A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

Abstract

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid's transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed "oligomers") with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed "PEG-ligands") as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azidealkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core-lipid anchor-PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to n core oligomer ) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core-PEG-ligand polyplexes formed by click modification of How to cite this article Loy DM, Klein PM, Krzysztoń R, Lächelt U, Rädler JO, Wagner E. 2019. A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship.

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

“…These postcleavage workarounds are necessary because DBCO undergoes an inactivating rearrangement under the strongly acidic conditions, 95% trifluoroacetic acid (TFA), used for peptide deprotection and cleavage from the resin. ,,, This rearrangement has been demonstrated in DBCO and related biarylazacyclooctynones and is thought to result from an acid-catalyzed 5- endo - dig cycloisomerization. , Longer peptides are unlikely to have a single primary amine or thiol for postcleavage couplings, so DBCO needs to be incorporated during Fmoc-SPPS. While DBCO can withstand lower TFA concentrations (<30%), ,, most commonly used protecting groups and resins require higher TFA for efficient removal.…”

“…The finding that (MeCN) 4 CuBF 4 prevents acid-catalyzed 5- endo - dig cycloisomerization in DBCO, and perhaps other alkyne-derived isomerization reactions, is an important observation. For example, several groups have reported synthetic limitations when working with various strained alkynes due to the 5- endo - dig cycloisomerization, − and protecting these strained alkynes with (MeCN) 4 CuBF 4 should increase the number of synthetic strategies available. This (MeCN) 4 CuBF 4 protection may also be effective in protecting DBCO peptides after resin cleavage and ether precipitation, as suggested by the 50 equiv (MeCN) 4 CuBF 4 data shown in Figure C.…”

Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

“…Additionally, impact on mitochondrial activity was measured in MTT assays (see Figure S4, Supplementary Material). As discussed in other work, forming nanoparticles by microfluidics leads to an increased control over the production process which in turn generates decreased hydrodynamic diameters and narrow dispersity [38,42,43]. Loy et al produced well-defined and reproducible polyplexes with controlled surface characteristics with the help of microfluidic a self-assembly based on electrostatic interaction [44].…”

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

“…For example, several groups have reported synthetic limitations when working with various strained alkynes due to the 5-endo-dig cycloisomerization, [82][83][84][85][86] and protecting these strained alkynes with (MeCN)4CuBF4 should increase the number of synthetic strategies available. This (MeCN)4CuBF4 protection may also be effective in protecting DBCO peptides after resin cleavage and ether precipitation, as suggested by the 10 and 50 equiv.…”

“…[83][84] Longer peptides are unlikely to have a single primary amine or thiol for post-cleavage couplings, so DBCO needs to be incorporated during Fmoc-SPPS. While DBCO can withstand lower TFA concentrations (<30%), 82,[85][86] most commonly used protecting groups and resins require higher TFA for efficient removal. We set out to investigate the stability of DBCO in standard TFA cleavage conditions using the model peptides H-C(StBu)GK(DBCO)ENTWY-R (R = NH2 1a, OH 1b, or NHNH2 1c; see Table S1 for all peptide sequences) and Ac-RRRYSTEVEK(DBCO)NV-NHNH2 2a (DBCO coupled to Lys side chains and Cys protected with StBu to prevent potential thiol-yne reactions).…”