2020ResearchNon-viral Gene Delivery

A High-Throughput Screening Platform to Identify Nanocarriers for Efficient Delivery of RNA-Based Therapies

Vitor Francisco, Catarina Rebelo, Artur Filipe Rodrigues, Josephine Blersch, Hugo Fernandes, Lino Ferreira29 September 2026

Summary

Non-viral vectors such as nanoparticles (NPs) show promise for RNA-based therapies, but suffer from limited cell targeting, poor cellular internalization, and inadequate endolysosomal escape capacity. The field lacks a systematic, high-throughput approach to identify effective polymeric nanocarriers that can deliver diverse RNA payloads (coding and non-coding) with spatiotemporal control. ### Nanoparticle Properties | Parameter | Method 1 | Method 2 | |---------------|--------------|--------------| | Average size (DLS) | 310 ± 192 nm | 405 ± 242 nm | | Zeta potential | 13.8 ± 10.1 mV | 12.7 ± 8.6 mV | |.

Keywords

Nanocarriers Polymeric mRNA siRNA Transfection Cellular uptake Nanoparticles

Purpose: Non-viral vectors such as nanoparticles (NPs) show promise for RNA-based therapies, but suffer from limited cell targeting, poor cellular internalization, and inadequate endolysosomal escape capacity. The field lacks a systematic, high-throughput approach to identify effective polymeric nanocarriers that can deliver diverse RNA payloads (coding and non-coding) with spatiotemporal control.

Hypothesis: A high-throughput screening methodology combining light-triggerable nanoparticle libraries with high-content imaging analysis can identify polymeric nanocarrier formulations that efficiently deliver different types of RNA molecules (siRNA, miRNA, mRNA) to specific cell populations, outperforming commercial transfection agents.

Aims: 1. Synthesize two light-responsive poly(amido amine) (PAA) polymer libraries using Michael-type addition, with photolabile groups positioned either as pendant moieties (Method 1) or integrated into the polymer backbone (Method 2) 2. Characterize nanoparticle physicochemical properties (size, zeta potential, light-triggered disassembly efficiency) and RNA complexation capacity for both small (siRNA/miRNA) and large (mRNA) RNA molecules 3. Develop and validate high-content imaging assays to evaluate NP internalization, cytotoxicity, and functional RNA delivery (GFP knockdown for siRNA; Cre recombinase-mediated GFP expression for mRNA) in engineered cell models 4. Benchmark lead formulations against commercial transfection reagents (Lipofectamine 2000, Lipofectamine RNAiMAX) and correlate NP properties with biological activity

Delivery system:

Component: Polymer Chemistry; Description: Poly(amido amine)s (PAAs) synthesized via Michael-type addition of bisacrylamide monomers (A-E: methylene bisacrylamide, hexamethylene bisacrylamide, cystamine bisacrylamide, dihydroxyethylene bisacrylamide, bisacryloyliperazine) with amine monomers (1-32: various diamines, polyamines, and functional amines including piperazine, pyrrolidine, imidazole groups)

Component: Photoresponsive Groups; Description: Method 1: NVOC (4,5-dimethoxy-2-nitrobenzyl chloroformate) attached as pendant side-chain (20% substitution) Method 2: P1 [(2-nitro-1,3-phenylene)bis(methylene)diacrylate] incorporated into polymer backbone (25% incorporation)

Component: Nanoparticle Formation; Description: Nanoprecipitation in water with zinc sulfate (1.0 M) added to induce NP assembly; size: 310 ± 192 nm (Method 1) and 405 ± 242 nm (Method 2); zeta potential: 13.8 ± 10.1 mV and 12.7 ± 8.6 mV respectively

Component: RNA Payloads; Description: • Small RNAs: siRNA (targeting GFP), miRNA, siRNA-Cy5 (fluorescently labeled for tracking) • Large RNAs: Cre recombinase mRNA (~1000 kDa), GFP mRNA

Component: Complexation Strategy; Description: • Small RNAs: Electrostatic surface binding to pre-formed NPs (100 μL NP at 400 μg/mL + 100 μL siRNA at 8 μg/mL, 2h incubation) • Large RNAs: Polyplex formation (polymer + mRNA mixed directly at various w/w ratios, 10 min complexation)

Component: Release Mechanism; Description: UV light-triggered (365 nm, 10 min, 100 mW/cm² for characterization; 1 mW/cm² for cell studies) photolysis of o-nitrobenzyl groups → o-nitrobenzaldehyde, causing NP disassembly and cytoplasmic RNA release

Component: Targeting; Description: Cell type-specific delivery based on NP composition; no active targeting ligands described

Approach:

Parameter: Cell Models; Details: • HeLa-GFP cells (CellBiolabs) — stable GFP expression for siRNA-mediated knockdown assays • Cre reporter fibroblasts (SC-1 cells with LoxP-flanked GFP cassette) — Cre mRNA delivery results in permanent GFP expression

Parameter: Culture Conditions; Details: DMEM with 10% FBS, 0.5% PenStrep; seeding density: 4000 cells/well (HeLa-GFP) or 3000 cells/well (fibroblasts) in 96-well plates

Parameter: Transfection Protocol; Details: • siRNA: 10 min transfection with NPs (20 μg/mL) pre-complexed with siRNA; medium replaced with 5% FBS + blasticidin (10 μg/mL); UV activation for 10 min immediately after transfection • mRNA: 4 h transfection with polyplexes (5 μg polymer/well, 50 ng mRNA/well); medium replaced with complete DMEM; analyzed at 48h

Parameter: Controls; Details: • Positive: Lipofectamine RNAiMAX® (siRNA) and Lipofectamine 2000® (mRNA) per manufacturer's protocols • Negative: Untreated cells; NPs without light activation; NPs without RNA

Parameter: Experimental Replicates; Details: Three independent experiments (n=3) for transfection studies

Parameter: Assay Readouts; Details: High-content imaging at 48h post-transfection: cell viability (Hoechst + PI staining), NP internalization (Cy5 fluorescence), GFP knockdown (HeLa-GFP cells), or Cre-mediated recombination (GFP expression in fibroblasts)

Parameter: Validation Techniques; Details: Flow cytometry, Western blot, RT-qPCR for selected formulations

Key methods:

Analysis Category: Nanoparticle Characterization; Methods: • Dynamic light scattering (DLS) — size, zeta potential, NP count • UV irradiation (365 nm, 10 min) — light-triggered disassembly (NP count decrease)

Analysis Category: RNA Complexation; Methods: • Cy5-tagged siRNA: fluorescence quantification after centrifugation (supernatant = non-complexed RNA) • mRNA: agarose gel retardation assay at various w/w ratios

Analysis Category: Cell-Based High-Content Imaging; Methods: • IN Cell Analyzer 2200 automated fluorescence microscope • Program A (HeLa-GFP): Nuclear segmentation (Hoechst), PI staining for dead cells, cytoplasmic GFP intensity quantification • Program B (Cre fibroblasts): Multi-target analysis for GFP-expressing cells; nuclear condensation removal for viability

Analysis Category: Molecular Biology Validation; Methods: • Flow cytometry: BD Accuri C6, PI staining, GFP fluorescence • Western blot: Anti-Cre recombinase, anti-GFP, anti-β-tubulin antibodies • RT-qPCR: SYBR Green, ΔΔCt method, GAPDH normalization; Cre, GFP, GAPDH primer sequences provided

Analysis Category: Statistical Analysis; Methods: GraphPad Prism (version 6.0h); results presented as Mean ± SEM

Key results: ### Nanoparticle Properties

Parameter: Average size (DLS); Method 1: 310 ± 192 nm; Method 2: 405 ± 242 nm

Parameter: Zeta potential; Method 1: 13.8 ± 10.1 mV; Method 2: 12.7 ± 8.6 mV

Parameter: Light-triggered disassembly (>50% count decrease); Method 1: 52.5% of formulations; Method 2: 82.7% of formulations

Parameter: siRNA complexation efficiency; Method 1: Slightly better than Method 2; Method 2: Variable, some low-zeta potential NPs complexed ~100%

siRNA Delivery Outcomes: - Cellular uptake not solely charge-dependent: Some low zeta potential formulations (e.g., E1, A1) showed high internalization, while some high zeta potential NPs (e.g., A15, P1D23) showed low uptake — suggesting composition-dependent uptake mechanisms - Limited correlation between NP physicochemical properties (disassembly efficiency, siRNA complexation, cellular uptake) and GFP knockdown activity for top 10 formulations of each library - Cell viability: <5% toxicity after UV exposure or NP disassembly (Fig. 6) - Best formulations outperformed Lipofectamine for GFP knockdown (fold increase shown in Fig. 8)

mRNA Delivery: - Polyplex formation required: mRNA complexation with pre-formed NPs required high w/w ratios (≥1:100 or 1:5 for P1E28/P1C5), while polyplexes showed retardation from 1:1 ratio - Cre mRNA delivery: Successful transfection of Cre reporter fibroblasts resulted in permanent GFP expression; one molecule delivered can produce detectable signal

Benchmarking vs. Conventional Techniques: - High-content imaging advantages: Single-cell resolution, multiplexed readouts (viability + uptake + functional delivery), less labor-intensive than Western blot or qPCR - RT-qPCR limitation: Detects total mRNA regardless of functional delivery (cannot distinguish translated from endosome-trapped mRNA) - Western blot limitation: Bulk protein quantification, time-consuming, no single-cell resolution

Interpretation: The authors conclude that their high-throughput screening platform successfully identifies light-triggerable nanocarrier formulations for efficient RNA delivery, with compositional elements (rather than surface charge alone) being the primary determinants of transfection efficiency. They emphasize that intracellular trafficking likely plays a crucial role not captured by simple physicochemical characterization. The methodology is presented as a versatile, scalable approach for developing delivery systems for RNA therapeutics across various disease contexts, with potential applications in cell targeting studies. The synthesis pathways are highlighted as inexpensive, simple to fabricate, and amenable to industrial scale-up.

10. Limitations (Explicitly Stated or Evident):

1. Intracellular trafficking not evaluated: The authors acknowledge that the limited correlation between NP properties and activity suggests intracellular trafficking (not assessed in this study) likely plays a crucial role — requiring future investigation

2. In vitro only: All screening and validation performed in cancer cell lines (HeLa) and engineered fibroblasts; no in vivo validation reported

3. mRNA delivery efficiency: Although demonstrated, the study shows mRNA transfection requires significantly different formulation conditions than siRNA (polyplex vs. surface binding, longer transfection time), indicating complexity for universal platforms

4. Endpoint timing: All assays measured at 48h; no time-course studies to assess RNA release kinetics or duration of effect

5. Library scale: Two libraries of ~110-160 polymers each — while high-throughput relative to conventional approaches, still represents a limited fraction of possible polymer space

6. Cell type specificity: Only two cell lines tested; targeting to primary cells or in vivo tissues not demonstrated

7. No toxicity profiling beyond viability: Limited assessment of immunogenicity, inflammatory responses, or off-target effects

8. UV light penetration limitations: Clinical translation of light-triggered systems may be constrained by tissue penetration of UV light

Report prepared based on the provided manuscript content. For full experimental details and supplementary information, please refer to the original publication.

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A High-Throughput Screening Platform to Identify Nanocarriers for Efficient Delivery of RNA-Based Therapies

Summary

Keywords

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