Comparison of thermally actuated retro-diels-alder release groups for nanoparticle based nucleic acid delivery

Abstract

The present study explores alternate pericyclic chemistries for tethering amine-terminal biomolecules onto silver nanoparticles. Employing the versatile tool of the retro-Diels-Alder (rDA) reaction, three thermally-labile cycloadducts are constructed that cleave at variable temperature ranges. While the reaction between furan and maleimide has widely been reported, the current study also evaluates the reverse reaction kinetics between thiophene-maleimide, and pyrrole-maleimide cycloadducts. Density Functional Theorem (DFT) calculations used to model and plan the experiments, predict energy barriers for the thiophene-maleimide reverse reaction to be greatest, and the pyrrole-maleimide barriers the lowest. Based on the computational analyses, it is projected that the cycloreversion rate would occur slowest with the thiophene, followed by furan, and finally pyrrole would yield the promptest release. These thermally-responsive linkers, characterized by Electrospray Ionization Mass Spectrometry,H and C NMR, are thiol-linked to silver nanoparticles and conjugate single stranded siRNA mimics with 5' fluorescein tag. Second harmonic generation spectroscopy (SHG) and fluorescence spectroscopy are used to measure release and rate of release. The SHG decay constants and fluorescence release profiles obtained for the three rDA reactions confirm the trends obtained from the DFT computations.

Combinatorial Delivery of miRNA‐Nanoparticle Conjugates in Human Adipose Stem Cells for Amplified Osteogenesis

“…Prior to investigating the osteogenic inductive ability of co‐transfecting miR‐21 and miR‐148b, the photothermal release of the miRNA mimic molecules from the surfaces of SNPs and GNPs was initially characterized. While gold nanoparticles were commercially procured, the hydroxypropyl cellulose (HPC)‐stabilized silver nanoparticles were synthesized in house as previously described and characterized . The latter yielded particles with an average diameter size of 75.05 ± 0.65 nm, while the gold nanoparticles' average diameter size was 78.8 ± 2.85 nm.…”

“…With any RNA delivery vehicle, a stable conjugation is crucial to protect the structure and function of therapeutics against opsonization and RNase degradation. Characterization of this system and its stability was described in our previous manuscript . By conjugating to the nanoparticle surface, we can enhance the stability of RNA and shield it from RNAse degradation and lysozyme hydrolysis.…”

“…While there have been several advances made in the design of nanoparticle systems, their release mechanisms are largely assumed or neglected. To design a nanoparticle vehicle with targeted ability to decage and release miRNA mimics, a thermally labile linker, responsive to the nanoparticles' photothermal phenomenon was developed that could cleave and release conjugated miRNA mimics at localized temperatures greater than 60 °C . The covalently bound linker was based on Diels–Alder chemistry which relies on the cycloadduct formation between a diene molecule and dienophile.…”

“…On the maleimide terminal, a hexanoic acid group was added which enabled the covalent crosslinking between carboxyl ends and the 5' amine‐modified miRNA mimics to yield a robust amide bond. At temperatures 60 °C or higher, it was demonstrated that the retro‐Diels–Alder reaction, i.e., the reversal of the cycloadduct, could be triggered . Using photo‐activation, sufficient localized heat was generated to thermally cleave the linker.…”

“…SHG is dipole forbidden in centrosymmetric media, like bulk solution, but can be generated at the surface of nanoparticles where the symmetry is broken. Recently, SHG has been used as an accurate method to determine molecular adsorption and chemical reactions at nanoparticle surfaces, ion‐transport processes in liposomes, and the release of oligonucleotides from the surface of plasmonic nanoparticles …”