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Solid Lipid Nanoparticles exhibit physical stability, protection of incorporated labile drugs from degradation, controlled release, excellent tolerability, and site-specific targeting. Hence a procedure for the preparation of drug-loaded (catechin) nanoparticles will be generated wherein the particles will be producing the Solid Lipid Nanoparticle by Water in Oil emulsion (W/O) method. The synthesis will follow standard procedure. The nanoparticles will be analyzed using the Atomic force microscopy. The efficiency of catechin loading and release will be studied by spectrophotometric method.
Key words: Catechin. Solid lipid nanoparticles. Tea.
In recent years, extensive studies have been carried out on tea and the tea constituents for their potential health benefits. It has multiple preventive and therapeutic effects. In addition to caffeine, tea also contains catechin. A cup of brewed green tea contains in the extract 30-40% catechins. After consumption, tea polyphenols are detected in both intact forms and as metabolites that could reach sub micromolar concentrations in blood plasma. Catechin is a flavan-3-ol, a type of natural phenol and antioxidant. It is a plant secondary metabolite, belongs to the group of flavan-3-ols (or simply flavanols). It is often considered to belong to the family of flavonoids.
Chemical structure of (+)-Catechin
Catechin structure shows two benzene rings (called the A- and B-rings) and a dihydropyran heterocycle (the C-ring) with a hydroxyl group on carbon 3. There are two chiral centers on the molecule on carbons 2 and 3. Therefore, it has four diastereoisomers. Two of these isomers are in trans configuration and are called catechin and the other two are in cis configuration and are called epicatechin.
The most common catechin isomer is the (+)-catechin. The other stereoisomer is (-)-catechin or ent-catechin. The most common epicatechin isomer is (-)-epicatechin (also known under the names L-epicatechin, epicatechol, (-)-epicatechol, l-acacatechin, l-epicatechol, epi-catechin, 2,3-cis-epicatechin or (2R,3R)-(-)-epicatechin). The catechins are abundant in teas derived from the green tea plant, as well in some cocoas and chocolates. Catechins are also present in some fruits, vegetables and wine, and are found in many other plant species. The health benefits of catechins have been studied extensively in using humans and animal models. In animal models there was reduction in atherosclerotic plaques. In vitro reduction in carcinogenesis was also seen. Flavanols like catechins, usually from tea or cocoa beans, are believed to keep arteries flexible, increase small vessel circulation, reduce blood pressure and protect against sunburns. Extensive scientific research and the epidemiological observations in recent years have showed that the polyphenolic compounds like catechins present in green tea may prevent a variety of cancer types. For example, EGCG and other tea polyphenols are well known for their antioxidant activities. It was also shown that the tea polyphenols inhibit carcinogen-induced DNA damage and tumor promoter-induced oxidative stress. These results are consistent with the commonly mentioned idea that tea prevents cancer because tea polyphenols are antioxidants. The pharmacokinetics of tea catechins have been studied in detail in humans and rodents. At present no suitable formulation has been devised to estimate the oral bioavailability in humans.
Several factors affect the bioavailability of tea polyphenols such as gastrointestinal degradation/metabolism, poor membrane permeability, and transporter-mediated intestinal secretion/efflux. Currently, nanotechnology is being utilized in various ways to treat cancer, including molecular imaging, early detection, targeted therapy, and cancer bioinformatics. Cancer related nano-devices include, but are not limited to, injectable nanovectors, such as liposomes; biologically targeted, nanosized magnetic resonance imaging contrast agents; and novel, nanoparticles-based methods. Over the last two decades, several researches have been carried out on a variety of nanoparticles, such as gelatin ceramic, liposomes, and micelles, for therapeutic use. For drug delivery, the core of the nanoparticles is loaded with the drug. Permeation and visibility are also used based on the intended applications. The surface can either be bare or conjugated to targeting ligands like polyethylene glycol, aptamer or antibody to prevent macrophage uptake of the nanoparticles.
In the recent years, nanotechnology has emerged as an attractive alternative method for the efficient drug delivery. The various advantages of using nanoparticles for drug delivery are: the particles are ultra small and their sizes can be controlled. The nanoparticles have large surface area to mass ratio which leads to improved drug efficacy and high reactivity. The nanoparticles can be used specifically for targeted drug delivery and this minimizes the side effects of the drug use. In this study we are using solid-lipid nanoparticles (SLNs). They have controllable pharmacokinetic parameters and can be engineered with three types of hydrophobic core designs: a homogenous matrix, a drug-enriched shell, or a drug-enriched core. The objectives of this project are: Preparation of SLN nanoparticles loaded with tea catechins. Drug release studies at in vitro conditions. Quantification of catechins and Anti oxidant assays: DPPH.
Materials required for SLN preparation
Polyoxyethylene(40) stearate(S-40), Polyoxamer188( S-68),Glycerol mono stearate chemicals were purchased from Sigma Aldrich, India. Hexane, Acetone, Ethanol solvants were purchased from Qualigens.
Preparation of Solid Lipid Nanoparticles:
In a beaker, 70mg of S-40 poly oxyethylene stearate and 30mg of F-68 poloxamer were weighed, and this was kept in the water bath at temperature 80°C for melting. To this clear oil phase, 5ml of GMS (25 mg) was added and slowly stirred. 1ml of MilliQ water of same temperature was added slowly and stirred until it completely turned into transparent. To this clear mixture, 100 µg/ml Tea catechins was added and stirred constantly. Thermodynamically stable W/O micro emulsion was formed. The W/O micro emulsion, was then dispersed in 5ml cold 0.8% S-40 aqueous solution (2-4 °C). Ultra sonication was carried out for 2 hours. The dispersion containing nanoparticles was then stored under appropriate conditions for further use.
Drug release studies for SLN:
Dialysis bags were soaked overnight in PBS buffer. The bags were taken and 5ml of SLN dispersion was poured in dialysis bags. The ends of the bags were sealed with clamps. In a 100ml conical flask, 50 ml of PBS (pH 7.4) buffer (dissolution medium) was taken; the sealed dialysis bags containing the SLN were soaked in the PB and kept on a shaker. From the flask, 1ml of the dissolution medium was pipetted out and fresh buffer was added. At regular timings (30mins, 1hr,2hr,3hr,4,5,6,7,8,12,24 and 48) samples were taken and fresh buffer was introduced immediately. The samples were collected and labelled and were later used to quantify the amount of catechin present using the vanillin reagent method.
Quantification of catechin (released from SLN) :
To prepare 70% sulphuric acid solution, 70ml of concentrated sulphuric acid was taken and the volume was made up to 100ml by adding 30ml MilliQ water. This was placed in ice bath (Ice cold solution). To this ice cold sulphuric acid solution, 1 gram of vanillin was added and dissolved. This is vanillin reagent.In another beaker, 0.1g of catechins dissolved in 100ml of MilliQ. This is standard catechin solution. From the above solution 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0,7ml, 0.8ml,0.9ml, 1.0ml were taken and these were made up to 1ml with MilliQ. 0.5ml of sample from each of the above test tubes were taken. To this, 1.6ml of vanillin reagent and 0.4ml of MilliQ is added. This was incubated for a period of 15 minutes. Spectrophotometric readings were taken at 500nm and recorded. Graphs were plotted to analyze results.
Anti oxidant Assay by DPPH Method:
To prepare sodium acetate buffer of pH 5.5, 35.7ml of glacial acetic acid solution (290 µl in 50 ml MilliQ) and 64.3ml of sodium acetate solution (1.36 g in 100ml MilliQ) was mixed. To 40ml of this sodium acetate buffer, 60ml of methanol is added. This is buffered methanol. To make DPPH working solution (0.1mM), 5ml of 1mM DPPH solution is taken and to that 45 ml of buffered methanol is added. From this DPPH working solution, 1ml is taken in a test tube. To this, 0.5 ml supernatant (from SLN release studies) is added and this is incubated at room temperature in a dark place for 30mins. Spectrophotometric studies are carried out at 517 nm.
Atomic force microscopy results:
Fig.2 Sample SLN:
The AFM used for obtaining the images of the SLN sample was operated in a static mode.
The tip voltage used was 0nV, with an error range of 14µm. From the Fig.2, we see that the
image size for first sample is 3.3µm. The data shows the size and morphology of the particle with the type of solvent used, i.e. water or acetone. The image scans obtained from the atomic force microscopy showed properly spaced individual particles in the nanometer range. It was verified that clumping and aggregation of particles was absent, which meant that the procedure followed for nanoparticle synthesis was appropriate and yielded the desired outcome.
Table 1. Vanillin reagent Method for Standard:
O.D at 500.nm
Fig.3 Standard curve of Vanillin method.
Table 2. Vanillin reagent Method for Released catechins:
Fig.4 Graph showing release studies using vanillin method
DPPH for Standard:
Fig.5 Graph showing DPPH for standard
Table 3. DPPH assay for Released Catechins:
% of Inhibition
Fig.6 Graph showing the DPPH assay for the samples.
The loaded SLN particles were studied for the rate of release of the catechin by using dialysis over a period of 8 hours and an interval of 30 minutes for sample extraction. The amount of sample used in the dialysis membrane was 5ml. The estimation of the released catechin was done using the vanillin reagent assay. The reliability of results was ensured by conducting the assay for a set of triplicates for each sample. The graph was plotted for O.D. versus concentration for the standard. The O.D. values for samples were estimated and used to determine the corresponding concentration of catechins released, using the graphs. As observed from the graph drawn between O.D. and the concentration of the catechins released, the sample which was collected at 5th hour showed maximum release of catechins, and 2nd and 1st hour samples also showed a good amount of catechin concentration. Whereas the last sample collected at 8th hour showed a least concentration of catechins released. A low standard deviation was obtained from which it can be inferred that release was consistent and uniform. The anti-oxidant study of catechin loaded SLN particles was carried out using DDPH. The readings were taken with respect to a negative control because the DPPH reaction with catechins reduces the DPPH radical and makes the strong purple colour of the radical reduce in intensity. Hence the nanoparticle treated DPPH solutions lose colour intensity facilitating a quantitative assay at 517 nm, to measure the radical scavenging anti-oxidant potential of the gelatine based nanoparticles loaded with catechins. On analysing the samples, which were collected at 2nd 3rd and 12th hours, good %inhibition was observed. We find that the percentage inhibition of radicals for SLN samples range from 4.289 to 27.366%. This suggests that the SLN particles have lower percentages of inhibition than the unloaded catechin. However, the variation in the range of the %inhibition values indicates that by changing the synthesis parameters like the solvent, the temperature, the surfactant ratio etc., we can change the anti-oxidant activity of the SLN as well, and modify it as the requirements of the drugs change.
Nanoparticles are being used extensively in the drug delivery systems for treatment and
therapeutic purposes. Hence the main objective of this project was to study the effects of loading a tea polyphenol, catechin into a solid lipid nanoparticle thereby achieving a nanoscale compound. The post synthesis studies of the samples obtained suggest that the drug releases at a uniform rate from the loaded SLN's. Also, the antioxidant properties of catechin were retained even after being loaded inside the SLN which implies that these particles if made compatible to inject in-vivo can be used for treatment of cancer.