Enhanced oral bioavailability of baicalein using phospholipid-coated nanoparticles: A novel drug delivery system

 Corresponding author: Saeem Ahmad Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Enhanced oral bioavailability of baicalein using phospholipid-coated nanoparticles: A novel drug delivery system Saeem Ahmad *, Manish Kumar Sahu, Dev Sharan Chaturvedi and Jeetendra Kushwaha Shanti College of Pharmacy Nowgong (M.P.) World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 Publication history: Received on 16 August 2025; revised on 22 September 2025; accepted on 24 September 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.23.3.0842 Abstract Phospholipid-coated nanoparticle formulation of Baicalein demonstrated superior pharmacokinetic properties, including slower absorption, prolonged circulation time, and increased bioavailability compared to free Baicalein. This formulation holds significant promise for enhancing the oral bioavailability and therapeutic efficacy of Baicalein, particularly for chronic diseases where sustained drug release is beneficial. Further research should focus on the longterm safety, efficacy, and clinical applicability of this nanoparticle formulation, including evaluations in animal models and eventual clinical trials. Additionally, exploring other lipid-based nanoparticle formulations could further improve the bioavailability and therapeutic outcomes of Baicalein and similar drugs with poor solubility. Characterized phospholipid-coated nanoparticles for the delivery of Baicalein, a flavonoid with limited bioavailability. The formulation process, utilizing the emulsification technique, allowed the formation of stable nanoparticles with optimal encapsulation efficiency (88%) and drug loading capacity (6.5%). The optimization of lecithin concentration (3%) resulted in the highest encapsulation and drug loading, demonstrating the significant role of lipid concentration in the formulation of effective drug delivery systems. Keywords: Baicalein; Phospholipid-Coated Nanoparticle; Bioavailability; Lipid 1. Introduction Nanoparticles are ultra-small particles that range in size from 1 to 100 nanometers (nm). To put this into perspective, a nanometer is one-billionth of a meter, much smaller than the wavelength of visible light and even smaller than many biological molecules like proteins. Nanoparticles possess unique physical, chemical, and biological properties due to their high surface area-to-volume ratio and quantum effects. Unlike their bulk material counterparts, the behavior of nanoparticles is significantly different; for instance, gold nanoparticles can appear red or purple due to their sizedependent optical properties, whereas bulk gold is always metallic yellow. The size range of nanoparticles, typically less than 100 nm, allows them to exhibit distinct characteristics that are advantageous for a variety of applications in medicine, electronics, and materials science (Li and Tan, 2018). 1.1. Types of Nanoparticles Nanoparticles are classified into different categories based on their composition, structure, and properties. The primary types include metal nanoparticles, metal oxide nanoparticles, carbon-based nanoparticles, polymer-based nanoparticles, and quantum dots. Each type has unique characteristics and applications that make them integral to various fields, such as medicine, electronics, environmental science, and material engineering. This section provides a comprehensive overview of these types, detailing their synthesis, properties, and uses, supported by extensive research references. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 476 1.2. Metal Nanoparticles 1.2.1. Gold Nanoparticles (AuNPs) Gold nanoparticles (AuNPs) are among the most extensively studied due to their unique optical, electronic, and thermal properties. Their size typically ranges from 1 nm to 100 nm. AuNPs can be synthesized via chemical reduction, where gold salts (e.g., HAuCl₄) are reduced using agents like citrate, resulting in the formation of nanoparticles with a controllable size and shape. The optical properties of AuNPs are defined by the phenomenon known as surface plasmon resonance (SPR), where free electrons resonate with incident light, giving them distinctive colors (e.g., red in solution). 1.2.2. Applications AuNPs are utilized in drug delivery, cancer therapy, biosensors, and as contrast agents in imaging. For example, in cancer treatment, AuNPs can be conjugated with targeting ligands to deliver drugs directly to cancer cells, minimizing damage to healthy cells (El-Sayed et al., 2020). 1.2.3. Silver Nanoparticles (AgNPs) Silver nanoparticles (AgNPs) are widely used due to their potent antimicrobial properties. They are synthesized using chemical, physical, and biological methods, with green synthesis gaining popularity for its eco-friendly approach. AgNPs exhibit strong antibacterial activity against a range of pathogens, including antibiotic-resistant strains. 1.2.4. Applications AgNPs are used in medical devices, wound dressings, water purification, and consumer products like textiles and cosmetics due to their ability to inhibit microbial growth (Rai et al., 2019). 1.2.5. Iron Nanoparticles (FeNPs) Iron nanoparticles (FeNPs) are notable for their magnetic properties, making them valuable in magnetic resonance imaging (MRI), targeted drug delivery, and environmental remediation. Zero-valent iron nanoparticles (nZVI) are effective in degrading pollutants due to their high reactivity. 1.2.6. Applications FeNPs are used in catalysis, wastewater treatment, and as MRI contrast agents. In cancer therapy, they are used for hyperthermia treatment, where they generate heat to kill cancer cells when exposed to an alternating magnetic field (Sun and Zhang, 2017). 2. Materials and Methods The methodology for the proposed research aimed at enhancing the oral bioavailability of Baicalein through a phospholipid-coated nanoparticle system was structured into multiple stages, each designed to address a specific aspect of formulation, characterization, and evaluation. The following sections describe the work that was conducted: 2.1. Formulation Development Phospholipid-coated nanoparticles were formulated using an emulsification method, where various concentrations and types of phospholipids (lecithin) were tested to optimize the formulation for drug loading capacity and stability. Lecithin was chosen for its ability to form stable nanoparticle coatings. The lecithin concentration in the organic solvent phase was varied from 2% to 4% (w/v). For Baicalein, the concentration in the formulation was set at 1% (w/v). Baicalein was dissolved in an appropriate solvent, and the lecithin was added to this solution under controlled conditions to create a nanoparticle suspension. The nanoparticles were formed using the solvent evaporation method. After preparation, the entrapment efficiency of Baicalein in the nanoparticles was evaluated by measuring the amount of free drug in the supernatant after centrifugation at 15,000 rpm for 30 minutes. The unentrapped Baicalein was separated and quantified using high-performance liquid chromatography (HPLC). The final formulation was optimized based on the drug loading capacity and the stability of the nanoparticle system. (Patil et al., 2016) World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 477 2.2. Characterization of Nanoparticles 2.2.1. Dynamic Light Scattering (DLS) Dynamic Light Scattering (DLS) was employed to determine the size and polydispersity index (PDI) of the nanoparticles. DLS works by analyzing the fluctuations in light scattering due to the Brownian motion of particles in suspension. The nanoparticle size is determined by measuring the rate at which the particles diffuse in the medium, and from this, the hydrodynamic diameter is calculated. The PDI provides information about the size distribution of the nanoparticles, where a PDI value of less than 0.3 indicates a narrow size distribution and high uniformity of the particles. The DLS measurements were performed at a scattering angle of 90° using a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were diluted to an appropriate concentration (typically 1–2 mg/mL) with distilled water before measurement. The measurements were taken in triplicate to ensure accuracy and reproducibility. The data were processed using the Zetasizer software to determine the mean particle size and PDI. (Li et al., 2019) 2.2.2. Zeta Potential Measurement The zeta potential was measured to evaluate the surface charge of the nanoparticles, which is a critical parameter for predicting the stability of nanoparticle suspensions. Zeta potential is a measure of the electrostatic repulsion between particles in a dispersion and reflects the potential energy of interaction between charged particles. Nanoparticles with a zeta potential greater than ±30 mV are considered stable because the high electrostatic repulsion between particles prevents aggregation. The zeta potential was measured using a Zetasizer Nano ZS (Malvern Instruments, UK), with the samples prepared at a concentration of 1 mg/mL in distilled water. (Patil et al., 2016) 2.2.3. Transmission Electron Microscopy (TEM) Transmission Electron Microscopy (TEM) was used to examine the morphology and shape of the phospholipid-coated nanoparticles. TEM is a high-resolution imaging technique that utilizes an electron beam to pass through thin samples, producing detailed images at the nanometer scale. For the TEM analysis, nanoparticle samples were prepared by depositing a small drop of the nanoparticle suspension onto a carbon-coated copper grid and allowing it to dry under ambient conditions. The samples were then observed using a JEOL JEM-1400 transmission electron microscope at an operating voltage of 120 kV. The TEM images allowed for direct visualization of the nanoparticles’ spherical shape, and the size distribution of the nanoparticles was confirmed by measuring the diameters of at least 100 nanoparticles from multiple TEM images. This method provided insight into the uniformity of the nanoparticles and allowed for confirmation of the size distribution observed in the DLS analysis. (Xie et al., 2018) 2.3. High-Performance Liquid Chromatography (HPLC) The encapsulation efficiency and drug loading capacity of Baicalein in the phospholipid-coated nanoparticles were determined using high-performance liquid chromatography (HPLC). The amount of unentrapped Baicalein was separated from the nanoparticles by centrifugation. The supernatant was collected, and the Baicalein content was quantified by HPLC. The HPLC system consisted of a Shimadzu LC-20AT liquid chromatograph equipped with a C18 column (250 mm × 4.6 mm, 5 μm particle size) and a UV detector set at 270 nm, which is the absorption wavelength of Baicalein. The mobile phase consisted of a mixture of methanol and water (60:40 v/v), and the flow rate was set to 1.0 mL/min. The encapsulation efficiency was calculated as the percentage of Baicalein entrapped in the nanoparticles relative to the total Baicalein added during the formulation process, while the drug loading capacity was calculated as the ratio of the amount of drug encapsulated in the nanoparticles to the total weight of the nanoparticle formulation. (Zhao et al., 2020) 3. Results 3.1. Formulation Development The formulation of phospholipid-coated nanoparticles was successfully carried out using the emulsification method. Various concentrations of lecithin (2%, 3%, and 4% w/v) were tested, and Baicalein was incorporated into the nanoparticle core at a concentration of 1% (w/v). The solvent evaporation technique allowed the nanoparticles to form with a stable lecithin coating. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 478 3.2. Characterization of Nanoparticles 3.2.1. Dynamic Light Scattering (DLS) The Dynamic Light Scattering (DLS) analysis of the phospholipid-coated nanoparticles yielded a mean hydrodynamic diameter of 215 ± 10 nm (Figure 7.1), a polydispersity index (PDI) of 0.22, and a zeta potential of +32 mV (Figure 7.2), indicating a positive surface charge. The measurements were performed at a scattering angle of 90° using a Zetasizer Nano ZS (Malvern Instruments, UK). The nanoparticles were diluted to a concentration of 1 mg/mL with distilled water, and the data were collected in triplicate to ensure precision and reproducibility. Figure 1 Particle Size Analysis of the phospholipid-coated Baicalein nanoparticles Figure 2 Zeta potential of the phospholipid-coated Baicalein nanoparticles 3.3. Transmission Electron Microscopy (TEM) The Transmission Electron Microscopy (TEM) analysis of the phospholipid-coated nanoparticles revealed well-defined, spherical-shaped nanoparticles with an average diameter of 210 ± 15 nm. The nanoparticles were uniformly dispersed, with no signs of aggregation, confirming the stability of the formulation. The size distribution was consistent with the results from the Dynamic Light Scattering (DLS) analysis, further validating the uniformity of the nanoparticles. The TEM images were obtained using a JEOL JEM-1400 transmission electron microscope at an operating voltage of 120 kV. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 479 Figure 3 TEM image of the phospholipid-coated Baicalein nanoparticles 3.4. High-Performance Liquid Chromatography (HPLC) The encapsulation efficiency of Baicalein in the phospholipid-coated nanoparticles was found to be 88%. The drug loading capacity was calculated to be 6.5%. These results were obtained by separating the unentrapped Baicalein through centrifugation and quantifying the drug content using high-performance liquid chromatography (HPLC). Table 1 Table for the optimization results Lecithin Concentration (%) Encapsulation Efficiency (%) Drug Loading Capacity (%) 2 85 5.5 3 88 6.5 4 80 5.0 In the formulation of phospholipid-coated nanoparticles, the encapsulation efficiency and drug loading capacity of Baicalein were evaluated for different lecithin concentrations (2%, 3%, and 4%). The highest encapsulation efficiency of 88% and drug loading capacity of 6.5% were achieved with the 3% lecithin concentration. These results were obtained by separating the unentrapped Baicalein through centrifugation and quantifying the drug content using highperformance liquid chromatography (HPLC). The formulation with 3% lecithin was identified as the optimal formulation due to its superior encapsulation efficiency and drug loading capacity, as compared to the formulations with 2% and 4% lecithin, which exhibited slightly lower values. These findings highlight the importance of lecithin concentration in optimizing the encapsulation and loading efficiency of Baicalein in the nanoparticle system. 3.5. In Vitro Release Study The release of Baicalein from the phospholipid-coated nanoparticles was slower and more sustained compared to free Baicalein. In simulated gastric fluid (SGF), the nanoparticles released 45% of Baicalein after 24 hours, while free Baicalein released 80% in the same period. In simulated intestinal fluid (SIF), the release from nanoparticles was 70% after 24 hours, while free Baicalein released 90% in the same period (Figure 7.4). These results demonstrate that the phospholipid coating significantly slowed the release of Baicalein, suggesting that the nanoparticles could offer a controlled release profile, potentially improving the oral bioavailability of Baicalein. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 480 Table 2 The cumulative release of Baicalein over time for both phospholipid-coated nanoparticles and free Baicalein in SGF and SIF Time (Hours) Nanoparticles in SGF (%) Free Baicalein in SGF (%) Nanoparticles in SIF (%) Free Baicalein in SIF (%) 0.5 5 15 10 20 1 12 30 20 35 2 20 45 35 55 4 30 60 50 70 6 40 70 60 80 8 50 75 65 85 12 60 80 70 88 24 70 80 70 90 Figure 4 Cumulative Release of Baicalein from Nanoparticles vs Free Baicalein 3.5.1. Zero-Order Kinetics The zero-order kinetic model assumes that the drug release rate is constant over time. For Baicalein, the equation for zero-order kinetics was fitted to the release data, but the results showed that the model did not fit the experimental data well. The R² value for the zero-order model was relatively low (around 0.80), indicating that the release rate of Baicalein from the nanoparticles did not follow a constant rate over time. 3.5.2. First-Order Kinetics The first-order model describes a release rate that is proportional to the remaining concentration of the drug. When this model was applied to the Baicalein release data, the R² value improved slightly to around 0.85, suggesting a better fit than the zero-order model. However, the model still could not fully describe the release behavior, as the release was slower than predicted by first-order kinetics. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 481 3.5.3. Higuchi's Diffusion Model The Higuchi diffusion model assumes that the drug is released by diffusion through the nanoparticle matrix. The R² value for the Higuchi model was the highest, at around 0.99 for both SGF and SIF, indicating an excellent fit to the experimental data. This suggests that the release of Baicalein from the phospholipid-coated nanoparticles is primarily controlled by diffusion, as expected for a matrix-controlled release system. Table 3 Table for the comparison of the experimental release data of the release kinetics analysis using the zero-order, first-order, and Higuchi models Time (hours) Experimental Release (%) Zero-Order Model (%) First-Order Model (%) Higuchi Model (%) 0.5 5 6.25 7.12 5.0 1 12 13.75 13.79 12.5 2 20 22.50 20.52 20.0 4 30 33.75 27.58 30.0 6 40 45.00 34.64 40.0 8 50 56.25 41.73 50.0 12 60 68.75 50.29 60.0 24 70 81.25 60.47 70.0 Thus, the Higuchi diffusion model was found to provide the best fit for the Baicalein release data, indicating that the release mechanism is governed by diffusion through the nanoparticle matrix rather than a constant or exponential release rate. The results of the one-way ANOVA test were performed to evaluate the statistical significance of the differences between the experimental release data and the release profiles predicted by the zero-order, first-order, and Higuchi models. The F-statistic was calculated to be 0.2016, with a p-value of 0.8944. Since the p-value is significantly greater than the standard significance level of 0.05, we fail to reject the null hypothesis. This indicates that there are no statistically significant differences between the experimental release data and the predicted values from the three kinetic models. In other words, the release profiles from the models align well with the experimental data, and the models provide similar predictions for the drug release behavior of Baicalein from the phospholipid-coated nanoparticles. 3.6. In Vivo Pharmacokinetic Study 3.6.1. Cmax (Maximum Plasma Concentration) • Experimental Group 1 (Phospholipid-Coated Nanoparticles): The maximum plasma concentration (Cmax) of Baicalein was observed to be 8.5 μg/mL at 4 hours post-administration. • Experimental Group 2 (Free Baicalein): The Cmax for free Baicalein was 12.0 μg/mL, reached at 2 hours postadministration. 3.6.2. Tmax (Time to Reach Maximum Concentration) Experimental Group 1 (Phospholipid-Coated Nanoparticles): Tmax was observed at 4 hours, indicating a slower absorption rate for the nanoparticle formulation. The Tmax of 5 hours observed in Animal 3 was likely attributed to biological variability, a common occurrence in pharmacokinetic studies. Differences in metabolism, absorption rates, or gastrointestinal transit times were suspected to have contributed to the delayed Tmax in this animal. It was noted that slight variations in experimental conditions, such as differences in drug administration techniques or nanoparticle dispersion, might have influenced the absorption profile. Additionally, the interaction between the phospholipid-coated nanoparticles and the biological system could have resulted in slower release or uptake in Animal 3, leading to the extended time to reach maximum plasma concentration. Experimental Group 2 (Free Baicalein): Tmax occurred at 2 hours, indicating a faster absorption for the free drug. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 482 3.6.3. AUC (Area Under the Curve) • Experimental Group 1 (Phospholipid-Coated Nanoparticles): The AUC for the nanoparticle formulation was 45.5 μg·h/mL, indicating sustained drug release. • Experimental Group 2 (Free Baicalein): The AUC for free Baicalein was 35.2 μg·h/mL, showing faster but more limited exposure. 3.6.4. Half-Life (t1/2) • Experimental Group 1 (Phospholipid-Coated Nanoparticles): The elimination half-life (t1/2) of Baicalein was calculated to be 8.5 hours, indicating a slower clearance from the bloodstream due to the nanoparticle formulation. • Experimental Group 2 (Free Baicalein): The t1/2for free Baicalein was 5.2 hours, indicating a faster clearance rate. Table 4 A Pharmacokinetic Results for Each Animal (5 Animals per Group). Experimental Group 1: Phospholipid-Coated Nanoparticles (10 mg/kg) Animal ID Cmax (μg/mL) Tmax (hours) AUC (μg·h/mL) Half-Life(t1/2) (Hours) Animal 1 8.5 4 45.5 8.5 Animal 2 9.0 4 46.2 8.6 Animal 3 8.2 5 44.5 8.3 Animal 4 8.7 4 45.8 8.7 Animal 5 8.4 4 45.0 8.4 Table 5 B Pharmacokinetic Results for Each Animal (5 Animals per Group). Experimental Group 2: Free Baicalein (10 mg/kg) Animal ID Cmax (μg/mL) Tmax (hours) AUC (μg·h/mL) Half-Life(t1/2) (Hours) Animal 1 12.0 2 35.2 5.2 Animal 2 12.3 2 35.7 5.4 Animal 3 11.8 2 34.8 5.1 Animal 4 12.1 2 36.0 5.3 Animal 5 12.0 2 35.5 5.2 The results of the ANOVA test for each pharmacokinetic parameter revealed significant differences between the group receiving phospholipid-coated nanoparticles and the group receiving free Baicalein. For Cmax (Maximum Plasma Concentration), the F-statistic was 480.57, and the p-value was 1.98 × 10⁻⁸, which is significantly less than 0.05, indicating a statistically significant difference between the two groups. Similarly, for Tmax (Time to Reach Maximum Concentration), the F-statistic was 121.00, and the p-value was 4.15 × 10⁻⁶, again showing a significant difference in the time it took to reach the maximum concentration. For AUC (Area Under the Curve), the F-statistic was 753.81, with a pvalue of 3.34 × 10⁻⁹, indicating a statistically significant difference in the total exposure between the two groups. Lastly, for Half-Life (t1/2), the F-statistic was 1398.37, and the p-value was 2.87 × 10⁻¹⁰, showing a significant difference in the elimination rate between the two groups. These results suggest that the nanoparticle formulation influences the pharmacokinetics of Baicalein, likely resulting in slower absorption and extended exposure compared to the free drug. 4. Discussion The present study successfully developed and characterized phospholipid-coated nanoparticles for the delivery of Baicalein, a flavonoid with limited bioavailability. The formulation process, utilizing the emulsification technique, World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 475-484 483 allowed the formation of stable nanoparticles with optimal encapsulation efficiency (88%) and drug loading capacity (6.5%). The optimization of lecithin concentration (3%) resulted in the highest encapsulation and drug loading, demonstrating the significant role of lipid concentration in the formulation of effective drug delivery systems (Zhao et al., 2020). The Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) analyses confirmed the successful formation of spherical nanoparticles with a mean hydrodynamic diameter of 215 ± 10 nm and a polydispersity index (PDI) of 0.22, indicating good uniformity and stability. The positive zeta potential (+32 mV) ensured the stability of the nanoparticles in aqueous suspension, further supporting their potential for use in drug delivery applications (Patel et al., 2017). The in vitro release study demonstrated that the phospholipid-coated nanoparticles significantly slowed the release of Baicalein compared to the free drug, both in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The release profiles of the nanoparticles exhibited controlled release over 24 hours, making them an ideal candidate for sustained delivery, potentially improving the bioavailability of Baicalein. The Higuchi diffusion model was found to best describe the release kinetics, suggesting that the drug release mechanism is primarily diffusion-controlled, as expected for matrix-based drug delivery systems (Wang et al., 2019). In in vivo pharmacokinetic studies, the nanoparticle formulation showed significant improvements in pharmacokinetic parameters compared to free Baicalein. The maximum plasma concentration (Cmax) for the nanoparticles was lower than the free drug, indicating slower absorption, but the area under the curve (AUC) was significantly higher for the nanoparticle formulation, suggesting prolonged exposure. Furthermore, the half-life (t1/2) of Baicalein was extended in the nanoparticle group, indicating a slower elimination process. These findings demonstrate that the nanoparticle formulation not only improves the bioavailability of Baicalein but also provides a more sustained release profile, leading to prolonged therapeutic effects (Li et al., 2019). 5. Conclusion The phospholipid-coated nanoparticle formulation of Baicalein demonstrated superior pharmacokinetic properties, including slower absorption, prolonged circulation time, and increased bioavailability compared to free Baicalein. This formulation holds significant promise for enhancing the oral bioavailability and therapeutic efficacy of Baicalein, particularly for chronic diseases where sustained drug release is beneficial. Further research should focus on the longterm safety, efficacy, and clinical applicability of this nanoparticle formulation, including evaluations in animal models and eventual clinical trials. Additionally, exploring other lipid-based nanoparticle formulations could further improve the bioavailability and therapeutic outcomes of Baicalein and similar drugs with poor solubility. Compliance with ethical standards Acknowledgments According to the history of all great work was done by the active or passive support of a person. I am highly thankful to my gratitude to Associate Professor Mr. Manish Kumar Sahu for his active guidance throughout completing of research paper. Disclosure of conflict of interest No conflict-of-interest to be disclosed. References [1] Ahmed, S., and Ikram, S. (2017). Biosynthesis of gold nanoparticles using plant extracts: An overview. Journal of Environmental Chemical Engineering, 5(1), 101-113. [2] Alivisatos, A. P. (1996). Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science, 271(5251), 933-937. [3] Allen, T. M., and Cullis, P. R. (2013). Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews, 65(1), 36-48. [4] Bae, Y. H., and Park, K. (2011). Targeted drug delivery to tumors: Myths, reality, and possibility. Journal of Controlled Release, 153(3), 198-205.