2009 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2009), October 4-6, 2009, Kuala Lumpur, Malaysia

The Effect of Preparation Parameters on the Size and Morphology of PLGA-Based Nanoparticles

*J. Sameni, **N.I. Bukhari, *N.A. Azlan, *T. Julianto, *A.B.A. Majeed

*Faculty of Pharmacy, Universiti Teknologi Mara, Malaysia **Faculty of Pharmacy, International Medical University, Bukit Jalil, Malaysia

Email: javadsameni@salam.uitm.edu.my Abstract-Nanoparticles now have various pharmaceutical and biomedical applications due to the many advantages such as improvement in drug bioavailability, ability to cross barriers after oral and parentral administration, and being an excellent drug carrier for insoluble drugs. However, size and morphological characteristics of nanoparticles are critical for drug release in the body, warranting understanding the parameters controlling the above characteristics. This paper describes, firstly, preparation parameters to optimize the size of polylactic-co-glycolic acid (PLGA)-based nanoparticles, and secondly, study the morphology of the same nanoparticles, formed by the emulsion solvent evaporation technique. The solvent evaporation technique involves an aqueous phase containing polyvinyl alcohol (PVA) as a stabilizing agent. After washing, the samples were freeze-dried and analyzed using particle size analyzer and scanning electron microscope. The preparation parameters studied included; homogenizing speed, homogenizing time, stabilizer concentration and temperature. Keywords: PLGA, Nanoparticles Preparation parameters.

I. INTRODUCTION Micro and nanoparticulate formulations have been

extensively explored in designing controlled release and sustained drug delivery systems. These particulate systems have various advantages compared to conventional dosage forms [1].

A well-engineered controlled drug delivery system provides release pattern resulting in an optimal drug concentration-time profile at the site of action and thus, improves therapeutic effects [2]. Controlled drug delivery systems may also reduce the need for frequent administrations thus increase patient’s compliance. Several nanoparticle systems are in use for controlling the drug release rate and in ensuring complete erosion to avoid the removal of empty remnants [3].

The fabrication of nanoparticles requires the use of polymeric materials possessing appropriate characteristics. Biocompatible and biodegradable polymers are preferred. Polylactic-co-glycolic acid (PLGA) is biodegradable and biocompatible polymer. Safety coupled with commercial

availability of PLGA in several monomer ratios and molecular weights have made it a preferred material for wide variety of drugs ranging from small-molecular-weight therapeutic agent to peptide hormones, antibiotics and chemotherapeutic drugs [4]. PLGA micro and nanoparticles [5, 6] have proven to be successful drug delivery systems for different classes of drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), anticancer drugs, peptides and steroid hormones.

In this study, the emulsion solvent evaporation technique was used which involved two different phases; an organic phase consisting of dichloromethane (solvent) and PLGA (polymer), and an aqueous phase which included distilled water and polyvinyl alcohol (PVA) (stabilizer).

Variables influencing the encapsulation process and the final nanoparticles include: (i) nature and solubility of drug being encapsulated; (ii) polymer concentration, composition and molecular weight; (iii) drug/polymer ratio; (iv) organic solvent; (v) concentration and nature of the stabilizer; (vi) temperature; (vii) stirring/agitation speed during emulsification process and; (viii) viscosity and volume ratio of the dispersed and continuous phase. However, the focus of the current study was on four factors, namely homogenizing time, homogenization speed, temperature during emulsification process and concentration of the stabilizer [7].

Homogenizing is one of the processes in preparing nanoparticles. This process generates droplets of the drug/matrix dispersion in the continuous phase for subsequent solvent removal. The homogenizer speed was the main parameter for controlling the drug/matrix dispersion droplet size in the continuous phase. Increasing the mixing speed generally resulted in decreased particles mean size, as it produced smaller emulsion droplets through stronger shear forces and increased turbulences [8]. The size of droplets is inversely related to the magnitude of shear stresses [9]. Therefore, increasing the energy density directly increases the shear stresses and results in more efficient droplet breakdown and hence a reduction of droplets size.

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2009 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2009), October 4-6, 2009, Kuala Lumpur, Malaysia

A surfactant such as PVA is generally added to the continuous phase to prevent coalescence of the drug dispersion droplets. Particle size was reduced with increase in stabilizer concentration [8].

Temperature of nanodispersion during homogenization controls the rate of volatile solvent removal from the solidifying nanospheres. Higher temperature facilitates the evaporation of the solvent from the continuous phase and thereby maintains a high concentration gradient for the solvent between the nanospheres and the continuous phase. The PLGA particles tend to be larger when prepared at higher temperature (38ºC), showing a wider size distribution and with lower particle density compared to those prepared at lower temperature (4ºC) [8].

In this study, the effect of homogenizing time, agitation and temperature of the emulsification process and stabilizer concentration were investigated on the size, particle size distribution and morphological characteristics of the prepared nanoparticles.

II. MATERIALS AND METHODOLOGY

A. Materials Polylactic-co-glycolic acid (PLGA) was purchased from

Boehringer Ingelheim (Germany) with ratio of 50:50. PVA (88%) and dichloromethane (DCM) was bought from Acros Organic (USA).

B. Preparation of PLGA nanoparticles

Batches of PLGA nanoparticles were prepared in triplicate by using oil in water (O/W) emulsion solvent evaporation technique. Different concentrations of stabilizer (PVA) were dissolved in 10 ml of distilled water and then stirred homogeneously to allow dissolution process. To 2 ml of DCM, 100 mg of PLGA was added. This solution was then poured into beakers containing different concentrations of the PVA solution (0, 0.2, 0.5, 0.7, 0.9, 1, 2.5 and 5%) separately. The mixture was homogenized at different homogenizing speeds (11000, 13000, 16000, and 22000 rpm). Homogenizing time was varied form 0.5, 1, 2 and 3 minutes. Then, 100 ml of distilled water (at 2ºC, 15ºC and room temperature) was added to each beaker, while stirring to allow particles to harden. The mixtures were stirred for 2 hours using magnetic stirrer for solvent evaporation. The resulting nanoparticles were collected by centrifugation (4000 rpm, 5 min), washed with distilled water (3 times) and then dried using freeze dryer. C. Particle size analysis

The size and size distribution of the particles were analyzed using Zetasizer Nano Series (Malvern Instruments Limited, UK). The suspension containing the sample was pipette into a low volume disposable sizing cuvette and analyzed using Zetasizer.

D. Scanning electron microscopy The size and morphological characteristics of the

nanoparticles were observed by using scanning electron microscope (SEM). The sample was mounted on the specimen stubs using double-sided adhesive tape, platinum-coated (10 nm) for 90 seconds using Auto Fine Coater (JFC 1600, Japan) and observed under SEM. E. Yield Nanoparticles dried using freeze-dryer were then weighed and the yield of particle preparation was calculated using the following formula:

III. RESULTS AND DISCUSSION The effect of homogenizing speed, stabilizer

concentration, homogenizing time and temperature on particle size, particle size distribution andmorphology of PLGA nanoparticles were investigated in this study and is discussed separately. A. Influence of homogenizing speed

Batches of nanoparticles using PLGA were prepared at 11000, 13000, 16000 and 22000 rpm. Increasing the mixing speed generally results in decreased particles mean size as it produces emulsion droplets through stronger shear forces and increased turbulence [8]. As the speed and/or duration of homogenization increases, the energy causing the droplet breakdown also increases resulting in decreased particle mean size.

Figure 1 indicates that homogenizing speed influences the particle size and polydispersity index (PDI). As expected, both particle size and PDI decrease as the homogenizing speed increases.

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Figure 1. The effect of homogenizing speed on mean size and PDI

of PLGA nanoparticles

Figure 2 shows SEM pictures of PLGA nanoparticles, captured at 5000x magnification using 10.0 kV. The nanoparticles prepared at 2°C with 1% of stabilizer concentration at the highest homogenizing speed produced are the smallest and with the lowest PDI, indicating the best

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2009 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2009), October 4-6, 2009, Kuala Lumpur, Malaysia

dispersion. When particles were prepared without using homogenizer (speed 0), very low amount of particles were obtained (Fig. 2E).

Figure 2. SEM images of PLGA nanoparticles with different homogenizing speed. A) 11000 rpm B) 13000 rpm, C) 16000 rpm D) 22000 rpm and E) 0 rpm B. Influence of stabilizer concentration

Figure 3 shows reduction in the particle mean size up to 824 nm when the stabilizer concentration was increased from 0 to 1%.

Smaller particles have a higher total interfacial area compared to the large particles, thus they require a higher concentration of the stabilizer. Therefore, the addition of higher stabilizer concentration to the solution results in increased particles size and PDI. An increase [10] and decrease [11, 12] in size of PLGA nanoparticles at high PVA concentration have been reported. These contradictory findings were clarified by Budhian who proposed two competing effects at high PVA concentration [9]. The size decreases due to enhanced interfacial stabilization while it increases due to increased viscosity of the aqueous phase.

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Figure 3. The effect of stabilizer concentration on the mean size and PDI of PLGA nanoparticles.

Scanning electron microscopy images are compared to

study the influence of PVA concentration on the particle size. Figure 4 shows PLGA nanoparticles prepared at 2°C and 22000 rpm with different PVA concentrations. The PLGA nanoparticles were larger when little or higher than 1% of PVA concentration was used.

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Figure 4. SEM images of PLGA nanoparticles at different PVA concentration. A) 0.5 % B) 0.9 % C) 1 % and D) 5%.

C. Influence of temperature

The PLGA nanoparticles tended to be larger when prepared at higher temperature with higher PDI and decreased particle density compared to those prepared at lower temperature (4ºC) [8]. In line with the above findings, the present study showed a larger particle size (870 nm) at room temperature while smaller size at 2ºC with a mean diameter of 824 nm.

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2009 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2009), October 4-6, 2009, Kuala Lumpur, Malaysia

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Figure 5. The effect of temperature on the mean size and PDI of

PLGA nanoparticles. SEM images (Fig 6) showed that PLGA nanoparticles

were spherical in shape with a smooth surface at different temperatures. Particle size and PDI was lower when the preparation temperature was reduced to 2ºC.

Figure 6. SEM images of samples with different temperature. (A) at 2ºC (B) at 15°C and (C) at room temperature. D. Influence of duration of homogenization

Short homogenizing time yields coarse particles due to less magnitude of shear stress applied. While at longer time, the increase in the energy density directly increases the shear stresses and results in more efficient droplet breakdown and hence a reduction of droplets size which eventually produces smaller particle size.

E. Yield

Figure 8 shows the yield of nanoparticles at the various PVA concentrations. The maximum yield of nanoparticles was obtained at 5% of PVA concentration. Nanoparticles had a yield of 50% at 1% concentration of PVA. The particles had low yield because of washing three times. The

observed decrease in the yield with decreasing PVA concentration could be due to the lower PVA density in the solution as well.

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Figure 7. The effect of homogenizer time on the mean size and PDI of

PLGA nanoparticles.

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Figure 8. Yield of the PLGA nanoparticles

In this study, only very small fraction of nanoparticles was obtained with 0% of PVA concentration. This shows that stabilizer is important in preparing particles and the PVA concentration can be manipulated to produce ideal nanoparticles with the desired size

IV. CONCLUSION

The homogenizing speed, homogenizing time, stabilizer concentration and temperature have been shown to influence the particles size, size distribution and surface morphology of PLGA nanoparticles. Increased homogenizing speed promoted particle size reduction due to higher shear stress applied.

Similarly, at higher PVA concentration, the size of nanoparticles was reduced. Temperature proportionally affected the size of nanoparticles, at higher temperature the nanoparticles size was larger than those produced at lower temperature. Thus, it is useful to consider the variables in formulating polymeric drug delivery system to ensure the desired characteristics of nanoparticles are achieved.

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2009 IEEE Symposium on Industrial Electronics and Applications (ISIEA 2009), October 4-6, 2009, Kuala Lumpur, Malaysia

ACKNOWLEDGMENT The authors wish to thank Ministry of Science,

Technology and Innovation, Malaysia for financial support under Science Fund project 02-01-01-SF0219. We also thank Mr. Karim of Microscopy Unit, Faculty of Pharmacy UiTM for helping and guiding us in completing our analysis using the scanning electron microscope.

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