to that of a bezafibrate solution. Over 28 hours, approximately 86.42% of the drug was released from the optimized BZ-CNPs formulation, demonstrating a controlled and sustained release profile as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB. In contrast, the bezafibrate solution exhibited a rapid burst release, with 82.68% of the drug being released within the first 5 hours. This significant difference in release patterns indicates that the nanoparticle formulation can provide prolonged drug release, which is essential for maintaining therapeutic concentrations over an extended period.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003e3.9.1 Release Kinetics of BZ-CNPs\u003c/h2\u003e\u003cp\u003eThe in vitro drug release data of BZ-CNPs were analyzed using various kinetic models, including Zero-order, First-order, Higuchi, and Korsmeyer\u0026ndash;Peppas, to determine the release mechanism. The Korsmeyer\u0026ndash;Peppas model showed the best fit (R\u0026sup2; = 0.9771), indicating its suitability in describing the release pattern. The release exponent (n\u0026thinsp;=\u0026thinsp;0.6139) suggested a non-Fickian (anomalous) diffusion mechanism, implying a combination of drug diffusion and matrix erosion. This sustained release behavior supports the potential of BZ-CNPs for prolonged therapeutic action in osteoporosis management[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Stability Studies\u003c/h2\u003e\u003cp\u003eThe stability of bezafibrate calcium nanoparticles (BZ-CNPs) was assessed under different storage conditions, and the results indicated that the nanoparticles maintained their stability over three months. As shown in Table VI, the nanoparticles demonstrated minimal changes in particle size, PDI, and zeta potential at various temperatures (4˚C, 25˚C, and 40˚C). The particle size increased slightly at higher temperatures, with the most significant change observed at 40˚C, where the size reached 302.6 nm, compared to the initial size of 242.1 nm at 4˚C.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe PDI also showed a slight increase at elevated temperatures, indicating a minor enhancement in particle distribution, although the changes were not significant enough to impact the formulation's overall stability. The zeta potential remained relatively stable, with a slight decrease in values across all temperatures. Initially, the zeta potential at 4˚C was \u0026minus;\u0026thinsp;32.7 mV, decreasing to -36.1 mV at 40˚C; however, these values still suggest that the nanoparticles maintained sufficient stability due to the repulsive forces between particles, which prevent aggregation.\u003c/p\u003e\u003cp\u003eRegarding the entrapment efficiency, there was only a slight reduction across all storage conditions, with the highest retention observed at 4˚C. The entrapment efficiency was 87.2% at the beginning and dropped minimally to 85.9% at 4˚C, indicating that the nanoparticles maintained a high degree of drug retention. In contrast, at higher temperatures (25˚C and 40˚C), the entrapment efficiency decreased more noticeably, but it remained within an acceptable range (80.6% at 40˚C).\u003c/p\u003e\u003cp\u003eThese findings suggest that the BZ-CNPs exhibit good stability at lower temperatures, with only slight changes observed in critical parameters like particle size, zeta potential, and entrapment efficiency. The results support the potential for long-term storage and application of BZ-CNPs, particularly when stored at 4˚C, where stability was most effectively preserved.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable VI\u003c/b\u003e Stability studies of nanoparticles under various storage conditions over time.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabf\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eStorage Conditions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInitial Value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e\u003cp\u003eAfter Three Months\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;2˚C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;2˚C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;2˚C\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticle size (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e242.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e254.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e287.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e302.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.302\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.387\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.449\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.486\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZeta Potential (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e-36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-38.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e-39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%Entrapment Efficiency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e87.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e85.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e82.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e80.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section2\"\u003e\u003ch2\u003e3.11. In vivo Studies\u003c/h2\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e\u003ch2\u003e3.11.1. Effect of Formulation on Body Weight of Rats\u003c/h2\u003e\u003cp\u003eThe body weight changes of rats over three weeks were monitored to evaluate the impact of the inducer and different treatments. Significant weight loss was observed in the inducer group (Group II), with a marked decrease in body weight from Week 1 to Week 3. The body weight of animals in Group II decreased from 302.14 g to 261.42 g, indicating that the dexamethasone-induced osteoporosis protocol caused substantial weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eIn comparison, Group I (normal control) exhibited gradual weight gain throughout the study period, with body weight increasing from 254.28 g to 267.85 g. Groups III (Zoledronic acid treatment) and IV (bezafibrate solution treatment) also displayed minor reductions in body weight; however, these changes were not statistically significant. Group III\u0026rsquo;s body weight remained relatively stable, ranging from 259.28 g to 260.00 g, while Group IV\u0026rsquo;s body weight experienced slight fluctuations, decreasing from 268.57 g to 260.00 g.\u003c/p\u003e\u003cp\u003eGroups V and VI, treated with bezafibrate-loaded calcium nanoparticles (BZ-CNPs) of different particle sizes, showed no significant changes in body weight, suggesting that the formulation did not have a detrimental impact on the animals\u0026rsquo; overall health. Specifically, Group V maintained consistent body weight, starting at 267.85 g and ending at 266.42 g, while Group VI showed a slight decline (257.85 g to 248.57 g), but this reduction was not significant when compared to the control group. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eOverall, these findings suggest that while the inducer and the bezafibrate solution formulation (Group IV) led to a noticeable reduction in body weight, the bezafibrate-loaded calcium nanoparticles in Groups V and VI did not significantly affect body weight, indicating their safety in terms of systemic weight changes (Table VII).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable VII\u003c/b\u003e Changes in body weight of experimental subjects throughout the in vivo study.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabg\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeek 1 (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWeek 2 (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWeek 3 (g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e254.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e258.57\u0026thinsp;\u0026plusmn;\u0026thinsp;57.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e267.85\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInducer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e302.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e285.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e261.42\u0026thinsp;\u0026plusmn;\u0026thinsp;12.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStandard\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e259.28\u0026thinsp;\u0026plusmn;\u0026thinsp;6.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e265.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e260.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e268.57\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e272.14\u0026thinsp;\u0026plusmn;\u0026thinsp;4.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e260.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e267.85\u0026thinsp;\u0026plusmn;\u0026thinsp;4.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e266.42\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e266.42\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e257.85\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e260.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e248.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section2\"\u003e\u003ch2\u003e3.11.2. Bone Mineral Density (BMD)\u003c/h2\u003e\u003cp\u003eGroup V, which was treated with bezafibrate-loaded calcium nanoparticles (BZ-CNPs), showed a BMD of 3.838\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 g/cm\u0026sup3;, which was comparable to the normal control group (Group I, 3.968\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 g/cm\u0026sup3;). This suggests that BZ-CNPs effectively improved bone density in osteoporotic rats. This was further supported by the data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, which visually demonstrated the positive impact of BZ-CNPs on bone health.\u003c/p\u003e\u003cp\u003eIn contrast, the inducer group (Group II) exhibited the lowest BMD of 2.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016 g/cm\u0026sup3;, indicating significant bone loss induced by dexamethasone. This result (Table VII) underscores the detrimental effects of osteoporosis and highlights the efficacy of BZ-CNPs in counteracting these effects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther treatment groups also showed improvements in BMD, with Group III (Zoledronic acid treatment) and Group VI (bezafibrate-loaded calcium nanoparticles with different formulations) exhibiting BMD values of 3.602\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 g/cm\u0026sup3; and 3.642\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013 g/cm\u0026sup3;, respectively. While these values were lower than those observed in Group I and Group V, they still indicated a therapeutic effect compared to the inducer group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec43\" class=\"Section2\"\u003e\u003ch2\u003e3.11.3. Bone Mineral Content Markers\u003c/h2\u003e\u003cp\u003eSerum calcium (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB) and phosphorus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC) levels are important indicators of bone mineral content. The control group had significantly higher serum calcium levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while in the Inducer group, the calcium levels were notably lower (7.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 mg/dL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Control), suggesting a condition like osteoporosis. The Standard treatment group showed moderate recovery (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer), and the Drug solution group also displayed some improvement. However, in the NP-1 and NP-2 treatment groups, calcium levels showed significant improvement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer), with values approaching those of the Control group (NP-1: 11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39, NP-2: 11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mg/dL), indicating that nanoparticle-based treatments effectively restored calcium homeostasis.\u003c/p\u003e\u003cp\u003eSimilarly, phosphorus levels in the Inducer group were significantly low (2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/dL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Significant recovery in phosphorus levels was observed in the NP-1 (3.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) and NP-2 (3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer), while no significant difference was seen in the Drug Solution and Standard groups when compared to the Inducer group [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section2\"\u003e\u003ch2\u003e3.11.4. Bone Resorption Biomarker\u003c/h2\u003e\u003cp\u003eSerum alkaline phosphatase (ALP), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD is a key biomarker of bone resorption. In the control group, the ALP level was 292.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 U/L, while in the Inducer group, the value was significantly elevated (725.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 U/L; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating severe bone resorption. Both the Standard treatment group (407.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 U/L) and the Drug solution group (567.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 U/L) showed a reduction in ALP levels, although these values remained considerably higher compared to the Control group [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The NP-1 group (291.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 U/L) significantly reduced ALP levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer; ns vs Control), while the NP-2 group (378.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 U/L) also demonstrated a statistically significant improvement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer). These results suggest that the anti-resorptive effect of NP-1 was slightly more effective than that of NP-2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section2\"\u003e\u003ch2\u003e3.11.5. Bone Formation Biomarker\u003c/h2\u003e\u003cp\u003eOsteocalcin, a marker of bone formation, is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE. In the control group, the osteocalcin level was 17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ng/mL, while in the Inducer group, it drastically decreased to 1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ng/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The Standard treatment group showed significant improvement (14.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ng/mL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Ind), whereas the Drug solution group (7.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ng/mL) exhibited less pronounced improvement. Both NP-1 (17.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ng/mL) and NP-2 (17.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ng/mL) groups restored osteocalcin levels to those similar to the Control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs Inducer; ns vs Control), indicating that both nanoparticle formulations substantially supported bone formation (Table VIII).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable VIII\u003c/b\u003e Biochemical parameters measured across experimental groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabh\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCalcium Level (mg/dL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhosphorous Level (mg/dL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAlkaline Phosphatase\u003c/p\u003e\u003cp\u003eLevel (mg/dL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOsteocalcin Level (mg/dL)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup I\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e292.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup II\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e725.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup III\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e407.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e14.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e567.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e7.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup V\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e291.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e17.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup VI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e378.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e17.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec46\" class=\"Section2\"\u003e\u003ch2\u003e3.11.6. Assessment of Oxidative Stress Biomarkers in Osteoporosis\u003c/h2\u003e\u003cp\u003eThe assessment of oxidative stress biomarkers in osteoporosis revealed notable variations (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) across the experimental groups. Group II exhibited the highest protein content (0.591\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 mg/mL), suggesting a greater concentration of proteins compared to other groups. However, this group also showed elevated levels of malondialdehyde (MDA) (0.460\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 nM/mg of protein content), indicating increased oxidative stress and lipid peroxidation. In contrast, Group I, which served as the control, had lower MDA levels (0.163\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016 nM/mg of protein content), suggesting minimal oxidative damage. Catalase [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] activity, which plays a crucial role in mitigating oxidative stress, was highest in Group I (0.204\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 nM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min/mg of protein content), reflecting a more efficient antioxidant defense. Group II, however, demonstrated significantly lower CAT activity (0.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 nM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min/mg of protein content), indicating a compromised ability to neutralize oxidative damage.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Superoxide dismutase (SOD) activity, another important antioxidant enzyme, was highest in Group III (4.045\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 U/mg of protein content), suggesting a strong defense against oxidative stress in this group. On the other hand, Group IV showed a reduced SOD activity (3.811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052 U/mg of protein content), which may indicate a less effective antioxidant response [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Overall, the findings highlight (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) the intricate relationship between oxidative stress and antioxidant defense in osteoporosis, suggesting that antioxidant enzyme activities such as CAT and SOD are crucial in modulating the extent of oxidative damage. These results also point to the potential benefits of antioxidant-based therapies to counteract the oxidative stress associated with osteoporosis. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec47\" class=\"Section2\"\u003e\u003ch2\u003e3.11.7. Histopathological Analysis\u003c/h2\u003e\u003cp\u003eThe histopathological examination of femur bones in rats revealed distinct variations among the experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). In the control group, the bone structure was normal, with a cortical bone thickness of 150 \u0026micro;m, well-preserved periosteum and endosteum, and healthy osteocyte lacunae containing nuclei. On the other hand, the osteoporosis-induced group showed a significant reduction in cortical bone thickness (119 \u0026micro;m), along with irregularities in the periosteum and endosteum, and a reduced number of osteocytes, many of which lacked nuclei. The Haversian canal in this group appeared enlarged and irregular [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The standard group displayed a cortical bone thickness of 132 \u0026micro;m, with slight improvements in bone architecture and a smaller Haversian canal compared to the osteoporosis-induced group. The group treated with smaller-sized BZ-CNPs (Group IV) exhibited a cortical bone thickness of 130 \u0026micro;m, slight irregularities in the periosteum and endosteum, and several lacunae devoid of osteocyte nuclei [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In contrast, the group treated with smaller-sized BZ-CNPs (Group V) showed a normal bone structure, with a cortical bone thickness of 156 \u0026micro;m and numerous osteocytes with well-defined nuclei, suggesting active bone regeneration [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Lastly, the group treated with larger-sized BZ-CNPs (Group VI) demonstrated moderate improvement, with a cortical bone thickness of 148 \u0026micro;m and a regular periosteum and endosteum, although the osteocyte count was lower than in Group V[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, bezafibrate-loaded calcium nanoparticles were created utilizing the co-precipitation approach to treat osteoporosis. It was found that bezafibrate was a white, crystalline, odorless powder with melting points of 182\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56˚C and 183.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34˚C. 99.92% pure. Functional groups were determined using Fourier Transform Infrared Spectroscopy (FTIR), showing characteristic peaks at reported frequencies. Compatibility studies confirmed no interaction between the drug and excipients. Box Behnken Design was used for optimization, and fifteen batches were developed. One optimized batch was selected based on desired responses, and further optimized batches were evaluated for various parameters. The optimized bezafibrate calcium nanoparticles showed a particle size of 242.1 nm, PDI of 0.302, -32.7 mV of zeta potential, and 87.2% drug entrapment efficiency with 82.7% drug release in 29 hours. Effects of temperature and heat on the lattice structure of the crystal structure were verified by DSC and XRD. The drug release kinetics were plotted, and the calcium nanoparticles followed the Korsmeyer Peppas model, indicating non-Fickian diffusion. Stability studies demonstrated that the calcium nanoparticles were more stable at 4˚C storage conditions. Biochemical tests showed the successful creation of calcium in bones and elevated levels of osteocalcin, indicating bone formation. Serum ALP levels were higher in the inducer groups compared to the control and test groups. Oxidative stress biomarkers show the effectiveness of BZ-CNP for anti-osteoporotic therapy. The current study's findings indicate that these generated BZ-CNPs effectively promote bone creation in the rat model of osteoporotic DEX-induced osteoporosis. Additionally, BZ-CNPs, a novel pharmaceutical treatment that has demonstrated exceptional therapeutic benefits for the effective management of osteoporosis with excellent selectivity for bone tissues, are anticipated to help cure senile osteoporosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cb\u003eEthics approval and consent to participate\u003c/b\u003e:\u003c/strong\u003e\u003cp\u003e All the procedures related to the animal study were approved by the Institutional Animal Ethics Committee (IAEC) with approval number BBDNIIT/IAEC/MAY/2024/07\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cb\u003eConsent for publication\u003c/b\u003e:\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003e\u003cb\u003eCompeting interests\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003e\u003cb\u003eConsent to Participate\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research did not receive a specific grant from funding agencies in the public, commercial, or not-for-profit sector\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: S KS, N D, S Y; Data curation: N D; Formal analysis: S KS, Alka; Investigation: S Y, Alka; Methodology: SY, ND; Roles/Writing - original draft: SY, Alka; and Writing \u0026ndash; review and editing: N D, SKS\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgments: The authors want to acknowledge the USIC facility at BBAU, CSIR-IITR\u003c/p\u003e\u003ch2\u003eAvailability of data and material:\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrince, R. L., et al. (2019). Adding Lateral Spine Imaging for Vertebral Fractures to Densitometric Screening: Improving Ascertainment of Patients at High Risk of Incident Osteoporotic Fractures. \u003cem\u003eJournal Of Bone And Mineral Research\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(2), 282\u0026ndash;289.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGera, S., \u0026amp; Sampathi, S. (2022). \u003cem\u003eTherapeutic Potential of Naringenin Nanosuspension: In Vitro and In Vivo Anti-Osteoporotic Studies.\u003c/em\u003e Pharmaceutics, 14: p. 1449.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarcucci, G. (2023). \u003cem\u003eOxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches\u003c/em\u003e. \u003cem\u003eAntioxidants (Basel)\u003c/em\u003e, 12(2).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlorencio-Silva, R. (2015). \u003cem\u003eBiology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells.\u003c/em\u003e Biomed Res Int, 2015: p. 421746.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClarke, B. (2008). Normal bone anatomy and physiology. \u003cem\u003eClinical Journal Of The American Society Of Nephrology\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(Suppl 3), S131\u0026ndash;S139.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJi, W., Chen, M., \u0026amp; Ji, H. (2022). \u003cem\u003eThe calcium supplementation effect of calcium-binding oligopeptides from bonito (Auxis thazard) hydrolysate in rats\u003c/em\u003e (p. 42). Food Science and Technology.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, J. M. (2020). \u003cem\u003eOsteoblast-Osteoclast Communication and Bone Homeostasis\u003c/em\u003e. \u003cem\u003eCells\u003c/em\u003e, 9(9).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTresguerres, F. G. F. \u003cem\u003eThe osteocyte: A multifunctional cell within the bone.\u003c/em\u003e 2024(1618\u0026thinsp;\u0026ndash;\u0026thinsp;0402 (Electronic)).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePonzetti, M., \u0026amp; Rucci, N. (2021). \u003cem\u003eOsteoblast Differentiation and Signaling: Established Concepts and Emerging Topics\u003c/em\u003e. \u003cem\u003eInternational Journal Of Molecular Sciences\u003c/em\u003e, 22(13).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark, E. (2020). \u003cem\u003eOsteoprotective Effects of Loganic Acid on Osteoblastic and Osteoclastic Cells and Osteoporosis-Induced Mice\u003c/em\u003e. \u003cem\u003eInternational Journal Of Molecular Sciences\u003c/em\u003e, 22(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBonewald, L. F. (2021). Chap. 7 \u003cem\u003e- Osteocytes\u003c/em\u003e. In D. W. Dempster, et al. (Eds.), \u003cem\u003eMarcus and Feldman's Osteoporosis (Fifth Edition)\u003c/em\u003e (pp. 135\u0026ndash;163). Academic.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDatt, N., Poonuru, R. R., \u0026amp; Yadav, P. K. (2022). Development and characterization of griseofulvin loaded nanostructured lipid carrier gel for treating dermatophytosis. \u003cem\u003eFood Hydrocolloids for Health\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 100074.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, M., et al. (2025). Colloidal Copper Nanoparticles Loaded with Vanillic Acid as Liquid Dressings: Development, Characterisation and Evaluation. \u003cem\u003ePharmaceutical Innovation\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(2), 40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede B\u0026ocirc;lla, L., et al. (2024). Calcium oxide nanoparticles: Biosynthesis, characterization and photocatalytic activity for application in yellow tartrazine dye removal. \u003cem\u003eJournal of Photochemistry and Photobiology A: Chemistry\u003c/em\u003e, \u003cem\u003e447\u003c/em\u003e, 115182.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRani, M., et al. (2024). Biowaste-derived nanocomposite of calcium oxide incorporated in nickel oxide for efficient removal of organic pollutants. \u003cem\u003eBiomass Conversion and Biorefinery\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(4), 6495\u0026ndash;6510.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNuruzzaman, M., et al. (2025). Effect of Heat Treatment on Polymorphism and Particle Size Distribution of Calcium Carbonate Nanoparticle Synthesized via Mechanochemical Process. \u003cem\u003eApplied Nano\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(2), 8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElmsmari, F., et al. (2021). Calcium hydroxide-loaded PLGA biodegradable nanoparticles as an intracanal medicament. \u003cem\u003eInternational Endodontic Journal\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e(11), 2086\u0026ndash;2098.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMada, S. B., et al. (2020). Milk-derived bioactive peptides with antiosteoporotic effect: A mini review. \u003cem\u003eFudma Journal of Sciences\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(3), 351\u0026ndash;357.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePizzino, G., et al. (2017). Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model. \u003cem\u003eFrontiers In Pharmacology\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 558.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLucinda, L. M. F., et al. (2017). Evaluation of the anti-osteoporotic effect of Ginkgo biloba L. in Wistar rats with glucocorticoid-induced-osteoporosis by bone densitometry using dual-energy x-ray absorptiometry (DEXA) and mechanical testing. \u003cem\u003eAnais Da Academia Brasileira De Ci\u0026ecirc;ncias\u003c/em\u003e, \u003cem\u003e89\u003c/em\u003e(4), 2833\u0026ndash;2841.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHonary, S., \u0026amp; Zahir, F. (2013). \u003cem\u003eEffect of Zeta Potential on the Properties of Nano-Drug Delivery Systems - A Review (Part 1)\u003c/em\u003e. \u003cem\u003eTropical Journal of Pharmaceutical Research\u003c/em\u003e, 12(2).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMuhammad, S. I. (2013). \u003cem\u003eBone mass density estimation: Archimede\u0026rsquo;s principle versus automatic X-ray histogram and edge detection technique in ovariectomized rats treated with germinated brown rice bioactives\u003c/em\u003e. \u003cem\u003eClinical Interventions in Aging\u003c/em\u003e, : pp. 1421\u0026ndash;1431.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBanji, D., et al. (2014). Role of Triticum aestivum aqueous extract in glucocorticoid induced osteoporosis in rats. \u003cem\u003eIndian Journal Of Experimental Biology\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(2), 153\u0026ndash;158.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIantomasi, T., et al. (2023). Oxidative Stress and Inflammation in Osteoporosis: Molecular Mechanisms Involved and the Relationship with microRNAs. \u003cem\u003eInternational Journal Of Molecular Sciences\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(4), 3772.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLemmerer, A., et al. (2009). Concomitant polymorphs of the antihyperlipoproteinemic bezafibrate. \u003cem\u003eCrystal Growth\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(6), 2646\u0026ndash;2655.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang, S., et al. (2019). Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris. \u003cem\u003eJournal of Central South University\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(1), 184\u0026ndash;195.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrajapati, P., et al. (2023). Azilsartan Ameliorates Skeletal Muscle Wasting in High Fat Diet (HFD)-induced Sarcopenic Obesity in Rats via Activating Akt Signalling Pathway. \u003cem\u003eArchives Of Gerontology And Geriatrics\u003c/em\u003e, \u003cem\u003e112\u003c/em\u003e, 105025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhine, E. E., et al. (2022). Synthesis and characterization of calcium oxide nanoparticles for CO2 capture. \u003cem\u003eJournal of Nanoparticle Research\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(7), 139.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRakić, T., et al. (2014). Comparison of full factorial design, central composite design, and box-behnken design in chromatographic method development for the determination of fluconazole and its impurities. \u003cem\u003eAnalytical Letters\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(8), 1334\u0026ndash;1347.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDashnyam, K., et al. (2020). Nanoscale Calcium Salt-Based Formulations As Potential Therapeutics for Osteoporosis. \u003cem\u003eACS Biomater Sci Eng\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(8), 4604\u0026ndash;4613.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaleki Dizaj, S., et al. (2016). Application of Box\u0026ndash;Behnken design to prepare gentamicin-loaded calcium carbonate nanoparticles. \u003cem\u003eArtificial cells nanomedicine biotechnology\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e(6), 1475\u0026ndash;1481.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, S., et al. (2021). Experimental investigation of microalgal harvesting with low cost bottom ash: Influence of temperature and pH with zeta potential and thermodynamic function. \u003cem\u003eEnvironmental Technology Innovation\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e, 101376.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCacua, K., et al. (2019). Surfactant concentration and pH effects on the zeta potential values of alumina nanofluids to inspect stability. \u003cem\u003eColloids Surfaces A: Physicochemical Engineering Aspects\u003c/em\u003e, \u003cem\u003e583\u003c/em\u003e, 123960.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbeywardena, M. R., et al. (2020). Surfactant assisted synthesis of precipitated calcium carbonate nanoparticles using dolomite: Effect of pH on morphology and particle size. \u003cem\u003eAdvanced Powder Technology\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(1), 269\u0026ndash;278.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEmami, J., Yousefian, H., \u0026amp; Sadeghi, H. (2018). Targeted Nanostructured Lipid Carrier for Brain Delivery of Artemisinin: Design, Preparation, Characterization, Optimization and Cell Toxicity. \u003cem\u003eJournal Of Pharmacy \u0026amp; Pharmaceutical Sciences : A Publication Of The Canadian Society For Pharmaceutical Sciences, Soci\u0026eacute;t\u0026eacute; Canadienne Des Sciences Pharmaceutiques\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(1s), 225s\u0026ndash;241s.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, R., et al. (2020). Electrosprayed Polymeric Nanospheres for Enhanced Solubility, Dissolution Rate, Oral Bioavailability and Antihyperlipidemic Activity of Bezafibrate. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 705\u0026ndash;715.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHabte, L. (2019). \u003cem\u003eSynthesis of Nano-Calcium Oxide from Waste Eggshell by Sol-Gel Method\u003c/em\u003e. \u003cem\u003eSustainability\u003c/em\u003e, 11(11).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKamaraj, S., et al. (2018). Curcumin drug delivery by vanillin-chitosan coated with calcium ferrite hybrid nanoparticles as carrier. \u003cem\u003eEuropean journal of pharmaceutical sciences\u003c/em\u003e, \u003cem\u003e116\u003c/em\u003e, 48\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, R., et al. (2020). The effects of additives on the porosity and stability of amorphous calcium carbonate. \u003cem\u003eMicroporous mesoporous materials\u003c/em\u003e, \u003cem\u003e292\u003c/em\u003e, 109736.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAfzal, A., et al. (2020). Molecular dynamics in the supercooled liquid and glassy states of bezafibrate and binary mixture of fenofibrate. \u003cem\u003eof Non-Crystalline Solids\u003c/em\u003e, \u003cem\u003e550\u003c/em\u003e, 120407.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMuhammad Mailafiya, M., et al. (2019). Cockle Shell-Derived Calcium Carbonate (Aragonite) Nanoparticles: A Dynamite to Nanomedicine. \u003cem\u003eApplied Sciences\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(14), 1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang, S., et al. (2009). Effects of nano calcium carbonate and nano calcium citrate on toxicity in ICR mice and onbone mineral density in an ovariectomized mice model. \u003cem\u003eNanotechnology\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(37), 375102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen, X., et al. (2024). Associations of global biomarkers of oxidative stress with osteoporosis, bone microstructure and bone turnover: Evidence from human and animal studies. \u003cem\u003eBone\u003c/em\u003e, \u003cem\u003e183\u003c/em\u003e, 117077.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahmani, D., et al. (2023). The beneficial effects of simultaneous supplementation of Lactobacillus reuteri and calcium fluoride nanoparticles on ovariectomy-induced osteoporosis. \u003cem\u003eBMC Complement Med Ther\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1), 340.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhajuria, D. K., Razdan, R., \u0026amp; Mahapatra, D. R. (2015). Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model. \u003cem\u003eEuropean Journal Of Pharmaceutical Sciences\u003c/em\u003e, \u003cem\u003e66\u003c/em\u003e, 173\u0026ndash;183.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuo, Z., et al. (2023). Investigation of the treatment potential of Raloxifene-loaded polymeric nanoparticles in osteoporosis: In-vitro and in-vivo analyses. \u003cem\u003eHeliyon\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(9), e20107.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElghareeb, M. M., et al. (2022). Estradiol and zinc-doped nano hydroxyapatite as therapeutic agents in the prevention of osteoporosis; oxidative stress status, inflammation, bone turnover, bone mineral density, and histological alterations in ovariectomized rats. \u003cem\u003eFrontiers in Physiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 989487.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bezafibrate, calcium nanoparticles, osteoporosis, repurposing, Box-Behnken design","lastPublishedDoi":"10.21203/rs.3.rs-7197944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7197944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoporosis is a progressive skeletal disorder characterized by an imbalance between bone resorption and bone formation, leading to reduced bone mineral density and increased fracture risk. Bezafibrate (BZ), a lipid-lowering fibrate, has recently gained attention as a promising repurposed therapeutic agent for osteoporosis due to its potential bone-protective effects. This study aimed to develop and evaluate a novel bezafibrate-loaded calcium nanoparticle (BZ-CNP) system to enhance its therapeutic efficacy against osteoporosis and assess its in vivo performance in an osteoporotic animal model. Calcium nanoparticles (CNPs), known for their biocompatibility and inherent bone-targeting capabilities, were formulated using the chemical precipitation method for efficient drug delivery to bone tissue. BZ-CNPs were optimized using Box-Behnken Design (BBD), and their physicochemical properties were thoroughly characterized. The therapeutic potential of the optimized formulation was evaluated in a dexamethasone-induced osteoporotic rat model. The optimized BZ-CNPs exhibited a particle size of 242.1 nm, a polydispersity index (PDI) of 0.302, and a zeta potential of −32.7 mV, indicating stable nanoscale dispersion. The entrapment efficiency was 87.2%, demonstrating efficient drug loading. In vivo and biochemical parameters results revealed a significant improvement in bone turnover markers, confirming the formulation's efficacy in reversing osteoporosis-induced bone loss. The developed Bezafibrate-loaded calcium nanoparticles represent a promising nanocarrier system for targeted delivery, offering enhanced therapeutic outcomes in osteoporosis management.\u003c/p\u003e","manuscriptTitle":"Nanoengineered Bezafibrate-Loaded Calcium Nanoparticles for Osteoporosis: A Repurposing Approach for Targeted Bone Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-07 09:33:38","doi":"10.21203/rs.3.rs-7197944/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T11:37:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T10:10:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213635727715635203347424310881076544966","date":"2025-08-29T00:51:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T07:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51137763729036534909959710331831569332","date":"2025-08-09T06:54:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124037070385873857487288516029432542558","date":"2025-08-06T15:12:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T06:45:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-04T06:39:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T13:57:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-07-23T15:13:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c4dbd79a-1c04-4b3a-969e-ad6c68052a36","owner":[],"postedDate":"August 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:04:13+00:00","versionOfRecord":{"articleIdentity":"rs-7197944","link":"https://doi.org/10.1007/s12668-025-02305-7","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-12-17 15:57:36","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-08-07 09:33:38","video":"","vorDoi":"10.1007/s12668-025-02305-7","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02305-7","workflowStages":[]},"version":"v1","identity":"rs-7197944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7197944","identity":"rs-7197944","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}