Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury

Abstract

In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.

Figure 1

GPC traces for PtBA 1 and PtBA-b-P(ODA-co-DA) 2.

Figure 2

(A) UV-vis spectra of iNOS PNA-SCK conjugates, (B). UV-vis spectroscopy of iNOS PNA-SCK-TAT conjugates.

Figure 3

TEM image of SCKs 9.

Figure 4

Dynabead competition assay to determine iNOS mRNA binding affinity of SCK-PNA conjugates. A) Biotinylated iNOS mRNA (10 pM) bound to streptavidin coated Dynabeads were incubated with 1 nM 5′-radiolabeled 240 or 480 antisense ODN and increasing concentrations of the corresponding SCK-PNA conjugate. After incubation for 4 h, the bound versus free fractions were separated by a magnet and the radioactivity in each fraction was quantified by liquid scintillation counting. B) Plots of the average fraction of mRNA bound by radioactively labeled ODN vs. the SCK-PNA conjugates from three experiments fit to the analytical expression 2) described in the experimental section from which the IC₅₀ was extracted. The data in Table 3 was obtained from averaging fits to each independent experiment (see supporting information).

Scheme 1

Synthesis of amphiphilic functional block graft copolymer, 7.

Scheme 2

Self assembly of the multi-functional block graft copolymer 7 into micelle 8, crosslinking into SCK 9, conjugation of Alexa Fluor 633 hydrazide 10, and functionalization with PNAs and TAT to give the final bio-synthetic hybrid nanostructure 13.