A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship
- Published
- Accepted
- Subject Areas
- Bioengineering, Biophysics, Biotechnology
- Keywords
- Gene silencing, siRNA delivery, Nanoparticle, Polyplex, Microfluidics, Folate receptor targeting
- Copyright
- © 2019 Loy et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship. PeerJ Preprints 7:e27669v1 https://doi.org/10.7287/peerj.preprints.27669v1
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 PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne 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 ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. 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 azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.
Author Comment
This is a submission to PeerJ Materials Science for review.
Supplemental Information
CO’s chemical structure.
Calculated molecular weight: 3081.07 Da
CON’s chemical structure.
Calculated molecular weight: 3235.24 Da
LA’s chemical structure and MALDI mass spectrum.
Measured in positive mode. Calculated molecular weight: 2929.16 Da. Mass found: 2930.73 Da.
LAE’s chemical structure and MALDI mass spectrum.
Measured in positive mode. Calculated molecular weight: 3187.39 Da. Mass found: 3181.00 Da.
DF’s chemical structure and MALDI mass spectrum
Measured in positive mode. Calculated molecular weight: 884.95 Da. Mass found: 881.89 Da.
DP3F’s chemical structure and MALDI mass spectrum.
Measured in positive mode. Calculated molecular weight: 1187.32 Da. Mass found: 1183.42 Da.
DP12F’s chemical structure and MALDI mass spectrum.
Measured in positive mode. Calculated molecular weight: 1484.67 Da. Mass found: 1479.96 Da.
DP24F’s chemical structure.
Calculated molecular weight: 2013.30 Da.
DP24F’s chemical structure.
Calculated molecular weight: 3141.65 Da
Channel design of the single meander channel.
Circles on the left represent inlets, a circle on the right an outlet. Liquids are pumped from left to right. The inserts a, b, and c present the details of the regions marked with squares in the channel sketch.
Channel design of the double meander channel.
Circles represent inlets, except the circle on the bottom of the left side, which is an outlet. Liquids are pumped from top left to bottom left. The inserts a, b, and c present the details of the regions marked with squares in the channel sketch.
DLS data of core (CO + siRNA) – lipid anchor polyplexes produced by bulk mixing.
The mixing order is denoted on the x-axis. At first, the components written in the first line were mixed. Second, the third component was added to the mixture by rapid pipetting. Whenever CO and siRNA were mixed together, was it in the first or second step, the mixture was incubated for 45 in before the next step. Shape and color indicate which lipid anchor was used: Blue circle: LA, orange cube: LAE. A: Mean hydrodynamic diameter (z-average). B: Mean polydispersity index (pdi). C: Mean zeta potential, measured in HBG pH 7.4. Statistics: A, B: Error bars correspond to 95 % confidence intervals. C: Error bars correspond to mean zeta deviations. N = 3.
DLS data of core (CO + siRNA) polyplexes produced at a T-junction.
siRNA is dissolved in HBG pH 7.4, CO is dissolved either in HBG pH 7.4 (panels 1 and 2) or HBG pH 7.4 with 50 % [v/v] acetone (panels 3 and 4). Subfigures are divided into four panels. Panel “hand”: Bulk mixed polyplexes for comparison. Remaining panels: Depict the remaining amount of acetone in the final formulation. For panels 2 and 4, solutions were pumped at equal flow rates, while solutions depicted in panel 3 were pumped with a flow rate (FR) ratio of 1:10 (CO:siRNA). A: Mean hydrodynamic diameter (z-average). B: Mean polydispersity index (pdi). Statistics: Error bars correspond to 95 % confidence intervals. N = 3.
Automated production of core (CO + siRNA) – lipid anchor – PEG-ligand polyplexes.
Solvent with or without acetone, double meander channel. First junction: CO:siRNA = 1:10, siRNA in HBG pH 7.4, CO in HBG pH 7.4 ± 50 % acetone. 1.0 ml/h total flow rate. Second junction: Core polylex:lipid anchor oligomer (±PEG-ligand) = 1:11, lipid anchor or lipid anchor – PEG-ligand oligomer in HBG pH 7.4 ± 50 % acetone, 1.1 ml/h total flow rate. Formulation key: core: core polyplex, LA/LAE: lipid anchors, Px: total number of ethylene oxide repetitions (in LA/LAE + PEG-ligand), F: folic acid. See Fig. 1A for detailed structures A: Mean hydrodynamic diameter (z-average) in nm. B: Mean polydispersity index (pdi). Grey spheres: CO was dissolved in HBG pH 7.4. Blue cubes: CO was dissolved in HBG pH 7.4 with 50 % acetone. Error bars correspond to 95 % confidence intervals; n = 3.
FRET control measurements of core (CO + siRNA) – lipid anchor polyplexes and their components.
Title of each panel indicates dye measured; Color indicates dyes used in this formulation. FRET: excites Atto488 (485 nm), measures Cy5 (680 nm). ‘formulation’ specifies formulation composition (e.g. ‘core + LA’: conventionally prepared core polyplex with 20 mol % LA lipid anchor oligomer added with microfluidics). Cy5 is coupled to siRNA’s sense strand. Atto488 is coupled via azide – alkyne click chemistry to the azide of LA or LAE oligomers. Measured fluorescence is divided by gain’s value to exclude amplifier effects.
Agarose gel shift assay.
Core (CO + siRNA) – lipid anchor (LA/LAE) – PEG-ligand (FolA) polyplexes. Key:Px: total amount of ethylene oxide repetitions (LA/LAE + PEG-ligand). Solvent: 100 % HBG or HBG + 90 % fetal bovine serum (FBS), up to 24 h incubation at 37 °C. Gel: 1 % agarose in 1x TBE buffer with 0.1 % GelRed®. 17 % loading buffer. Runtime: 1 h, 80 V. t: Time from formulation until measurement; t > 0: incubated at 37 °C. First row: 100 % HBG pH 7.4, other rows: 10 % HBG pH 7.4, 90 % FBS.
Ethidium bromide displacement assay ± heparin stress.
Core (CO + siRNA) – lipid anchor (LA/LAE) polyplexes were incubated with ethidium bromide and increase of fluorescence relative to siRNA with dye alone was measured. Resistance to anionic stress was investigated with the addition of heparin. Key: LA: core (CO + siRNA) + 20 mol % LA; LAE: core (CO + siRNA) + 20 mol % LAE; none: core (CO + siRNA) alone. Sample’s mean fluorescence minus the mean fluorescence of the negative control (HBG alone) is reported. Error bars correspond to 95 % confidence intervals; n = 3.
MTT assay of core (CO + siRNA) – lipid anchor (LA/LAE) – PEG-ligand polyplexes.
All polyplexes were prepared using microfluidics. Values are calculated relative to values of buffer treated cells. Colors indicate type of siRNA used: Light color: control siRNA, saturated color: siGFPLuc siRNA. ‘core polyplex’ (green bars): core polyplex formulation used for all subsequent modifications with 20 mol % lipid anchors and lipid anchor-PEG-ligands. Formulation key: P12: core polyplex with unmodified lipid anchor. Px: PEGx, F: Folate. Detailed PEG-ligand description in Fig. 1A. A: Polyplexes with LA lipid anchor (blue bars). B: Polyplexes with LAE lipid anchor (orange bars). Statistics: Error bars correspond to 95 % confidence intervals. N = 5.
Luciferase activity assay of core (CO + siRNA) – lipid anchor – PEG-ligand polyplexes
All polyplexes were prepared using microfluidics. Luciferase enzyme activity is measured in relative light units (RLU) and is shown relative to values of buffer treated cells. siGFPLuc siRNA was used in all samples. Core polyplexes with 20 mol % lipid anchors and lipid anchor - PEG-ligands were used. PEG-ligand Formulation key: P12: core polyplex with unmodified lipid anchor. Px: PEGx, F: Folate. Detailed PEG-ligand description in Fig. 1A. Color key: Blue bars: polyplexes with LA, orange bars: polyplexes with LAE. Statistics: Tips of horizontal lines indicate compared samples. Samples were compared with a two-sided student’s t – test with HOLM correction. N = 5. Key: NS: not significant at α = .05; ***: α < .001. Error bars correspond to 95 % confidence intervals.
Gel shift of core (CO + siRNA) – lipid anchor – PEG-ligand (FolA) polyplexes at t = 0 h
Solvent: with (top) and without (bottom) 90 % FBS in HBG. Order: (left to right) siRNA, Core polyplex (CO + siRNA), core polyplex + lipid anchor (LA), core polyplex + LA + PEG-ligand (DF), core polyplex + LA + PEG-ligand (DP3F), core polyplex + LA + PEG-ligand (DP12F) , core polyplex + LA + PEG-ligand (DP24F), core polyplex + LA + PEG-ligand (DP48F) , core polyplex + LAE, core polyplex + LAE + PEG-ligand (DF) , core polyplex + LAE + PEG-ligand (DP3F) , core polyplex + LAE + PEG-ligand (DP12F) , core polyplex + LAE + PEG-ligand (DP24F) , core polyplex + LAE + PEG-ligand (DP48F).
Gel shift of core (CO + siRNA) – lipid anchor – PEG-ligand (FolA) polyplexes at t = 1 h
Solvent: 90 % FBS in HBG. Incubation temperature: 37 ° C, Order: (left to right) siRNA, Core polyplex (CO + siRNA), core polyplex + lipid anchor (LA), core polyplex + LA + PEG-ligand (DF), core polyplex + LA + PEG-ligand (DP3F), core polyplex + LA + PEG-ligand (DP12F) , core polyplex + LA + PEG-ligand (DP24F), core polyplex + LA + PEG-ligand (DP48F) , core polyplex + LAE, core polyplex + LAE + PEG-ligand (DF) , core polyplex + LAE + PEG-ligand (DP3F) , core polyplex + LAE + PEG-ligand (DP12F) , core polyplex + LAE + PEG-ligand (DP24F) , core polyplex + LAE + PEG-ligand (DP48F).
Gel shift of core (CO + siRNA) – lipid anchor – PEG-ligand (FolA) polyplexes at t = 4 h
Solvent: 90 % FBS in HBG. Incubation temperature: 37 ° C, Order: (left to right) siRNA, Core polyplex (CO + siRNA), core polyplex + lipid anchor (LA), core polyplex + LA + PEG-ligand (DF), core polyplex + LA + PEG-ligand (DP3F), core polyplex + LA + PEG-ligand (DP12F) , core polyplex + LA + PEG-ligand (DP24F), core polyplex + LA + PEG-ligand (DP48F) , core polyplex + LAE, core polyplex + LAE + PEG-ligand (DF) , core polyplex + LAE + PEG-ligand (DP3F) , core polyplex + LAE + PEG-ligand (DP12F) , core polyplex + LAE + PEG-ligand (DP24F) , core polyplex + LAE + PEG-ligand (DP48F).
Gel shift of core (CO + siRNA) – lipid anchor – PEG-ligand (FolA) polyplexes at t = 24 h
Solvent: 90 % FBS in HBG. Incubation temperature: 37 ° C, Order: (left to right) siRNA, Core polyplex (CO + siRNA), core polyplex + lipid anchor (LA), core polyplex + LA + PEG-ligand (DF), core polyplex + LA + PEG-ligand (DP3F), core polyplex + LA + PEG-ligand (DP12F) , core polyplex + LA + PEG-ligand (DP24F), core polyplex + LA + PEG-ligand (DP48F) , core polyplex + LAE, core polyplex + LAE + PEG-ligand (DF) , core polyplex + LAE + PEG-ligand (DP3F) , core polyplex + LAE + PEG-ligand (DP12F) , core polyplex + LAE + PEG-ligand (DP24F) , core polyplex + LAE + PEG-ligand (DP48F).
