Formulation, Characterization and in vitro Evaluation of Methotrexate Solid Lipid Nanoparticles


Jameel Ahmed Mulla*1, Sarasija Suresh2 and Imtiyaz Ahmed Khazi3

1Department of Pharmaceutics, K.L.E. Society’s College of Pharmacy, Hubli, India

2Department of Pharmaceutics, Al-Ameen College of Pharmacy, Bangalore, India

3Department of Chemistry, Karnataka University, Dharwad, India

*Corresponding Author E-mail:



Solid lipid nanoparticles (SLNs) of methotrexate were produced by microemulsion method in an acidic aqueous system. The SLNs were composed of low melting fatty acid (glyceryl monostearate), surfactants (Egg lecithin and tween 80) and water. All the formulations were subjected to particle size analysis, zeta potential, drug entrapment efficiency and in vitro release studies. The SLNs formed were in nano-size range with maximum entrapment efficiency. Formulation with 253 nm in particle size and 85.12% of drug entrapment was subjected to scanning electron microscopy (SEM) for surface mophology, differential scanning calorimetry (DSC) for thermal analysis and short term stability studies. SEM confirms that the SLNs are circular in shape. The drug release behavior from SLN suspension exhibited biphasic pattern with an initial burst and prolonged release over 24 h.


KEYWORDS: Solid lipid nanoparticles (SLNs) Methotrexate (MTx) Particle size analysis Entrapment efficiency In vitro release study




Solid lipid nanoparticles (SLNs), introduced in 1991, to combine advantages and to avoid disadvantages of other colloidal carriers has attracted increased attention in recent years, and is regarded as an alternative carrier system to traditional colloidal systems, such as emulsions, liposomes and polymeric microparticles and nanoparticles1-3. Proposed advantages include, possibility of controlled drug release and drug targeting, increased drug stability, high drug payload, incorporation of lipophilic and hydrophilic drugs feasible, no biotoxicity of the carrier, avoidance of organic solvents, no problems with respect to large scale production and sterilization4.


Many of pharmaceutical researchers have prepared SLNs as an alternative colloidal therapeutic systems, utilizing different approaches like modified high shear homogenization and ultrasound techniques1, emulsification-diffusion method5, solvent injection method6, solvent diffusion method7, microemulsion method8 hot homogenization technique9.


SLN were prepared by the dispersion of warm oil-in-water (o/w) microemulsions in cold water; solid lipids with low melting points were used as internal phase of the microemulsion. Considering the droplet structure as the structural organization of o/w microemulsion, liquid oil nanodroplets are present in the warm o/w microemulsion; a rapid quenching of the warm o/w microemulsion in cold water permits the crystallization of oil nanodroplets present in the microemulsion forming the solid nanospheres10.


Methotrexate (MTx) has a broad range of cytostatic activity, especially when given in high doses with folinic acid rescue. MTx is an antifolate that competively binds to dihydrofolate reductase (DHFR) which inhibits precursors of DNA and RNA and inhibits cell replication. It is an important drug in treatment of an acute lymphoblastic leukemia (ALL), choriocarcinoma, related trophoblastic tumors and psoriasis11. This research paper aims to prepare, characterize and evaluate the methotrexate loaded solid lipid nanoparticles.




Methotrexate (MTx) was provided by Unimed Technologies Ltd (Gujarat, India), glyceryl monostearate (GMS) from Loba Chemie Pvt Ltd (Mumbai, India), tween 80 by Merck Ltd (Mumbai, India), egg lecithin from Himedia (Mumbai, Indi a) and Millipore water by Millipore (India) Pvt. Ltd (Bangalore, India). Other chemicals are of analytical grades.

Table 1: Compositions of Mtx-SLN formulations

Formulation code

Concentration of drug  (mg)


of lipid  (%)

Concentration of surfactants, 1:1 (%)





















Table 2: Particle size, polydispersity index and zeta potential

Formulation code

Average diameter (nm)*

Polydispersity index*

Zeta potential (mV)*













* mean ± SD., n = 3



Solubility of MTx in solid lipid:

One of the most important factors that determine the loading capacity of the drug in the lipid is the solubility of drug in melted lipid. Briefly, 10mg of MTx was taken in wide mouth screw capped bottles. The solid lipid was separately heated above its melting point. This lipid melt was gradually added in portions to MTx with continuous stirring using cyclomixer. The amount of molten lipid required to solubilize the MTx was noted visually. The end point of the solubility study was the formation of clear solution of molten lipid. The solid lipid was selected on the basis of the solubilizing potential and the acceptability by topical, peroral and parenteral route12.


Preparation of SLN:

MTx-SLNs formulations were prepared from a warm o/w microemulsion technique10. Drug and GMS were melted at 70 0C. Warm acidic aqueous solution of egg lecithin was added to melted lipid-drug mixture (at 70 0C) in presence of co-surfactant, tween 80 under stirring. The warm microemulsion was then added carefully drop wise into ice cold water (2-3 0C) with continuous stirring (T25 basic Ultra Turrax, IKA, USA). The ratio between the microemulsion and the dispersion medium was about 1:10. The SLN dispersion was subjected to ultra sonication for a period of 10 min. The SLN dispersions were then lyophilized.


Figure 1: Solubility of MTx in solid lipids

Table 3: Effect of time of storage (at 450C / 75%RH) on particle size and entrapment efficiency

Formulation code

Particle size (nm)*

Entrapment efficiency (%)*


Zero day

One month

Zero day

One month






* mean ± SD., n = 3


Table 4: Effect of sterilization on particle size and entrapment efficiency

Formulation code

Particle size (nm)*

Entrapment efficiency (%)*










* mean ± SD., n = 3


Measurement of size and zeta potential:

Size and zeta potential of SLN were measured by Photon Correlation Spectroscopy (PCS) using zetasizer 3000 HSA (Malvern, U.K.). Samples were diluted appropriately with the aqueous phase of the formulation for the measurements and the pH of diluted samples ranged from 6.8 to 7.0. Zeta potential measurements were done at 25 0C and the electric field strength was around 23.2 V/cm. The zetasizer measures the zeta potential based on the Smoluchowski equation

Where, ζ’ is zeta potential, ‘UE is electrophoretic mobility, η’ is viscosity of the medium and ε’ is dielectric constant.


Determination of drug entrapment efficiency (EE %):

The entrapment efficiency of the drug was determined by measuring the concentration of free drug in the dispersion medium9. The samples were centrifuged at 4000 rpm for 30 minutes. The amount of free drug was determined in the clear supernatant by UV spectrophotometry at 303 nm using supernatant of non-loaded nanoparticles as basic correction. The amount of incorporated drug was determined as a result of the initial drug minus the free drug. The entrapment efficiency could be calculated by the following equation13.


Figure 2: Entrapment efficiency (%) Data expressed as mean ± SD., n = 3

Table 5: Nonlinear fits for MTx released from SLNs




First order, ln( Q0-Q) vs. t

Higuchi Plot, Q vs. √t

First order



y = -7.8534x + 12.271

y = 0.9097x + 0.4606




y = -10.576x + 15.8

y = 0.9115x + 0.2186




y = -15.752x + 24.632

y = 0.9327x - 0.493



Q0 = drug to be released at zero time (mg), Q = amount of drug released at time t (mg), t = time in hours



Figure 3: Scanning electron microscopy of MTx-GMS


Scanning electron microscopy (SEM):

The morphological examination of SLN was performed by Scanning electron microscopy (Joel JSM 840A, Japan). Cleaned brass specimen studs were used for mounting the samples. Wet solvent paint was applied on these studs and while the paint was wet, the pellets were placed on each stud and allowed to dry. The sample was observed by SEM.


Differential scanning calorimetry (DSC):

Differential scanning calorimetry (DSC) analysis of the bulk lipid and nanoparticles was conducted using a differential scanning calorimeter (DSC 60; Shimadzu) set at a heating rate of 100C/min.


Short-term stability study:

The selected SLN formulation was stored at 40ºC / 75% RH in Newtronic Temperature / Humidity Control Chamber QLH-2004, for a period of one month and average particle size and entrapment efficiency were determined.


Effect of sterilization:

To observe the effect of sterilization on particle size and entrapment efficiency, selected SLN formulation was autoclaved at 121 0C for 20 min.


In vitro release study:

In vitro release studies were performed using modified Franz diffusion cells9 having a surface area of 2.545 cm2 and 75 ml of capacity. Dialysis membrane (LA 401) having pore size 2.4 nm, molecular weight cutoff 12000 – 14000 (HIMEDIA), was used. Membrane was soaked in distilled water for 12 hours before mounting in cell. Methotrexate formulation equivalent to 5 mg of drug was placed in the donor compartment and the receptor compartment was filled with dialysis medium (phosphate buffer of pH 7.4, 75ml). The content of the cell was stirred with the help of magnetic stirrer at 37 0C. At fixed time intervals; 1ml of the sample was withdrawn from the receiver compartment through side tube. Fresh phosphate buffer of pH 7.4 was placed to maintain constant volume. Samples were analyzed by UV spectrophotometrically at 303 nm.


Statistical analysis:

Statistical analysis was performed with SPSS 13.0 software package. Results are expressed as the mean±standard deviation (X±SD). Statistically significant differences were determined using the Student’s t-test and analysis of variance (ANOVA) with      p < 0.05 as a minimal level of significance14.


Figure 4: Differential scanning calorimetry of GMS bulk and SLN



Solubility of MTx in solid lipid:

Solubility studies (Fig. 1) indicated that amongst the glyceryl monostearate (GMS), tripalmitin (TRIP), tristearin (TRIS) and trilaurin (TRIL), GMS effectively solubilized MTx. The solubilizing potential coupled with already reported biocompatibility and acceptability of GMS for topical, peroral and parenteral route has favored its selection for the present study15.


Particle size and zeta potential:

The mean particle size, polydispersity index and zeta potential of colloidal carriers are important characteristics of SLNs from which the stability of drug-loaded SLNs can be predicted. Average particles size, polydispersity and zeta potential are shown in Table 2.  All the formulations had shown particle in nanosize range (163-253 nm) with narrow size distribution (polydispersity index = 105-121). Besides production parameters, lipid matrix, surfactant blend and viscosity of lipid and aqueous phase influence the outcome of the procedure. Leaving all other parameters constant, in this study the only variable was composition of lipid matrix varying from 2.5% to 7.5%. Effect of lipid concentration on particle size and entrapment efficiency is recorded in Table 2. The results revealed that increasing the lipid content over 5–7.5% results in larger particle size16. The choice of the emulsifiers and their concentration is of great impact on the quality of the SLN dispersion17. Investigating the influence of the emulsifier concentration on the particle size of SLN dispersions, we obtained best results with 2.5% egg lecithin. High concentrations of the emulsifier reduce the surface tension and facilitate the particle partition. The decrease in particle size is connected with a tremendous increase in surface area. The SLNs are stabilized with surfactant mixtures (egg lecithin/tween 80) to have lower particle sizes and higher storage stability. Use of surfactant mixtures with the aim to have lower particle size are also reported by Siekmann, B., Westesen, K., 1994; and Olbrich, C., Muller, R.H., 1999. The measurement of the zeta potential allows predictions about the stability of colloidal aqueous dispersions19. Usually, particle aggregation is less likely to occur for charged particles with high zeta potential due to electric repulsion20. In general, lipid nanoparticles are negatively charged on the surface21. The determination of zeta potential was performed in aqueous SLN stored at room temperature. The values are shown in Table 2.


Drug entrapment efficiency (EE %):

According to Professor Muller the prerequisite to obtain a sufficient loading capacity was a sufficiently high solubility of the drug in the lipid melt. Relative higher drug EE% was one of the major advantages of SLNs1. Figure 2 shows the drug EE% of MTx-GMS.  The loading capacity of SLN was found to be satisfactorily high. The data showed EE% as high as 85.12% for some formulation. For SLN formulations, the entrapment efficiency is lower for the sample with lower lipid concentration (Fig. 2). It has to be noticed that during the cooling process, the lipid solidifies and the drug is distributed into the shell of the particles, if the concentration of the drug in the melted lipid is well below its saturation solubility22.


Scanning electron microscopy (SEM):

SEM image of the MTx-GMS derived from Joel JSM 840A has been presented in Fig. 3. SEM confirms that the MTx-GMS are circular in shape. They are smooth and well separated on the surface.


Differential scanning calorimetry (DSC):

The thermal curves of GMS bulk lipid and nanoparticles showed endothermic peaks at 60.5 0C and 57 0C respectively (Fig. 4). The melting endothermic peaks of the nanoparticles appeared at slightly lower temperature. The decrease in melting temperature of nanoparticle formulated GMS lipid compared with the bulk has been attributed to their small size and presence of surfactants.


Figure 5: In vitro release of methotrexate from SLNs with different concentration of GMS


Short term Stability study:

After one month storage at 40ºC / 75% RH, it has been found that particle size of MTx-GMS increases by 6nm and entrapment efficiency was lowered by 1.67 % (Table 3). Transitions of dispersed lipid from metastable forms to stable form might occur slowly on storage due to small particle size and the presence of emulsifier that may lead to drug expulsion from solid lipid nanoparticles. Therefore lowered entrapment efficiency observed on storage may be due to expulsion during lipid modification9.


Effect of sterilization:

Effect of sterilization on particle size and entrapment efficiency was shown in Table 4. In selected MTx-GMS formulation, size of particles increases almost two times after sterilization, but still they are in nanosize. It was found that sterilization by autoclaving has least effect on entrapment efficiency. Therefore, sterilization by autoclaving can performed for SLNs of GMS stabilized with lecithin and tween 80.


In vitro release study:

Many research groups used vertical or flow-through Franz diffusion cells and dialysis bag/tubes for the study of drug release from solid lipid and polymeric nanoparticles and niosomes23-30. In order to evaluate the controlled release potential of the investigated formulations, the diffusion of MTx from the lipid particles was investigated over 24 h. Each sample was analyzed in triplicate. The results are shown in Fig. 5. The release rate of MTx depends on the total concentration of MTx in the formulation. MTx is released more quickly when using lower concentration because of the drug-enriched shell model proposed for these particles (MTx-GMS-1). Due to the large drug loading in MTx-GMS-3, the degree of diffusion can be decreased13.


Percentage of methotrexate released from SLNs up to 24 h were in the following order; MTx-GMS-1 (96.2 %), MTx-GMS-2 (91.4 %) and MTx-GMS-3 (78 %).  The release pattern revealed that there was an initially burst effect followed by a prolonged release of drug. This is because the drug may be located primarily in the shell of the particles. Other factors contributing to a fast release are large surface area, high diffusion coefficient (small molecular size), low matrix viscosity and short diffusion distance of the drug. The drug enriched core is surrounded by a drug free lipid shell. Due to the increased diffusional distance and hindering effects by surrounding solid lipid shell, the drug has a sustained release profile.


The formulations were further subjected to release kinetic studies. The release data was fitted into first order and Higuchi equations. Release of drug from almost all the SLNs followed Higuchi equation better than the first order equation (Table 5).



Solid lipid nanoparticles represent a particulate system, which can be produced with an established technique, microemulsion process allowing production on industrial scale. It can be achieved after the selection of optimal formulation and process parameters. The size distribution of SLN revealed a mono-dispersed profile in distilled water. In vitro release of methotrexate from SLNs in the phosphate buffer of pH 7.4, exhibited a biphasic pattern with an initial burst and prolonged release over 24 h.



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Received on 10.02.2009       Modified on 08.04.2009

Accepted on 12.05.2009      © RJPT All right reserved

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 685-689