Pharmacological characterization of chemically synthesized monomeric phi29 pRNA nanoparticles for systemic delivery

Abstract

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Figure 1

The structure and characterization of chemically synthesized bipartite packaging RNA (pRNA) (P1/P2). (a) The structure of a typical monomeric pRNA nanoparticle consisting of P1 and P2 RNAs. Folate and Alexa647 can be optionally conjugated to the 5′-ends of both P1 and P2. (b) Native PAGE analysis of P1, P2, unligated P1/P2, and ligated P1/P2. (c). Dimerization of pRNA-Ab′/pRNA-Ba′, as analyzed by TBM native PAGE as described previously.

Figure 2

The packaging RNA (pRNA) nanoparticle did not induce an interferon response in vitro. (a) KB cells were transfected with pRNA (50 nmol/l) (2′-F modified versus nonmodified), small interfering RNA (siRNA) (50 nmol/l), and polyinosinic:polycytidylic acid (poly I:C) 1 (µg/ml) using Fugene-HD. The RNAs were harvested 24 hours later for semiquantitative reverse transcriptase (RT)-PCR analysis for the interferon (IFN) responsive genes expressions. (b) Human peripheral blood mononuclear cell (PBMC) were incubated with 50 nmol/l pRNA or 1 µg/ml poly I:C. TLR-3, 7, and 9 gene expression were analyzed as in a. KT-107 and KT-108 are pRNA monomers that differ in the sequence of the 5′/3′ helical region. (c) Production of tumor necrosis factor-α (TNF-α) in the mouse macrophage cell line RAW-647 after incubation with different concentrations of pRNA and poly I:C. Three hours postincubation, the culture media were analyzed for TNF-α by enzyme-linked immunosorbent assay (ELISA). (d) Activation of Toll-like receptor (TLR)-3 pathway by pRNAs were measured using HEK-Blue-hTLR3 reporter cell line. pRNA differing in the length (29 nucleotides or 22 nucleotides) and the extent of modifications (FF, 2′-F modified helical region; NN, nonmodified helical region; all pRNA constructs used had a modified intermolecular interaction domain) in the 5′/3′ region were tested. After 24 hours incubation, the media were analyzed for TLR-3 activation. Poly I:C was used in all these assays as positive control.

Figure 3

The folate-packaging RNA (pRNA) nanoparticle targets FR⁺ tumors upon systemic administration. (a) HeLa xenograft tumor-bearing nude mice were injected with 15 nmol (~24 mg/kg) of KT-105 (Folate-AlexaFluor647-labeled pRNA nanoparticle) through the tail-vein (right). The control mice were injected with either phosphate-buffered saline (PBS) (left) or with folate (intraperitoneal (i.p.), 10 mg/kg) 10 minutes before KT-105 injection (middle). The mice were euthanized 24 hours after injection and whole-body imaging was conducted using IVIS Lumina station. (b) Following whole-body imaging, the mice were dissected and the major organ were isolated for imaging. H, heart; I, intestine; K, kidney; L, lung; Lv, liver; M, muscle; S, spleen; T, tumor. (c) KB xenograft tumor-bearing nude mice were injected with 15 nmol (24 mg/kg) (lower panel) or 3.75 nmol (upper panel) (~6 mg/kg) of KT-105 through the tail vein. And organ were isolated and imaged as described above.

Figure 4

Plasma concentration of packaging RNA (pRNA) nanoparticle upon systemic administration. Tumor-bearing mice (n = 3) were intravenously (i.v.) injected with 15 nmol of KT-105 (Folate-AlexaFluor647 pRNA nanoparticle). Blood was collected at different time points postadministration (5 minutes, 30 minutes, 2 hours, 5 hours, and 24 hours) through the lateral saphenous vein. The serum was isolated and the serum concentration of the nanoparticle was determined using capillary gel electrophoresis (CGE). The semi-log plot of the plasma concentration versus time is shown.

Figure 5

White blood cells counts in mice after packaging RNA (pRNA) nanoparticles injection. Clinical pathology analysis was conducted for the C57B/6 mice upon 1-week repeat intravenous (i.v.)-administrations of pRNA versus polyinosinic:polycytidylic acid (poly I:C) at the indicated doses. Total cell counts (left) and differentials (right) were displayed.