Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors

Abstract

Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications.

Fig. 1

Characterization of nanoparticles. (a), (b), and (c): schematics, morphology – as captured by SEM, and size distribution of multifunctional nanoparticles conjugated with a single peptide X (a), two peptides X and Y (b) and three peptides X, Y, and Z (c). (d) Controlled release of siRNA from nanoparticles loaded with siRNA against PLK1. (e) Controlled release of QC and ENX from nanoparticles loaded with siRNA against PLK1. (f) Components of a functional nanoparticle loaded with siRNA against PLK1 and their individual functions.

Fig. 2

Effect of peptides and small molecule enhancers for DNA and siRNA delivery. (a) Sequences for the biotinylated peptides used in screening for the optimal transfection enhancement effect. (b) Conjugation of mTAT and incorporation of TCHD and CQ or QC enhances the gene delivery efficiency of nanoparticles. (c) Transfection efficiency of nanoparticles conjugated with all peptides listed in (a). (d) Combination of two peptides for gene delivery. (e) Incorporation of ENX enhances knockdown efficiency of nanoparticles encapsulated with siRNA.

Fig. 3

Nanoparticles for delivery of siRNA against PLK1 to A549 cells. (a) Conjugation of a single peptide or two peptides enhances gene delivery to A549 cells. (b) Distribution of nanoparticles loading with a fluorescence dye, Coumarin-6, with different surface modifications. (c) The effect of replacing mHph1 with iRGD on gene delivery efficiency. PLGA-PLL and PLGA-PLL-PEG-iRGD were mixed at the ratios indicated for display of the peptide iRGD. (d) Comparison of delivery efficiency and cytotoxicity of siRNA against PLK1, using nanoparticles and RNAiMAX, on A549 cells.

Fig. 4

Systemic delivery of siRNA against PLK1, using nanoparticles, for inhibition of tumor growth in vivo. (a) Antitumor effects of nanoparticles loaded with siRNA against PLK1. (b) Expression of the PLK1 gene in residual tumors. (c) H&E staining of the control tumor (left) and the siPLK1 treated tumor (right); 40× magnification. (d) TUNEL staining of the control tumor (left) and the siPLK1 treated tumor (right); 20× magnification.