Preparation and Characterization of Solid Lipid Nanoparticles-Based Gel for Topical Delivery
Abstract
Solid lipid nanoparticles (SLNs) have been extensively investigated for effective delivery of both hydrophilic and lipophilic drugs by topical route. There are several scalable techniques for the preparation of SLNs such as homogenization, microemulsion template, and solvent emulsification diffusion. This chapter describes step-wise methodology for the preparation and characterization of SLNs using solvent emulsification diffusion method. Tretinoin, a lipophilic entity, was chosen as a model drug. The critical aspects and the important interpretations with respect to the preparation and characterization of SLNs are reported in “Notes” section.
Key words Solvent-emulsification diffusion, Solid lipid nanoparticles, Tretinoin, Topical route
1 Introduction
Solid lipid nanoparticles (SLNs) are submicron colloidal nanocar- riers (50–1000 nm) comprising of the drug either encapsulated or in matrix form with lipid particles. SLNs have witnessed a global regulatory acceptance due to their high safety profile and are hence preferred as an alternative to polymeric nanoparticles for the deliv- ery of the lipophilic and hydrophilic drugs. SLNs have potential applications in drug delivery as they exhibit several advantages such as drug loading of both lipophilic and hydrophilic moieties, improved stability, biodegradability, ease in scale-up, and cost- effectiveness. Till date, several methods are described in the litera- ture for SLN preparation, including high-pressure homogeniza- tion, i.e., hot homogenization [1, 2] and cold homogenization [3], microemulsion template technique [4, 5], melt dispersion technique [6], ultrasonication technique [7, 8], double emulsion technique [9, 10], solvent emulsification-evaporation technique [11], solvent emulsification-diffusion technique [12, 13]. Herein, we are discussing, solvent emulsification-diffusion tech- nique for preparation of solid lipid nanoparticles [14]. Solvent emulsification-diffusion technique was mainly used for polymeric nanocarriers, however, its use for fabrication of solid lipid nanopar- ticle was first explored by Trotta et al. [15]. Solvent emulsification- diffusion technique is an easily scalable technique, requires less physical stress and ensures loading of both hydrophilic and lipo- philic drugs. The preparation of SLNs using the solvent emulsification-diffusion technique involves preparation of a solvent-in-water emulsion using a “partially” water-miscible sol- vent containing the lipid in rational amounts. Upon transferring the transient oil-in-water emulsion into water, lipophilic material dissolved in the organic solvent solidifies instantaneously due to diffusion of the organic solvent from the droplets to the continuous phase [15]. A schematic representation of the solvent emulsification-diffusion technique is depicted in Fig. 1 and detailed preparation and its characterization protocol are discussed in the following section.
2 Material
2.1 Preparation of Solid Lipid Nanoparticles
1. Drug: Tretinoin (see Note 1).
2. Solid lipid: Glyceryl monostearate; Compritol 888 ATO®; Dynasan 116® and Cutina CBS® (see Note 2).
3. Surfactants/Stabilizer: Epikuron 200®; Tween 20 and Tween 80 (see Note 3).
4. Partial water miscible solvents: Benzyl alcohol (see Note 4).
5. Preservative: Methyl paraben, Propyl paraben.
6. Chelating agent: Disodium EDTA.
7. Antioxidant: Butylated hydroxytoluene.
8. Distilled water or Milli-Q reagent grade water.
9. Water shaker bath with controlled temperature.
10. Controlled temperature water bath.
11. Cyclomixer.
2.2 Characterization of Solid Lipid Nanoparticle
2.2.1 Particle Size Analysis
2.2.2 Entrapment Efficiency
2.2.3 Morphological Studies Using Scanning Electron Microscopy
2.3 Preparation of Gel
1. Particle size analyzer (Photon Correlation Spectroscopy; Beck- man Coulter N4 plus, Wipro, India) (see Note 5).
2. To dilute the sample: water (Milli Q®).
1. Ultrafilter: Nanosep®, (MWCO100KD) (see Note 6).
2. Centrifuge (Eltek TC 4100, Mumbai, India) for separation of encapsulated and unencapsulated drug.
3. UV-spectrophotometric method (Shimadzu UV-1650, Shimadzu Analytical Pvt. Ltd. India) for analysis of filtrate.
1. Cameca SU-SEM probe (resolution: upto 40oA; magnifica- tion: upto 40,000×; accelerating voltage: upto 30 kV; fully integrated EDS/WDS system).
2. SLN dispersion for analysis.
1. Gelling polymer: Carbopol Ultrez 10®, Carbopol® 940, and Carbopol® ETD 2020.
2. pH modifier: Triethanolamine.
2.4 Evaluation of Gel
2.4.1 Drug Content
1. Methanol to dissolve formulation component (see Note 7).
2. UV -spectrophotometric method (Shimadzu UV-1650, Shi- madzu Analytical Pvt. Ltd. India) for analysis of dissolved formulation.
2.4.2 Spreadability 1. TA-XT Texture Analyzer (Stable Micro Systems, New Delhi, India) comprising spreadability study related component such as heavy duty base plate, male cone, female cone, load cell 5 kg.2. SLN dispersion gel for analysis.
2.4.3 Rheological Studies
2.4.4 In Vitro Permeation Studies
1. Brookefield Synchro-Lectric Viscometer (Model RVT) with helipath stand.
2. SLN dispersion gel for analysis.
1. Franz diffusion cell cells with a surface of 3.14 cm2 and a receptor volume of 10 ml.
2. Permeation medium: pH 7.4 buffer (see Note 8).
3. HPLC system (Jasco PU-2080 Plus Intelligent (Jasco, Japan) equipped with a Jasco UV-2075 Intelligent UV/vis detector (Jasco, Japan), a Rheodyne 7725 injector (Rheodyne, USA), a Jasco Borwin Chromatography Software (version 1.50) inte- grator software and a Hi-Q-Sil C (4.6 mm × 250 mm and 10 μm particle size) column. Mobile phase: methanol: acetoni- trile: pH 6.8 phosphate buffer (65:20:15, v/v) at a flow rate of 1.2 ml/min.
3 Method
3.1 Screening of Lipid
The lipids are selected on the basis of their drug solubilizing capac- ity. Solubility of drug in lipid is of paramount importance as it governs loading of drug in the formulation. The commonly employed method for solubility determination is equilibrium solu- bility studies. However, the same cannot be employed for lipids. Thus, the method proposed by our research group [16, 17] was utilized.
1. Weigh and add 10 mg of drug individually in screw-capped tubes (see Note 9).
2. Heat the solid lipid (glyceryl monostearate/compritol 888 ATO/dynasan 116/cutina CBS) above its melting point.
3. Add the molten lipid gradually in a tube containing drug under continuous stirring with aid of a cyclomixer.
4. Inspect visually the amount of lipid required to solubilize the drug in a molten state and identify the solid lipid that solubi- lizes the highest amount of drug.
3.2 Screening of Surfactant
The surfactants are screened for their emulsification capacity. The screening method is as below; 1. Weigh and add drug and screened lipid in a capped tube con- taining partially miscible solvent (water-saturated benzylalcohol maintained at 55 0.5 ◦C) (see Notes 10 and 11). Vortex it for 3 s to achieve a homogenous organic phase (see Note 9).
2. Weigh and add surfactant (Tween 80) in a tube containing Milli Q® water (benzyl alcohol-saturated water maintained at 55 0.5 ◦C) (see Note 12). Vortex it for 3 s to achieve a homogeneous aqueous phase (see Note 9).
3. Gradually add an aqueous phase into an organic phase using an overhead stirrer at 850 × g for 2 min to form the primary emulsion.
4. Precipitate the drug-loaded solid lipid nanoparticles by adding the preformed emulsion into an aqueous phase containing a mixture of surfactant (Tween 80 and Tween 20) maintained at 55 0.5 ◦C and continuously stir for 20 min using an overhead stirrer to extract the benzyl alcohol into the continuous phase.
5. The surfactant which gives stable emulsion with smaller particle size will be considered for preparation of SLN.
3.3 Preparation of Solid Lipid Nanoparticle (SLN) System Using Solvent Emulsification- Diffusion (SED) Method
1. Weigh and add lipid (glyceryl monostearate), surfactant (Epi- kuron 200), drug, and anti-oxidant (butylated hydroxy tolu- ene) in a capped tube containing 2 g of partially miscible solvent (water saturated benzyl alcohol maintained at 55 0.5 ◦C) (see Notes 10 and 11). Vortex it for 3 s to achieve a homogenous organic phase (see Note 9).
2. Weigh and add 200 mg of surfactant (Tween 80), preservative (methyl paraben, propyl paraben) in a tube containing Milli Q® water (benzyl alcohol-saturated water maintained at 55 0.5 ◦C) (see Note 12). Vortex it for 3 s to achieve a homogeneous aqueous phase (see Note 9).
3. Gradually add aqueous phase into an organic phase using an overhead stirrer at 3000 rpm for 2 min to form the primary emulsion.
4. Precipitate the drug-loaded solid lipid nanoparticles by adding the preformed emulsion into an aqueous phase containing a mixture of surfactant (Tween 80 and Tween 20) maintained at 55 0.5 ◦C and continuously stir for 20 min using an overhead stirrer to extract the benzyl alcohol into the continuous phase.
5. No further purification will be required for removal of benzyl alcohol and surfactant from SLN dispersion if the amount present in the dispersion is within the acceptable limits for topical formulation (see Note 13).
3.4 Characterization of SLN Dispersion
3.4.1 Particle Size Analysis
1. To study the particle size of SLN dispersion, dilute the SLN dispersion with water (Milli Q®) (see Note 14).
2. Add the diluted sample in the cuvette and measure the particle size and polydispersion by photon correlation spectroscopy (PCS) using Zetasizer Nano Series or Brookhaven Instruments standard setup (see Note 15).
3.4.2 Entrapment Efficiency
The entrapment efficiency (EE), which corresponds to the percent- age of drug encapsulated within and adsorbed on to the nanopar- ticles can be determined by measuring the concentration of free drug in the dispersion medium.
1. To determine total drug content, dissolve SLN dispersion by adding 1 ml of methanol and filter the solution using 0.22 μm membrane. Measure the concentration of drug using the vali- dated UV-spectroscopic method.
2. To determine the encapsulation efficiency, add SLN dispersion (approximately 500 μl) to the Nanosep® centrifuge tube fitted with an ultrafilter (MW cut-off 100KD) and centrifuge it at 18,650 × g for 40 min at 25 0.5 ◦C.
3. Determine the free drug (i.e., drug non-associated with the solid lipid nanoparticles) by measuring the concentration of drug in the supernatant using the validated UV-spectroscopic method.
4. The encapsulated drug in solid lipid nanoparticles (encapsula- tion efficiency, EE) can be calculated from the ratio between the difference of the total and the free drug concentrations (TD and FD, respectively) divided by the total concentration, multiplied by 100 (Eq. 1).
3.4.3 Morphological Studies
3.5 Preparation of SLN Dispersion Gel
To study the morphology of the SLN dispersion, place drops of dispersion over the aluminium grid and completely dry them at ambient temperature, thereby leaving only a thin layer of particles on the grid. Observe the developed gird under scanning electron microscope (magnification: 20,000×; accelerating voltage:20.0 kV) at 25 2 ◦C.
1. Slowly disperse gelling polymers (viz., Carbopol Ultrez 10®, Carbopol® 940 and Carbopol® ETD 2020) in SLN dispersion under rapid overhead stirring and allow to hydrate for 20–30 min (see Note 16).
2. After complete hydration of gelling polymer, add dropwise triethanolamine, a pH modifier till the pH of 7 0.2 is achieved to obtain the SLN-based gel formulation (see Note 17).
3. The gelling polymer which offers compatibility with nanopar- ticulate dispersion and superior texture (feel and spreadability) is finalized for gel formulation.
3.6 Evaluation of Gel
3.6.1 Drug Content
1. Dissolve approximately 1 g of SLN dispersion gel by adding 1 ml of methanol and filter the solution using a 0.22 μm membrane.
2. Analyze the filtrate using Shimadzu UV-1650 PC UV-vis spec- trophotometer managed by Shimadzu UV probe version
2.10 at a wavelength of 344 nm.
3.6.2 Spreadability 1. Perform spreadability study using TA-XT texture analyzer and express the results as firmness (positive force) and stickiness (negative force).
2. Calibrate the instrument for height. The height calibration is required for spreadability study as it ensures the fix-distance between the male cone and female cone, such that a perfect fit can be ensured.
3. To perform calibration, fix female cone in the base plate and male cone to load cell holder. Allow the male cone to move downwards and touch the base of the female cone. Once the base is touched, the male cone reverts to a distance of 25 mm.
4. After calibration of the instrument, place 2.0 g of test com- pound (SLN dispersion gel) in female cone using a curved spatula to ensure no air bubble is entrapped within it.
5. Allow male cone to move downwards with a distance of 23 mm.
6. The force required for the male cone to travel test compound till the base of the female cone is considered as firmness and the force required for a male cone to detach from the gel is consid- ered as stickiness.
3.6.3 Rheological Studies
1. Perform rheological evaluation of SLN dispersion gel using Brookfield Synchro-Lectric Viscometer (Model RVT) using T-C spindle.
2. Place the sample in a beaker and allow to equilibrate for 5 min.
3. Place the spindle in a beaker containing test sample and mea- sure the dial reading at different spindle speed (0.5, 1, 2.5 and 5 rpm).
4. Note the dial reading by lowering the spindle speed from 5 to
0.5 rpm.
5. Viscosity in centipoise can be calculated by multiplication of dial reading with the factor as specified in Brookfield viscome- ter catalogue. The factor value varies with the spindle type.
3.6.4 In Vitro Permeation Studies
1. In vitro skin permeability is best measured using Franz diffu- sion cell comprising of two compartments, i.e., receptor com- partment and donor compartment separated by the permeation barrier (skin) (see Note 18).
2. Perform in vitro skin permeation studies on excised abdominal skin obtained from Wistar rat (Age: 3 months; Weight range: 200–250 g) with prior animal ethical committee permission (see Note 19).
3. Insert magnetic needle in receptor compartment and mount the skin in between receptor and donor compartment.
4. Fill the receptor compartment with 10 ml of modified perme- ation medium (i.e., pH 7.4 phosphate buffer containing albu- min) which simulates the physiological condition and allow to equilibrate for 2 h.
5. Add test compound (SLN dispersion gel) in donor compart- ment (typically 0.1–0.5 g) with curved spatula enabling the gel film to cover the entire skin surface evenly.
6. Cover the diffusion cells with aluminium foil to prevent light exposure as the drug is light sensitive.
7. Remove sample (0.3 ml) of fluid from receptor compartment at
1, 4, 6, 8, and 12 h interval and replace the equivalent amount with fresh permeation medium.
8. Determine the concentration of drug withdrawn from receptor compartment using validated HPLC method (see Note 20).
9. The total quantity of drug that diffuses through to the receptor compartment in time “t” during the steady state and the flux at steady state, Js [μg/(cm2 h)] can be calculated using linear portion of the correlation between the accumulated quantity of drug that diffused through the skin by unit area and time.
4 Notes
1. Both lipophilic and hydrophilic drugs can be delivered using SLN system. Tretinoin is a model drug which exhibits high lipophilicity and poor water solubility [14]. Thus, Tretinoin is an ideal candidate for incorporation into a lipid nanoparticles.
2. The lipid which exhibits highest drug solubility is considered for preparation of SLN. The high solubility will ensure high drug encapsulation within the system [14].
3. The surfactants act as emulsifying agents which slow down the inevitable separation of two phases as well as lower down the particle size. In some cases, one surfactant is not sufficient and may result in coalescence upon standing. Thus, the combina- tion of surfactants is preferred which acts by forming film at the
interface with sufficient viscosity and thereby prevent aggrega- tion upon standing [14, 15].
4. The solvent which exhibits partial miscibility in water is consid- ered for preparation of SLN by the solvent-emulsification dif- fusion method [14, 15].
5. Other nanoparticle analyzers such as Malvern Instruments/ Horiba Scientific should also work, provided they can measure the size range less than 100 nm.
6. Nanoseps® with different cut off are available and can be selected based on particle size of nanoparticles and molecular weight of the drug entity. For example, The nanoparticle with particle size of 30–60 nm and molecular weight in range of 300–600 kDa requires Nanoseps® 100 kDa which exhibits pore size of 10 nm [18].
7. The solvent which completely dissolves the solid lipid nanopar- ticulate system is selected for the preparation of sample for drug content analysis.
8. The pH at which drug exhibits maximum solubility is consid- ered for flux study and the volume of media should be atleast three times to the drug solubilizing capacity. This ensures maintenance of sink condition [19].
9. The tube should be capped tightly to avoid the loss of material during vortexing, and to prevent the evaporation of solvent afterwards.
10. Mutual saturation of water miscible solvent, i.e., benzyl alcohol and water is a very critical step for preparation of stable SLN. Missing this step may result in the generation of microparticles [14].
11. Temperature (55 ◦C) of water saturated benzyl alcohol is ver- y critical, as the solubility of lipid varies with solvent temperature [14].
12. Temperature (55 ◦C) of benzyl alcohol-saturated water is very critical, so as to achieve the equilibrium with the organic phase [14].
13. Purification of SLN dispersion using dialysis is performed if the solvent concentration is not within the acceptable limits [14].
14. The usual dilution will be decided on the basis of instrument parameters. For example, (in Zetasizer the count rate should be in between 150 and 450 kcps). In general, the dilution ranges between 10 and 1000 folds.
15. The diameters usually observed for nanoparticles prepared using preformed polymers range between 10 and 1000 nm. Polydispersity indexes lower than 0.2 indicate homogeneous systems presenting a satisfactory narrow particle distribution.
16. Hydration time may vary with the type of gelling polymer (15 min to 4 h). The complete hydration of polymer is neces- sary to get gel with optimum viscosity.
17. Excess of pH modifier may result in gritty appearance or reduced viscosity of final gel formulation.
18. A variety of other diffusion cells is also available including automated cells with flow-through receptor chambers.
19. Some laboratories use cadaver skin. Skin from at least two donors should be used, with skin from each donor used in equal numbers of cells.
20. For HPLC test, all samples are required to be filtered through a filter membrane (0.2 μM) to avoid blocking of the column.