Original article
Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design

https://doi.org/10.1016/j.ejmech.2012.01.001Get rights and content

Abstract

The objective of this study was the preparation, physico-chemical characterization and statistical optimization of cationic solid lipid nanoparticles (SLN) prepared by the PIT method as potential carrier for gene therapy, emphasizing the application of factorial design in such a kind of studies. The preliminary screening from a physico-chemical point of view on three cationic lipids (CTAB, DDAB and DOTAP), selected on the basis of their different chemical structure and increasing lipophilicity, allowed us to select SLN with DOTAP, due to its higher zeta potential and smaller particle size. Afterward, a 22 full factorial experimental design was developed in order to study the effects of two independent variables (amount of DOTAP and concentration of lipid matrix) and their interaction on mean particle size and zeta potential values. The factorial planning was validated by ANOVA analysis; the correspondence between the predicted values of size and zeta and those measured experimentally confirmed the validity of the design and the equation applied for its resolution. The factorial design showed a significant influence of the independent variables on the selected parameters; in particular, a higher effect of DOTAP was observed on zeta potential value. Different dilutions of the optimized SLN containing 7% w/w of cutina CP and 1% w/w of DOTAP, with size and zeta potential values respectively of 462.9 nm and 50.8 mV, were in vitro examined to evaluate the possible cytotoxicity on two models of cell cultures: human prostate cancer androgen-non-responsive DU-145 cells and primary cultures of rat astrocytes.

Graphical abstract

Highlights

► PIT method allows to produce cationic SLN using DOTAP as cationic lipid. ► The factorial design demonstrated the significative effect of lipids concentration on size and zeta values. ► SLN with 7% w/w of Cutina CP and 1% w/w of DOTAP has the best properties for gene delivery. ► A lower cytotoxicity on rat astrocytes compared to DU-145 cells was observed.

Introduction

Advances in gene therapy and nanotechnology resulted in the development and application of drug delivery systems as non-viral gene carrier molecules [1], [2]. However, it should be useful to produce positively charged systems able to transport gene material into the cell with a satisfied transfection effectiveness. Important efforts have already been focused on the design of carriers for the transport of DNA or RNA into the cells, such as lipoplexes, polyplexes and cationic lipid nanoparticles (SLN) [3], [4], [5]. Increasing attention has been paid to the use of SLN for gene therapy, due to their widespread technological advantages, including their large scale production and the use of materials that are generally accepted as safe [1]. The different methods applied to prepare SLN can be distinguished in two fundamental groups: high-energy and low energy emulsification processes. High-energy methods, such as high-pressure homogenization and ultrasound technique, involve the use of specific devices such as high-pressure homogenizers or sonifiers, and only a very low amount of the mechanical energy produced is used for emulsification [6]. On the other side, low energy procedures, which include solvent-diffusion technique and the phase inversion temperature (PIT) method, allow to obtain nanoparticles with a minimum energy. In particular, the PIT method makes use of the intrinsic physico-chemical behavior of the formulation, that is precisely the phase transitions induced by changing temperature (PIT) taking place during the emulsification [7], [8]. The aim of the present work was the preparation and statistical optimization of cationic SLN produced by the PIT method as potential gene delivery systems. We want to emphasize the preliminary characterization of the prepared systems from a physico-chemical point of view and the feasibility to apply a full factorial design to achieve the maximum amount of information in such a kind of studies, thus reducing both time and costs. For this reason, a preliminary screening on three cationic lipids (CTAB, DDAB and DOTAP) selected on the basis of their different chemical structure and increasing lipophilicity (Table 1) was developed. The physico-chemical and morphological characterization allowed us to select DOTAP as the best cationic compound among those tested, considering parameters as particle size, polidispersity and zeta potential value. A 22 full factorial design was developed to evaluate the effects of two independent variables (amount of DOTAP and concentration of cetyl palmitate) and their interaction on mean particle size and zeta potential. In addition to traditional experimentation, factorial design, first reported by Box and Wilson [9] is a very useful tool to achieve a suitable mathematical model for the optimization of the formulation parameters, limiting the number of the experiments. The factorial analysis also allows to obtain the maximum amount of information from the experimental data and, moreover, to establish the influence of multiple factors on the formulation properties [10], [11]. The system selected for its zeta potential and mean particle size values, was also studied in order to evaluate its effect on cell viability utilizing two cell models: DU-145, androgen-non-responsive human prostate cancer cells, and primary cultures of rat astrocytes.

Section snippets

Materials

Cetyl palmitate (Cutina CP) was a gift from Cognis S.p.a. Care Chemicals (Como, Italy). Glyceryl Oleate (Tegin O) and Oleth-20 (Brij 98) were purchased from A.C.E.F. S.p.a. (Piacenza, Italy). Cetrimide (CTAB) and Dimethyldioctadecylammonium bromide (DDAB) were provided by Sigma Aldrich (Milan, Italy). N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) was purchased from Genzyme Pharmaceuticals LCC (Swiss).

SLN preparation

All formulations were prepared by the phase inversion temperature

Results and discussion

Before the experimental design was constructed, preliminary formulation studies were performed on three different cationic lipids to verify their ability to determine SLN formation by the PIT method. The influence of the cationic lipid on mean particle size (ZAve), polidispersity index (PDI) and zeta potential (ZP) was also evaluated.

Conclusions

Results of our study show that cationic solid lipid nanoparticles can be successfully prepared by the PIT method as potential gene delivery systems. In accordance with our results, we can emphasize the feasibility to apply the full factorial design as a very useful tool for the statistical optimization of the formulation, achieving the maximum information for the preliminary physico-chemical characterization. The preliminary screening demonstrated that the feasibility to obtain SLN is

References (35)

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    Citation Excerpt :

    ZP increased proportionally as the concentration of the two lipids increased. A study by Carbone et al showed that the magnitude of the ZP is directly proportional to the concentration of lipids used in the preparation (Carbone et al., 2012). The interaction effects of the terms on the ZP are shown in the 3D response surface plots and the 2D contour plots (Fig. 2).

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