A Method For Preparing Rebamipide-Loaded Nanoparticles In A Nanoparticle Drug Delivery System

Abstract

This invention is related to nanoparticle-based drug delivery system for Rebamipide, enhancing its therapeutic efficacy through improved stability and bioavailability. The method involves preparing a precursor phase with Rebamipide and a polymer, alongside an aqueous phase with a complementary polymer. The phases are combined under controlled homogenization, followed by rotary evaporation, sonication, and heating, yielding stable Rebamipide-loaded nanoparticles in a fine powder. The invention includes formulations optimized for bioavailability, utilizing specific polymers like gelatin, PEG 4000, and sodium alginate, tailored to Rebamipide’s solubility needs. Validation techniques, including UV spectrophotometry, solubility studies, thermal analyses, and zeta potential measurements, ensure quality and stability. The formulation process and analytical methods collectively provide a robust framework for developing an effective Rebamipide nanoparticle delivery system, with potential applications in enhanced therapeutic treatments.

Specification

Description:Figure 1 illustrates the Structure of Rebamipide. This invention provides a comprehensive method for creating Rebamipide-loaded nanoparticles designed to improve the drug's bioavailability and therapeutic performance. The process begins by formulating a precursor phase, where a selected polymer is dissolved in a solvent that optimally supports Rebamipide's solubility. This solution is thoroughly mixed to ensure uniformity before adding Rebamipide to create a homogenous blend, which forms the precursor phase. Alongside this, an aqueous phase is prepared by dissolving an aqueous-compatible polymer in water, with an amount equivalent to the Rebamipide quantity used to maintain consistency.
The two phases are then integrated carefully: the aqueous phase undergoes controlled stirring through homogenization, while the precursor phase is introduced gradually, drop by drop, to ensure the formation of a stable nanoparticle dispersion. The solution is subjected to rotary evaporation to reduce its volume by approximately three-quarters, concentrating the formulation. Following this, sonication is applied for 5-10 minutes, which reduces the particle size, creating a finer nanoparticle consistency. Finally, the solution is placed in a controlled water bath to allow for complete solvent evaporation, leaving behind dried Rebamipide nanoparticles.
The invention specifies a preferred Rebamipide-to-polymer ratio between 1:1 and 1:2, selected to balance solubility and optimal drug loading for therapeutic use. Additionally, the homogenization step is maintained for a recommended 20 minutes under mechanical stirring to support uniform particle dispersion. The final nanoparticle product is a fine powder achieved through the combined effects of sonication and controlled heating, resulting in nanoparticles that substantially enhance Rebamipide's bioavailability and therapeutic efficacy. This method represents a reliable approach for developing Rebamipide nanoparticles with superior stability and performance for drug delivery applications.
Figure 2 illustrates the formulation process for Rebamipide nanoparticles aims to enhance the drug's therapeutic efficacy by utilizing a nanoparticle drug delivery system. Here is a step-by-step breakdown of the formulation procedure:
Step 1 - Preparation of Precursor Phase: The process begins with weighing both Rebamipide (the drug) and a precursor polymer at a selected ratio, typically 1:1 or 1:2, depending on the desired drug loading and solubility. The precursor polymer is then dissolved in an appropriate solvent-either organic or aqueous-chosen to best dissolve Rebamipide. This mixture is stirred thoroughly to achieve a consistent solution, ensuring that the drug can be easily incorporated into the nanoparticle matrix. Once the solution is well-mixed, the drug is added to this precursor solution, forming the initial phase of the nanoparticle formulation.
Step 2 - Preparation of Aqueous Phase: Next, an aqueous phase is prepared by dissolving a specific aqueous-compatible polymer in water. The quantity of this polymer matches the weight of the drug to ensure proper consistency. This phase provides the hydrophilic environment needed to create a stable dispersion of the precursor phase when they are combined.
Step 3 - Homogenization: In this step, the aqueous phase is stirred continuously using a mechanical stirrer to maintain uniformity. The precursor phase is added gradually, drop by drop, into the aqueous phase while stirring, which allows the drug-polymer complex to disperse as nanoparticles within the aqueous environment. This homogenization step is critical for the even distribution of nanoparticles and is maintained for approximately 20 minutes to ensure stability in the dispersion.
Step 4 - Vaporization: Following homogenization, the combined solution undergoes vaporization using a rotary evaporator (Rota vapor). This process reduces the solution volume by around three-quarters, concentrating the nanoparticle suspension. Rotary evaporation helps remove excess solvent and partially solidifies the nanoparticle structure, making it more suitable for subsequent processes.
Step 5-Sonication: The solution is then subjected to sonication for 5-10 minutes. Sonication applies high-frequency sound waves to the mixture, which further reduces the size of the nanoparticles by breaking down larger particles and enhancing uniformity. This step contributes to creating a fine particle size that aids in the drug's enhanced bioavailability and stability.
Step 6 - Heating: Once sonicated, the solution is transferred into a China dish and heated using a thermostat-controlled water bath. This heating continues until all residual solvent evaporates completely, leaving behind the dried Rebamipide nanoparticles. Proper control of temperature during this step prevents degradation of the drug and helps retain the integrity of the nanoparticles.
Step 7- Collection and Storage: After drying, the nanoparticles are carefully scraped from the dish using an appropriate tool. They are then transferred into a container, such as a zip-lock cover or another suitable container, for safe storage. Proper storage conditions are necessary to maintain nanoparticle stability and ensure their therapeutic efficacy remains intact until use.
Formulation 1 consists of a precursor phase and an aqueous phase, both crucial for creating Rebamipide nanoparticles. In the precursor phase, the key ingredients include 100 mg of gelatin, 100 mg of Rebamipide, and sufficient water to make a total volume of 20 ml. To begin, the gelatin and Rebamipide are weighed in a 1:1 ratio. The gelatin is then dissolved in either water or an organic solvent compatible with Rebamipide, ensuring a homogeneous mixture. This step is vital as it allows the gelatin to serve as a matrix for encapsulating the Rebamipide, thereby enhancing its release profile in the body.
The aqueous phase of Formulation 1 contains 10 ml of PEG 4000 and enough water to make a total volume of 100 ml. Here, PEG 4000 is dissolved in water to stabilize the nanoparticles during the formulation process. The presence of this hydrophilic polymer is essential for achieving a stable dispersion of nanoparticles.
Formulation 2 follows a similar structure but employs different materials. The precursor phase consists of 100 mg of sodium alginate, 100 mg of Rebamipide, and 12.5 ml of water. Sodium alginate is selected for its unique properties that improve drug encapsulation and controlled release. After mixing the drug with sodium alginate and water, a homogenous precursor solution is created. The aqueous phase in this formulation includes 11.9 ml of triethanolamine combined with NaOH and enough water to make a total of 100 ml. This combination serves as a gelling agent and stabilizer, contributing to the overall integrity of the nanoparticles.
To quantify the amount of Rebamipide in the formulations, a standard calibration curve is established. This is achieved by preparing a stock solution of 100 mg of Rebamipide in 100 ml of Dimethyl Formamide (DMF) buffer, resulting in a concentration of 1000 µg/ml. A series of standard solutions at varying concentrations (5, 10, 15, 20, and 25 µg/ml) are created through dilution, and their absorbance is measured at 227 nm using a UV-Vis spectrophotometer. A calibration curve is then plotted with concentration on the X-axis and absorbance on the Y-axis, providing a reference for quantifying drug concentration in future experiments.
To evaluate the solubility of Rebamipide, solubility studies are conducted using various solvents, including water, DMSO, DMF, ethanol, methanol, propylene glycol, and phosphate buffer. These studies are critical for selecting the most suitable solvent for optimal drug delivery, as they help determine how well Rebamipide dissolves in different environments.
Fourier Transform Infrared (FTIR) spectroscopy is employed to characterize the pure drug. FTIR spectra are obtained using a thermos-IR 200 FTIR spectrophotometer with the potassium bromide pellet method. By collecting a background spectrum under identical conditions, researchers can analyze the functional groups present in Rebamipide, further understanding its chemical properties.
Thermal analysis of pure Rebamipide is performed using Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). In this process, the DSC is calibrated with indium, and samples weighing between 15-30 mg are analyzed under a nitrogen atmosphere at a heating rate of 10 °C/min. This analysis provides insights into the thermal stability and melting characteristics of Rebamipide, which are critical for understanding its behavior during formulation.
The surface morphology of the formulated nanoparticles is examined using Scanning Electron Microscopy (SEM). The samples are scanned with an electron beam at an acceleration potential of 10 kV, capturing images in secondary electron mode. This imaging allows researchers to assess the size, shape, and distribution of the nanoparticles, essential for ensuring uniformity in the formulation.
Dissolution studies are conducted using a USP dissolution tester, where samples equivalent to 100 mg of the nanoparticles are placed in HCl buffer (pH 1.2) at a stirring speed of 50 rpm and maintained at 37 ± 0.5 °C. At predetermined time intervals (5, 15, 30, 45, and 60 minutes), 5 ml samples are withdrawn and filtered for analysis. The absorbance of the filtered samples is measured at 227 nm after appropriate dilution, providing a quantitative assessment of the drug release profile from the nanoparticles.
For the assay of drug content, 100 mg of the gelatin formulation obtained through the solvent evaporation method is dissolved in 1000 ml of HCl buffer (pH 1.2). A 10 ml aliquot of this solution is diluted to 10 ml, and the absorbance is measured at 227 nm using a UV-Vis spectrophotometer. This assay is crucial for determining the actual drug content in the formulations.
The Entrapment Efficiency (EE) is calculated to evaluate the effectiveness of the formulation in retaining the drug within the nanoparticles. The drug-loaded nanoparticles are subjected to centrifugation at 5000-6000 rpm for 30 minutes, and the concentration of non-bound drug in the supernatant is measured using a UV spectrophotometer. The percentage of drug entrapment efficiency is calculated using the formula:

This metric provides valuable insights into the success of the nanoparticle formulation.
The Zeta Potential (ZP) is measured to assess the stability of the nanoparticle formulations. ZP indicates the electrostatic charge of the particles in suspension, influencing their stability and propensity to aggregate. Measurements are taken using a Malvern Zetasizer Nano ZS at 25 °C with a detection angle of 173°. The findings aid in optimizing the formulations and predicting their long-term stability, with a ZP value of ±30 mV or greater indicating good stability. This comprehensive characterization process ensures the development of effective nanoparticle delivery systems for Rebamipide, enhancing its therapeutic efficacy and bioavailability.
In the validation studies as illustrated in Figure 3, it was found that the estimation of Rebamipide by spectrophotometric method at 227nm has good reproducibility, at the various concentration 5- 25µ/ml.
Table 1:validation studies
S.NO Concentration Wavelength Absorbance
1 5µg/ml 227 0.01363
2 10µg/ml 227 0.03296
3 15µg/ml 227 0.05106
4 20µg/ml 227 0.07115
5 25µg/ml 227 0.08825

Solubility Studies of Rebamipide: Solubility studies are a crucial aspect of pharmaceutical formulation, providing insights into how effectively a drug can dissolve in various solvents. For Rebamipide, a series of solvents were evaluated to determine their solubility profiles, which can significantly influence formulation strategies and the overall effectiveness of drug delivery systems. Rebamipide demonstrates high solubility in DMSO (Dimethyl Sulfoxide), making it an ideal solvent for laboratory settings where a wide range of organic compounds need to be dissolved. This high solubility suggests that DMSO could be utilized effectively for initial formulations or as a vehicle for drug delivery, enhancing the drug's availability for therapeutic applications. Similarly, DMF (Dimethylformamide) also shows high solubility for Rebamipide. DMF is commonly employed in organic synthesis and pharmaceutical formulations, and its ability to solubilize Rebamipide indicates its potential as a solvent in drug preparation processes.
Another solvent tested is propylene glycol, in which Rebamipide is soluble. Propylene glycol is frequently used in food and pharmaceutical applications due to its low toxicity. Its ability to dissolve Rebamipide suggests that it could be utilized in formulations where a non-toxic solvent is preferable, thereby potentially enhancing the drug's bioavailability. On the other hand, Rebamipide's solubility in water is classified as sparingly soluble. This limited solubility is an important consideration, particularly for oral formulations, as it may affect the drug's absorption and therapeutic efficacy.
Ethanol and methanol exhibit slight solubility for Rebamipide, indicating that these solvents can dissolve only small amounts of the drug. While ethanol and methanol are common solvents in pharmaceutical formulations, their limited solubility for Rebamipide may pose challenges for developing effective formulations that require higher concentrations of the drug. Finally, the studies reveal that Rebamipide is insoluble in phosphate buffer. This finding is significant, as phosphate buffers are widely used in pharmaceutical formulations to maintain pH stability. The lack of solubility in this buffer suggests that alternative formulations may be necessary when designing drug delivery systems involving Rebamipide.
In conclusion, the solubility studies reveal a varied profile of Rebamipide's solubility across different solvents, ranging from high solubility in DMSO and DMF to insolubility in phosphate buffer. Understanding these solubility characteristics is vital for formulating effective drug delivery systems, as the choice of solvent can greatly impact drug release, bioavailability, and therapeutic efficacy. These insights will guide further development and optimization of formulations aimed at enhancing the delivery and effectiveness of Rebamipide.
Table 2: Solubilities studies
S.NO Solvents Solubility
1 DMSO Highly soluble
2 DMF Highly soluble
3 Propylene Glycol Soluble
4 Water Sparingly soluble
5 Ethanol Slightly soluble
6 Methanol Slightly Soluble
7 Phosphate buffer Insoluble

Figure 4 illustrates the FTIR Analysis of Rebamipide. The FTIR analysis of Rebamipide highlights the presence of various functional groups, including alkenes, aromatics, nitro groups, alcohols, and alkyl halides. Each of these functional groups contributes to the overall chemical behavior of Rebamipide, influencing its solubility, stability, and biological activity. Understanding the functional groups present in Rebamipide through FTIR provides valuable insights into its potential therapeutic mechanisms and helps in guiding the development of effective formulations for drug delivery.
Fourier Transform Infrared (FTIR) spectroscopy is a powerful analytical technique used to identify functional groups in a compound by measuring the absorption of infrared light at various wavelengths. The FTIR spectrum of Rebamipide reveals several key peaks corresponding to specific functional groups, which provide insight into the chemical structure and characteristics of the compound.Peak at 3001.24 cm⁻¹: This peak is associated with alkenes and aromatics. The presence of this peak indicates the existence of double bonds or unsaturated structures within the molecular framework of Rebamipide. Aromatic compounds typically display characteristic peaks in this range, suggesting that Rebamipide contains aromatic rings in its structure, which are crucial for its biological activity.
Peak at 1593.20 cm⁻¹: This peak corresponds to aromatic C-C stretching vibrations. The detection of this peak further supports the presence of aromatic groups in Rebamipide. The aromatic structure contributes to the compound's stability and can influence its interaction with biological targets, which is essential for its therapeutic efficacy.
Peak at 1292.31 cm⁻¹: This peak is indicative of nitro compounds, specifically associated with the N-O symmetric stretch. The presence of a nitro group in Rebamipide is significant, as it can play a role in the compound's pharmacological activities. Nitro groups are often associated with increased reactivity, which may enhance the drug's efficacy.
Peak at 1004.91 cm⁻¹: This peak relates to alcohols, carboxylic acids, esters, and ethers. The identification of this peak suggests that Rebamipide contains hydroxyl (-OH) or ether functionalities, which are important for solubility and interaction with biological systems. These groups can influence the drug's pharmacokinetics, affecting its absorption and distribution in the body.
Peak at 754.17 cm⁻¹: This peak is associated with alkyl halides, specifically the C-Cl stretching vibration. The presence of alkyl halides indicates that Rebamipide may have chlorinated carbon chains, which can affect the compound's lipophilicity and, subsequently, its ability to cross biological membranes.
Figure 5 illustrates the FTIR Analysis of Rebamipide and Gelatin. The FTIR analysis of the Rebamipide and gelatin mixture reveals various functional groups, including alkanes, amines, alkyl halides, and aliphatic amines. These functional groups play critical roles in determining the physicochemical properties of the formulation, such as solubility, stability, and drug release characteristics. Understanding these interactions is essential for optimizing drug delivery systems and enhancing the therapeutic efficacy of Rebamipide in its gelatin nanoparticle formulation.
Fourier Transform Infrared (FTIR) spectroscopy is a crucial tool for characterizing the functional groups present in a compound. In the analysis of the mixture of Rebamipide and gelatin, several key peaks were identified, each corresponding to specific functional groups. This analysis aids in understanding the interactions between Rebamipide and gelatin, as well as the overall chemical structure of the resulting formulation.
Peak at 2987.74 cm⁻¹: This peak is indicative of alkanes, specifically related to C-H stretching vibrations. The presence of this peak suggests that both Rebamipide and gelatin contain alkane structures. Alkanes are typically hydrophobic and can influence the solubility and stability of the drug formulation. This hydrophobic character can play a role in the encapsulation of Rebamipide within gelatin, potentially enhancing its therapeutic efficacy.
Peak at 1602.85 cm⁻¹: This peak corresponds to 1° amines, associated with N-H bending vibrations. The detection of this peak indicates that Rebamipide and/or gelatin may contain primary amine functional groups. Amines are known to participate in hydrogen bonding, which can enhance the solubility and stability of the formulation. This feature may also contribute to improved drug release characteristics.
Peak at 1294.24 cm⁻¹: This peak is associated with alkyl halides, specifically related to the C-H wagging vibrations. The presence of this peak suggests that there are alkyl halides in the formulation, likely originating from Rebamipide. This functionality can impact the lipophilicity and overall interaction of the drug with biological membranes, thus influencing its bioavailability. Peak at 1041.56 cm⁻¹: This peak relates to aliphatic amines, specifically associated with C-N stretching vibrations. The identification of this peak implies that aliphatic amines are present in the formulation. Aliphatic amines are known for their ability to form hydrogen bonds and interact with other molecules, which can enhance the stability and release properties of the drug-loaded gelatin nanoparticles. Peak at 572.86 cm⁻¹: This peak corresponds to alkyl halides, specifically associated with the C-Br stretching vibrations. The presence of this peak indicates that brominated compounds may be present in the formulation, likely related to the structure of Rebamipide. The presence of halogens can affect the overall reactivity and interaction of the drug with biological systems.
Figure 6 illustrates the FTIR Analysis of Rebamipide and Sodium Alginate. The FTIR analysis of the Rebamipide and sodium alginate mixture reveals several key functional groups, including α, β-unsaturated esters, aromatics, alkyl halides, and alkenes. These functional groups play essential roles in determining the physicochemical properties of the drug formulation, including solubility, stability, and release characteristics. Understanding these interactions is vital for optimizing drug delivery systems and enhancing the therapeutic efficacy of Rebamipide in sodium alginate formulations.
Fourier Transform Infrared (FTIR) spectroscopy is an effective analytical technique used to identify the functional groups present in chemical compounds. In the case of the mixture of Rebamipide and sodium alginate, several significant peaks were observed, each corresponding to specific functional groups. This analysis provides insights into the interactions between Rebamipide and sodium alginate, which are crucial for developing effective drug delivery systems.
Peak at 1726.29 cm⁻¹: This peak is indicative of α, β-unsaturated esters, specifically relating to the C=O stretching vibrations. The presence of this peak suggests that Rebamipide and/or sodium alginate contains carbonyl groups characteristic of ester functionalities. This functional group plays a significant role in determining the solubility and reactivity of the drug formulation, as esters can facilitate interactions with biological membranes, potentially enhancing drug absorption.
Peak at 1591.27 cm⁻¹: This peak corresponds to aromatics, specifically associated with C-C stretching vibrations within a ring structure. The detection of this peak indicates the presence of aromatic rings in the formulation, likely from the structure of Rebamipide. Aromatic compounds are known for their stability and can influence the overall behavior of the drug within the delivery system, affecting its release kinetics and bioavailability.
Peak at 1207.44 cm⁻¹: This peak is associated with alkyl halides, specifically related to the C-H wagging vibrations. The identification of this peak suggests that alkyl halides may be present in the formulation, likely due to the chemical structure of Rebamipide. Alkyl halides can affect the lipophilicity and overall interactions of the drug with biological systems, which can be beneficial for optimizing its delivery.Peak at 979.84 cm⁻¹: This peak is indicative of alkenes, specifically relating to the =C-H bending vibrations. The presence of this peak suggests that there are alkene functionalities in the formulation, which can influence the physical and chemical properties of the drug. Alkenes are known to contribute to the reactivity of compounds and can affect the stability of the drug in the delivery system.
Peak at 557.43 cm⁻¹: This peak corresponds to alkyl halides, specifically related to the C-Br stretching vibrations. The identification of this peak indicates that brominated compounds are present in the formulation, likely associated with the structure of Rebamipide. The presence of halogens can impact the overall reactivity and interactions of the drug, affecting its pharmacokinetic properties. Figure 7 illustrates the dissolution Studies of Rebamipide Nanoparticles. The dissolution study of Rebamipide nanoparticles showed a gradual increase in absorbance over time, indicating the drug's release from the nanoparticle formulation. At 0 minutes, the absorbance was 0.0003, reflecting minimal dissolution as the drug remained in its solid form. After 5 minutes, the absorbance rose to 0.0010, indicating the nanoparticles began releasing Rebamipide into the medium. By 15 minutes, the absorbance increased to 0.0017, confirming a significant release of the drug.

Table 2: Solubilities studies
Time Wavelength Absorbance
0 min 227 0.0003
5min 227 0.0010
15min 227 0.0017
30min 227 0.0024
45min 227 0.0032
60min 227 0.0040
At the 30-minute mark, the absorbance reached 0.0024, demonstrating an accelerating release rate. By 45 minutes, the absorbance had increased to 0.0032, suggesting an effective and sustained drug release. Finally, at 60 minutes, the absorbance peaked at 0.0040, representing the highest concentration of dissolved Rebamipide during the study, indicating successful drug release from the nanoparticle formulation. Dissolution studies are a critical component of pharmaceutical formulation development, as they provide insight into the release profile of a drug from its formulation.
In this case, the dissolution studies were conducted to assess the release of Rebamipide from its nanoparticle formulation over a specified time frame. The data collected includes the absorbance of the solution at a wavelength of 227 nm at various time intervals, which is indicative of the concentration of Rebamipide present in the solution due to its UV absorbance properties.The trend in absorbance values over time indicates a consistent increase in the dissolution of Rebamipide from its nanoparticle formulation. The gradual rise in absorbance signifies that the nanoparticles are capable of sustaining a controlled release of the drug into the dissolution medium.
The data suggest that the formulation is effective in promoting the dissolution of Rebamipide, which is crucial for enhancing its bioavailability. This release profile indicates that the nanoparticles are facilitating a slow and steady release of the drug, potentially improving therapeutic efficacy while minimizing potential side effects associated with rapid drug release.
, Claims:We claim
1. A method for preparing Rebamipide-loaded nanoparticles in a nanoparticle drug delivery system, comprising:
a) Formulating a precursor phase by dissolving a polymer in a solvent suitable for Rebamipide solubility and mixing thoroughly;
b) Adding Rebamipide to the precursor solution to create a homogenous precursor phase;
c) Preparing an aqueous phase by dissolving an aqueous polymer in water, maintaining an equivalent weight to the Rebamipide used;
d) Homogenizing the aqueous phase under continuous stirring and adding the precursor phase drop by drop to achieve a nanoparticle dispersion;
e) Reducing the solution volume via rotary evaporation to achieve a 3/4 reduction;
f) Sonicating the solution for 5-10 minutes to further reduce particle size; and
g) Heating the solution in a controlled water bath until complete solvent evaporation, resulting in dried Rebamipide nanoparticles.
2. The method of Claim 1, wherein the ratio of Rebamipide to precursor polymer is selected from a range of 1:1 to 1:2, based on solubility and therapeutic loading requirements.
3. The method of Claim 1, wherein the homogenization process is conducted for a duration of 20 minutes, under controlled mechanical stirring, to ensure stable dispersion of Rebamipide nanoparticles.
4. The Rebamipide nanoparticle formulation as claimed in claim 1, wherein the particle size is reduced to a fine powder through sonication and heat-drying, and wherein the nanoparticles provide enhanced bioavailability and therapeutic efficacy of Rebamipide.
5. A formulation for Rebamipide-loaded nanoparticles in a drug delivery system comprising:
a) A precursor phase containing 100 mg of Rebamipide and 100 mg of gelatin, combined with water up to 20 ml; and
b) An aqueous phase containing 10 ml of PEG 4000 with water up to 100 ml, mixed to produce a nanoparticle dispersion for improved therapeutic efficacy of Rebamipide.
6. A method of preparing Rebamipide nanoparticles as in Claim 1, comprising:
a) Dissolving the precursor phase polymer in water and adding Rebamipide;
b) Mixing the aqueous phase polymer in water, then combining both phases under homogenization for nanoparticle formation; and
c) Reducing the dispersion via evaporation, followed by sonication and heating to yield dried nanoparticles.

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