Curcumin is a safe and natural therapeutic moiety. The aim of the study is to design and develop a molecule containing mesoporous silica nanoparticles loaded with curcumin. Curcumin-loaded mesoporous silica nanoparticles will be evaluated using physicochemical and analytical techniques. Fourier transform infrared analysis confirms the successful incorporation of the curcumin into the mesoporous silica cells, where the quaternary carbon has an absorption band at 1521.22 cm-1 and CH2 at 2858.4 cm-1. X-ray diffraction analysis confirms drug entrapment within the silica nanoparticle as well as there is no extra peak of drug in the final hexagonal channel of the loaded nanoparticle. Scanning electron microscopy and particle size analysis techniques have shown that the size of the unloaded and loaded particles is different. MCM-NPs-C-CUR were successfully synthesized and further research will be conducted to investigate their therapeutic profile.
Research Article
Curcumin Loaded Mesoporous Silica Nanoparticles: Its Synthesis, Characterization, and Evaluation
https://doi.org/10.21203/rs.3.rs-2578348/v2
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Silica Nanoparticle
Curcumin Nanoparticle
Mesoporous Silica
Curcuma Longa
Curcumin has a wide range of therapeutic benefits and is known as an ideal candidate for the treatment of neurological disorders. It is extracted from the rhizomes of turmeric (Curcuma longa). In a healthy human body, cellular processes produce free radicals that enter cells and cause oxidative stress. Antioxidant molecules can heal the free radicals as well as the oxidative stress in the body. (Apel et al., 2004) Mitochondrial dysfunction - an imbalance of reactive oxygen species and reduced energy levels in cells and tissues - implicates a variety of pathways in neurochemical and neuro-behavioural disorders. (Forster et al., 1996; Navarro et al., 2002) Therefore, multimodal therapies have pleiotropic effects against age-related neurodegeneration. (Muller et al., 2007) Evidence suggests that progressive oxidative damage impairs central neurofunctional mechanisms. (Muller et al., 1989 and Banion et al., 1994) Gene expression studies have suggested a positive relationship between oxidative damage and biological ageing. This relationship depends on regulation of oxidative stress genes. (Fraser et al., 2005) This is an original research approach to the design of a novel formulation containing curcumin-loaded mesoporous silica nanoparticles for treating various diseases in the body.
2.1 Materials
Cetyltrimethylammonium bromides (CTAB, ≥99%) from Otto Chemie Pvt Ltd, tetraethyl orthosilicate (TEOS, ≥98%) from Alfa Aesar Chemicals Ltd., sodium dihydrogen orthophosphate dehydrates (≥98%) from Fisher Scientific, and curcumin (a gift sample) from Karnataka Antibiotic were purchased. Deionized water, sodium hydroxide, concentrated hydrochloric acid, poloxamers, methanol (≥99.5%), ethanol (≥99%), and acetonitrile HPLC grade were also used.
2.2 Methods
2.2.1 Synthesis of Mesoporous Silica Nanoparticles (MCM-NPs)
The silica loaded nanoparticles were synthesized using a slight modification of the method described by Hom et al, 2010; and Chen et al, 1993. Briefly, MCM-NPs-C was prepared by dissolving 2.8 g CTAB, 3.9 ml 2.0 M sodium hydroxide and 100 ml ultrapure water at 80oC for 30 min. Then 3.3 g TEOS was added rapidly with stirring (600 rpm for 4 min) at 80oC for 2.5 h to form a white precipitate. The sample was diluted thrice with approximately 300 ml of distilled water, filtered, and washed with 100 ml of methanol and 1 ml of concentrated hydrochloric acid to remove all CTAB surfactant. This procedure was repeated two times. The solid product was collected after centrifugation at 2000 rpm (CPR-30 Plus, REMI India). The process was carried out with different concentrations of the above chemicals to obtain different types of mesoporous silica nanoparticles (MCM-NPs) and the concentration of the chemicals and name of the sample are indicated in Table 1.
Table 1 Concentration of the chemicals
|
Sample Name |
CTAB (gm) |
2.0M NaOH (mL) |
H2O (mL) |
TEOS (gm) |
|
MCM-NPs-A |
1.5 |
1.7 |
120 |
2.1 |
|
MCM-NPs-B |
1.2 |
1.3 |
110 |
1.9 |
|
MCM-NPs-C |
2.8 |
3.9 |
100 |
3.3 |
|
MCM-NPs-D |
2.0 |
2.5 |
140 |
3.1 |
MCM-NPs – Mesoporous Silica Nanoparticle, CTAB - Cetyltrimethylammonium bromides, TEOS- tetraethyl orthosilicate
2.2.2 Preparation of Curcumin Loaded MCM-NPs-C
The nanoparticle sample MCM-NPs-C loaded with curcumin was confirmed by FTIR. Simply dissolve 10 mg of curcumin in 6 ml of ethanol and stir until a homogeneous mixture is obtained. Then at room temperature, 50 mg of dried MCM-NPs-C was added while stirring vigorously (400 rpm) and kept away from light for 20 hours. Afterwards, the MCM-NPs-C-CUR (curcumin-loaded MCM-NPs) were centrifuged continuously at 6000 rpm for 40 minutes and the supernatant was collected. The final solid product, MCM-NPs-C-CUR, was dried using vacuum drying.
2.2.3 Calculation Percentage Drug Loading and Encapsulation efficiency:
The collected supernatant was screened by ultraviolet (UV) spectrophotometer for measurement of unbound drug and calculation of encapsulation and drug loading using given formulae.
The drug was investigated for various physicochemical properties such as physical appearance, melting point and solubility in different solvents which were compared with the standards. Many characterization studies were carried out for the physicochemical properties of the prepared sample such as Fourier Transform Infrared (FTIR) spectra, particle size, zeta potential, polydispersity index, scanning electron microscopy (SEM) and X-ray diffraction analysis.
3.1 Physical appearance
The physical appearance of the powdered drug was assessed by pouring it onto light and dark backgrounds and the results were compared with those reported in the literature.
3.2 Determination of melting point
The capillary melting method was used to calculate the melting point of the sample. The sample was placed in a capillary with one end closed and then placed in Remi's melting point apparatus. The temperature at which the first molecule of the drug was transformed into a liquid was measured and compared with previous research.
3.3 Determination of solubility
The solubility of curcuminoids was tested in different solvents; 50 mg of drug was dissolved in 10 ml of solvent in a 10 ml solubility bottle. The bottle was properly capped and placed on a water bath shaker set at 37 degrees Celsius for 24 hours. Samples were taken manually and filtered through 0.45μm filter paper using a UV spectrophotometer (Lab India 3000+, Mumbai, India) and the absorbance of the solution after appropriate dilutions was recorded at 425 nm.
3.4 Fourier transform infrared analysis
An FTIR spectrophotometer (IR Affinity-1, Shimadzu, Japan) was used to quantify the sample. FTIR analysis is the most powerful technique for qualitative compound identification. The main application of FTIR spectrophotometry is the determination of drug identity by spectral comparison with an authentic sample and an unknown sample. IR scanning of the samples was performed between 4000 and 400 cm1. Briefly, the sample was mixed with an appropriate amount of KBr and converted into pellets using a KBr press with a hydraulic pressure of 15 tonnes. The observed spectral drug peak was compared with the literature. (Das et al., 2007)
3.5 Preparation of calibration curve of pure drug
Curcumin (100 mg) was accurately weighed and dissolved in phosphate buffer (pH 6.8) in a 100 ml volumetric flask. The drug was not completely soluble in phosphate buffer (pH 6.8), so methanol was used as a co-solvent to dissolve the drug and the volume was made up to 100 ml with phosphate buffer (pH 6.8). A stock solution of 100 μg/ml was prepared by diluting the solution (10 ml) to 100 ml with phosphate buffer (pH 6.8). From the stock solution, aliquots of 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml and 4.0 ml were transferred to 10 ml volumetric flasks and the volume made up to 10 ml with phosphate buffer (pH 6.8). The absorbance of these solutions was measured using a UV spectrophotometer (Lab India 3000+, Mumbai, India) at 425 nm.
3.6 Particle size Zeta potential and polydispersity index
The mean particle size, zeta potential and size distribution of the prepared NPs were determined by photon correlation spectroscopy (Nano ZS, Malvern, UK). All measurements were performed in triplicate with a maintained 900 and room temperature was maintained throughout the analysis. The sample was diluted with deionized water prior to measurement. The mean particle size, obtained as hydrodynamic diameter, was reported using the intensity distribution by cumulative analysis, and the zeta potential of the prepared NP (MCM-NPs-C-CUR) was determined using an electrophoretic cell with electric field, using the equipment described above. Zeta potential measurements were performed in duplicate after three independent experiments in automatic mode, using an average of 10 measurements for each sample within the duplicates.
3.7 Scanning Electron microscopy (Surface morphology)
The surface morphology and shape of the prepared samples (MCM-NPs-C-CUR) were analyzed by SEM (S-3400 N, Hitachi, Japan). (Huang et al., 2014)
3.8 Powder X-Ray diffraction analysis
The X-ray diffraction of the solid nature of the powder samples (MCM-NPs-C and MCM-NPs-C-CUR) was analyzed using an X-ray diffractometer (X'pert PRO, Panalytical B.V., The Netherlands) activated with the CuKα target. Data were obtained at a 2-diffraction angle of 50 to 50 0, with a scan rate of 40 mA per minute and a step size of 0.020, and X-rays were generated at 40 mA and 45 kV. The analysis process was repeated three times to confirm the results. (Zhang et al., 2007)
4.1 Physiochemical properties
The X-ray diffraction of the solid nature of the powder samples (MCM-NPs-C and MCM-NPs-C-CUR) was analyzed using an X-ray diffractometer (X'pert PRO, Panalytical B.V., The Netherlands) activated with the CuKα target. Data were obtained at a 2-diffraction angle of 50 to 50 0, with a scanning speed of 40 mA per minute and a step size of 0.020, and X-rays were generated at 40 mA and 45 kV. The analysis was repeated thrice to confirm the results.
Table 2 Physical characterization of the drug
|
Color |
Pale yellow |
|
State |
Crystalline Powder |
4.2 Melting point
The melting point was determined by the capillary fusion method. The observed melting point was compared with the literature, confirming that the drug used in the present study was in its pure form. The data are presented in Table 3.
Table 3 Melting point study of drug
|
Method |
Observed |
Reported |
|
Capillary fusion method |
148ºC |
149-150ºC |
4.3 Quantitative and qualitative estimation of solubility of Curcumin
The solubility of the drug was determined and the results are shown in Table 4. It is more soluble in phosphate buffer (pH 6.8) than in distilled water or 0.1 N HCl (pH 1.2).
Table 4 Solubility study of drug
|
Solvent |
Solubility (mg/ml) |
Remark |
|
Distilled water |
0.187 |
- |
|
0.1N HCl (pH 1.2) |
0.283 |
- |
|
Phosphate buffer pH 6.8 |
0.841 |
++ |
- practically insoluble, ++ slightly soluble
4.4 Preparation and optimization of MCM-NPs-C-CUR
Using Stober's synthesis method, mesoporous silica nanoparticles with ETP (MCM-NPs-C-CUR) were successfully prepared and optimized.
4.5 FT-IR analysis
The FT-IR spectra of MCM-NPs-C, curcumin and MCM-NPs-C-CUR are examined (Figure 1). There are peaks of O-H (3357.30 cm1) and H2O (1740.27 cm1) in the spectrum of MCM-NPs-C. It confirms the presence of many OH groups, which play a major role in the adsorption of ETP by forming hydrogen bonds, and other bands related to Si-O-Si (1066.86 cm-1), Si-OH (946.36 cm-1) and Si-O (445.14 cm-1). Furthermore, the infrared spectrum of curcumin and MCM-NPs-C-CUR shows the characteristic peak of C=C stretching (1485.7 and 1484.5 cm-1), which reinforces the aromatic phenyl ring. The absorption band of C=O (1766.5 and 1762.5) shows the characteristic stretching frequencies in both the final formulation and the drug. Another frequency of curcumin due to C-H vibrations is observed at 1071.20 in MCM-NPs-C-CUR. The quaternary carbon with an absorption band at 1521.22 cm1 and CH2 at 2858.4 cm1 confirms that the curcumin is successfully incorporated into the mesoporous silica cells of MCM-NPs-C-CUR.
4.6 Particle size Zeta potential and polydispersity index
The observed particle size, ζ-potential and polydispersity index are summarized here. The results showed that MCM-NPs-C-CUR and MCM-NPs-C were spherical with the same structural features. The average size (Figure 2) of MCM-NPs-C-CUR and MCM-NPs-C nanoparticles was observed to be 240 ±2 nm and 208 ±2.5, respectively, along with ζ-potential values of -28.6 ±2.5 and -34.6 ±2, and pdi values of 0.44 ±0.07 and 0.35 ±0.05, respectively. The smaller size of the blank and the larger size of the loaded nanoparticles indirectly confirmed the drug loading. The size and morphology were further evaluated using electron microscopy.
4.7 Percent drug loading and entrapment efficiency
After loading the curcumin on the MCM-NPs-C, the loading percentage and the entrapment efficiency were calculated using the formulas already discussed, and they found the drug loading to be 38.27%. The entrapment efficiency was also calculated and it was observed that the curcumin was entrapped up to 76.11% in the prepared silica nanoparticles. The reason for the high entrapment efficiency was the porous nature of the prepared silica nanoparticles.
4.8 Electron microscopy
Scanning electron microscopy (SEM, S-3400 N, Hitachi, Japan) was used to compare the morphological difference in the surface between MCM-NPs-C and MCM-NPs-C-CUR, and the observed difference was an almost spherical shape (Figure 3), with MCM-NPs-C, and the shape was slightly distorted after loading with curcumin. Thus, the result is clear evidence of the loading of curcumin on the prepared nanoparticles. The main purpose of performing SEM is to determine the difference due to loading and changes in the surface.
4.9 Powdered X-ray diffraction analysis
The X-ray pattern of the sample was observed and the observation of pure drug, then MCM-NPs-C and loaded MCM-NPs-C-CUR is shown in the figure. As a result, the diffraction pattern of MCM-NPs-C and MCM-NPs-C-CUR shows a peak near 3.50 to 5.20 and 240, demonstrating that the structure of MCM-NPs-C has a well-defined hexagonal pattern and no separate crystalline phase is observed in the final loaded formulation (Figure 4). Furthermore, the absence of an additional peak of the drug in the final hexagonal channel of MCM-NPs-C-CUR is encouraging evidence of drug entrapment within the silica nanoparticles.
Neurological disorders can be treated with spinal surgery, medication, and drug therapy. Herbal medicines have minimal or no side effects in the treatment process and curcumin is also approved as a safe molecule in the USA. Curcumin is extracted from the roots of turmeric (Curcuma longa) and has a wide range of activities including antioxidants, treatment of Alzheimer's disease, treatment of skin conditions, anti-inflammatory properties, lowering the risk of cardiovascular disease, immune system support and treatment of neurochemical disorders. In this study, we investigated the ability of the curcumin-loaded nano formulation to cross the blood-brain barrier and the ability of curcumin to cure neurological disorders. Here, we analysed the prepared formulations by FTIR, SEM and X-ray differentiation; the results show that the curcumin-loaded nanoparticles are successfully incorporated into the mesoporous silica cells, and size determination techniques analyse the sizes of unloaded and loaded particles as different from each other. Drug entrapment within the silica nanoparticle: There is no additional peak of the drug in the final hexagonal channel of the loaded nanoparticle. The result is therefore clear evidence for the loading of curcumin onto the prepared nanoparticle. The formulation was successfully prepared and assigned to a further study to assess its potency parameter for improved therapeutic value.
The present research article reports the synthesis of curcumin-loaded lipid-coated mesoporous silica nanoparticles. The developed nanoparticles had high drug entrapment and had significant result.
Acknowledgements
We would like to thank Shri Venkateshwara University, Gajraula.
MCM-NPS-C Mesoporous Silica Nanoparticles
MCM-NPs-C-CUR Curcumin loaded Mesoporous Silica Nanoparticles
UV Ultra violet
FTIR Fourier transform infrared analysis
SEM scanning electron microscopy
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No competing interests reported.