Research paperHigh loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: Quality by design optimization and characterization
Graphical abstract
The loading of PLGA nanoparticles with siRNA was optimized using design of experiments and the particles provided an siRNA burst release followed by a triphasic sustained release.
Introduction
The biodegradable and biocompatible polymer poly(dl-lactide-co-glycolide acid) (PLGA) has been used for decades for the delivery of a variety of drug molecules including small molecular weight compounds, peptides and proteins [1], [2]. It is approved for human use by the Food and Drug Administration (FDA), and several PLGA-based formulations have received worldwide marketing approval [3], [4]. The polymer has also caught interest for the delivery of nucleic acids like small interfering RNA (siRNA), and there is an increasing enthusiasm for exploring matrix systems like PLGA-based particles for siRNA delivery as an alternative to commonly used cationic and/or non-biodegradable gene delivery systems to avoid toxicity associated with these [5], [6]. In particular, PLGA nanoparticles have attracted much attention since they are assumed to meet the criteria required for successful siRNA delivery: (i) They are sufficiently small for efficient tissue penetration and cellular uptake, (ii) the siRNA can be entrapped into the PLGA matrix, which offers physical protection against RNase activity as well as a favorable colloidal stability of the system, and (iii) the PLGA particles generally possess an excellent controllable and alterable degradation profile and thus drug release span from days to years depending on the molecular weight, the composition of the block copolymer and the structure of the nanoparticles [6].
Previous reports have shown that PLGA nanoparticles are safe and efficient carriers for the delivery of plasmid DNA [7], [8]. However, from a technical point of view, it is difficult to load smaller nucleic acid molecules like siRNA into PLGA nanoparticles obtaining a high loading and encapsulation efficiency with the use of the classical preparation methods like the double emulsion solvent evaporation method. As other low molecular weight compounds, siRNA easily leaks from the inner water phase into the outer water phase during preparation due to its relatively low molecular weight, its hydrophilic character and the electrostatic repulsion forces present between the phosphate groups in the siRNA backbone and the anionic acid groups in the PLGA polymer. As a result, siRNA is usually incorporated into the PLGA nanoparticles with very low encapsulation efficiency and a limited loading percent, which represents a significant bottleneck for the further development of PLGA nanoparticles for siRNA delivery.
A common strategy to increase the loading is to complex siRNA with cationic excipients like dioleyltrimethylammoniumpropane (DOTAP) [9], polyethyleneimine (PEI) [10] or polyamines [11] to enhance the affinity between the siRNA and the particle matrix, thereby increasing the loading percent and the encapsulation efficiency of siRNA. However, such cationic excipients are often associated with toxicity and can retard the release of siRNA. It is therefore relevant to optimize the loading by other means and of ultimate importance to systematically investigate the effects of formulation and process parameters on the siRNA encapsulation efficiency into PLGA nanoparticles, since it has been shown that certain formulation and process variables significantly affect the encapsulation efficiency of macromolecular drugs into PLGA particles [12], [13], [14].
Design of experiments (DOE) is an efficient method for evaluating the effects of formulation and process parameters on response variables, and for subsequent optimization of these parameters with respect to the final specifications [15], [16]. Therefore, DOE is commonly adopted in pharmaceutical research since it provides the possibility of obtaining maximal information from a minimal number of experiments.
In the current study, siRNA-loaded PLGA nanoparticles were prepared by the double emulsion solvent evaporation technique [17]. The formulation and preparation process parameters were rationally optimized by a thorough understanding of the effect of various parameters including (i) the volume ratio between the inner water phase and the oil phase, (ii) the PLGA concentration, (iii) the sonication time for the primary emulsification, (iv) the siRNA load and (v) the amount of acetylated bovine serum albumin (Ac-BSA) added to the inner water phase to stabilize the primary emulsion. The experiments were planned by DOE, and parameters were optimized with respect to high siRNA loading percent and encapsulation efficiency without compromising the particle size. The optimal formulations were used to study the in vitro release behavior of siRNA from the PLGA nanoparticles, the surface composition of the nanoparticles by time-of-flight secondary ion mass spectrometry (ToF-SIMS), the protection achieved against nucleases, and finally the biological activity of the encapsulated siRNA.
Section snippets
Materials
Enhanced green fluorescent protein (EGFP) and firefly luciferase (FLuc) Dicer substrate asymmetric duplex siRNAs were provided by Integrated DNA Technologies BVBA (Leuven, Belgium) as dried, annealed, purified and desalted duplexes. Sequences were as follows: EGFP, sense 5′-pACCCUGAAGUUCAUCUGCACCACcg-3′, antisense 5′-CGGUGGUGCAGAUGAACUUCAGGGUCA-3′, FLuc sense 5′-pGGUUCCUGGAACAAUUGCUUUUAca-3′, antisense 5′-UGUAAAAGCAAUUGUUCCAGGAACCAG-3′ [10]. Lower case letters are 2′-deoxyribonucleotides and
Optimization of the siRNA-loaded PLGA nanoparticle formulation
The purpose of the FFD was to identify parameters that have a significant effect on the analyzed response variables. The choice of the five process parameters was based on previous experiments [17]. The response parameters were the siRNA encapsulation efficiency and the particle size.
Discussion
A successful nanoparticulate delivery system should, apart from possessing a favorable safety profile, have a high loading capacity in order to reduce the quantity of both the drug and the excipient material to be used for therapy. This is in particular important for costly biopharmaceuticals like siRNA. As the main validation parameter in the present study was the encapsulation efficiency of the siRNA, focus was on the process variables relevant for the formation and stabilization of the first
Conclusion
This study demonstrates that it is possible to increase the encapsulation efficiency without the use of cationic co-excipients to more than 60–70% of biologically active siRNA by the choice of optimized formulation parameters without compromising the particle size and the negative particle zeta potential. By statistical design of experiments, the most important parameters were found to be the concentration of PLGA and the w1/o phase ratio. In addition, the co-excipient Ac-BSA seems to have a
Acknowledgements
We are grateful to Maria Læssøe Pedersen and Stefania Baldursdottier for their excellent technical assistance. Brian Sproat is kindly acknowledged for the synthesis of the siRNA. We thank the Alfred Benzon Foundation (D.C.), the Danish Research Council for Technology and Production Sciences Grant No. 26-02-0019 (D.M.K.J.), Drug Research Academy and the Danish Agency for Science, Technology and Innovation for financial support. In addition, this paper has been carried out with financial support
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These authors contributed equally.
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Present address: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.