Comparison of Modular PEG Incorporation Strategies for Stabilization of Peptide–siRNA Nanocomplexes

Abstract

Nanoparticulate systems have shown great promise in overcoming the considerable trafficking barriers associated with systemic nucleic acid delivery, which must be addressed in order to unlock the full potential of technologies such as RNAi and gene editing in vivo. In addition to mediating the cytoplasmic delivery of nucleic cargo and shielding it from nuclease degradation and immunostimulation, nucleic acid-containing nanomaterials delivered intravenously must also be stable in the bloodstream after administration in order to avoid toxicity and off-target delivery. To this end, the hydrophilic molecule polyethylene glycol (PEG) has been deployed in many different nanoparticle systems to prevent aggregation and recognition by the reticuloendothelial system. However, the optimal strategy for incorporating PEG into self-assembled nucleic acid delivery systems to obtain nanoparticle stability while retaining important functions such as receptor targeting and cargo activity remains unclear. In this work, we develop substantially improved formulations of tumor-penetrating nanocomplexes (TPNs), targeted self-assembled nanoparticles formulated with peptides and siRNA that have been shown to mitigate tumor burden in an orthotopic model of ovarian cancer. We specifically sought to tailor TPNs for intravenous delivery by systematically comparing formulations with three different classes of modular PEG incorporation, namely PEG graft polymers, PEG lipids, and PEGylated peptide – each synthesized using straightforward bioconjugation techniques. We found that addition of PEG lipids or PEGylated peptide carriers led to formation of small and stable nanoparticles, but only nanoparticles formulated with PEGylated peptide carriers retained substantial activity in a gene silencing assay. In vivo, this formulation significantly decreased accumulation in off-target organs and improved initial availability in circulation, compared to the original non-PEGylated particles. Thus, from amongst a set of candidate strategies, we identified TPNs with admixed PEGylated peptide carriers as the optimal formulation for systemic administration of siRNA, based on their performance in a battery of physicochemical and biological assays. Moreover, this optimized formulation confers pharmacologic advantages that may enable further translational development of tumor-penetrating nanocomplexes, highlighting the preclinical value of comparing formulation strategies, and the relevance of this systematic approach for the development of other self-assembled nanomaterials.

Nanoparticle delivery of immunostimulatory oligonucleotides enhances response to checkpoint inhibitor therapeutics

“…This effect allows us to overcome delivery challenges common in many studies of anticancer therapeutics, including immunostimulatory agents, without being limited to direct intratumoral administration. PEGylated formulations of these peptides were synthesized as previously described (49) Poly(I:C) comprises short strands of polyinosine homopolymer annealed to short strands of polycytidine homopolymer, with an average size from 0.2 kb to 1 kb.…”

“…Our goal was to build nanoparticle systems that have immunomodulatory activity. We drew inspiration from previous work in our group using tandem peptides comprised of an N-terminal myristoyl coupled to transportan, a cell penetrating peptide (48), and one of an array of C-terminal homing domains to form nanocomplexes with oligonucleotides (39,40,49). Based on this prior work, we hypothesized that we could form similar nanocomplexes with oligonucleotide-based immunostimulatory agents (Fig.…”

Personalized RNA Medicine for Pancreatic Cancer

“…mirVana miRNA inhibitor against hsa-miR-21-5p and nontargeting controls were obtained from Thermo Fisher Scientific. PEGylation method of the tandem peptide was described previously (49).To form the TPNs in solution, miRNA inhibitor, PEGylated pTP-iRGD, and pTP-iRGD were resuspended in nuclease-free water and mixed with the miRNA inhibitor, PEGcontaining component, and peptide in 1:2.5:15 molar ratios, first by thoroughly mixing the miRNA with the PEGylated pTP-iRGD and by subsequently mixing in the peptide to create a concentrated solution of TPNs that were adjusted to the desired dilution and buffer composition with appropriate diluent. Binding of iRGD-TAMRA on cells was done at 4 C on ice.…”

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes

“…Based on a tandem peptide nanocomplex system, [52][53][54][55][56][57][58] previously developed by our lab to achieve targeted delivery of siRNA to tumors, we hypothesized a simple and modular delivery approach, CRISPR-GPS (Guiding Peptide Sequences). We discovered that it can co-deliver native or modified sgRNA and Cas9 protein to various cell types in vitro with comparable efficiency as lipofectamine® RNAiMAX, and may also offer the capacity to target specific cell types in vivo.…”