Visitors   Views   Downloads

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

View preprint
RT @Dome_TLA: My very first preprint has been published today in @PeerJPreprints https://t.co/Ua79s1vtby ! I am so excited and happy :-) #B…
29 days ago
My very first preprint has been published today in @PeerJPreprints https://t.co/Ua79s1vtby ! I am so excited and happy :-) #Bioengineering #Biotechnology #nanoparticles #microfluidics
NOT PEER-REVIEWED
"PeerJ Preprints" is a venue for early communication or feedback before peer review. Data may be preliminary.

Supplemental Information

CO’s chemical structure.

Calculated molecular weight: 3081.07 Da

DOI: 10.7287/peerj.preprints.27669v1/supp-2

CON’s chemical structure.

Calculated molecular weight: 3235.24 Da

DOI: 10.7287/peerj.preprints.27669v1/supp-3

LA’s chemical structure and MALDI mass spectrum.

Measured in positive mode. Calculated molecular weight: 2929.16 Da. Mass found: 2930.73 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-4

LAE’s chemical structure and MALDI mass spectrum.

Measured in positive mode. Calculated molecular weight: 3187.39 Da. Mass found: 3181.00 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-5

DF’s chemical structure and MALDI mass spectrum

Measured in positive mode. Calculated molecular weight: 884.95 Da. Mass found: 881.89 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-6

DP3F’s chemical structure and MALDI mass spectrum.

Measured in positive mode. Calculated molecular weight: 1187.32 Da. Mass found: 1183.42 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-7

DP12F’s chemical structure and MALDI mass spectrum.

Measured in positive mode. Calculated molecular weight: 1484.67 Da. Mass found: 1479.96 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-8

DP24F’s chemical structure.

Calculated molecular weight: 2013.30 Da.

DOI: 10.7287/peerj.preprints.27669v1/supp-9

DP24F’s chemical structure.

Calculated molecular weight: 3141.65 Da

DOI: 10.7287/peerj.preprints.27669v1/supp-10

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-11

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-12

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-13

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-14

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-15

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-16

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-17

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-18

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-19

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.

DOI: 10.7287/peerj.preprints.27669v1/supp-20

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).

DOI: 10.7287/peerj.preprints.27669v1/supp-21

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).

DOI: 10.7287/peerj.preprints.27669v1/supp-22

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).

DOI: 10.7287/peerj.preprints.27669v1/supp-23

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).

DOI: 10.7287/peerj.preprints.27669v1/supp-24

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Dominik M Loy conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, performed the computation work, authored or reviewed drafts of the paper, approved the final draft.

Philipp M Klein conceived and designed the experiments, contributed reagents/materials/analysis tools, approved the final draft.

Rafał Krzysztoń conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Ulrich Lächelt conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.

Joachim O Rädler authored or reviewed drafts of the paper, approved the final draft.

Ernst Wagner conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.

Data Deposition

The following information was supplied regarding data availability:

Data is available at Figshare,

https://figshare.com/s/24e1ceb3254c87357df6

Funding

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) SFB1032 (projects B1 Rädler and B4 Wagner), and the Cluster of Excellence Nanosystems Initiative Munich (NIM). Rafał Krzysztoń was supported by German Research Foundation (DFG) through the Graduate School of Quantitative Biosciences Munich (QBM) [GSC 1006]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Add your feedback

Before adding feedback, consider if it can be asked as a question instead, and if so then use the Question tab. Pointing out typos is fine, but authors are encouraged to accept only substantially helpful feedback.

Some Markdown syntax is allowed: _italic_ **bold** ^superscript^ ~subscript~ %%blockquote%% [link text](link URL)
 
By posting this you agree to PeerJ's commenting policies