Journal of the American Chemical Society (JACS)2021ResearchNon-viral Gene Delivery

One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA

Dapeng Zhang, Elena N. Atochina-Vasserman, Devendra S. Maurya, Ning Huang, Qi Xiao, Nathan Ona, Matthew Liu, Hamna Shahnawaz, Houping Ni, Kyunghee Kim, Margaret M. Billingsley, Darrin J. Pochan, Michael J. Mitchell, Drew Weissman, Virgil Percec29 September 2026DOI 10.1021/jacs.1c05813

Summary

Four-component lipid nanoparticles (LNPs) represent the leading non-viral vectors for mRNA delivery, but they have limitations including: (1) segregation of the neutral ionizable lipid as droplets in the LNP core, reducing transfection efficiency to ~1-2%; (2) the "PEG dilemma" where PEGylation increases circulation time but decreases cellular uptake and endosomal escape; and (3) instability at temperatures above -70°C. A one-component system. ### IAJD Libraries & DNP Formation | Parameter | Result | |---------------|------------| | Total IAJDs synthesized | 54 (6 libraries) | | In vitro active DNPs | 44/54 (81%) | | In vivo active DNPs | 31/54 (57%) | | DNPs.

Keywords

mRNA Lipid nanoparticle Transfection Nanoparticles Microfluidics Endosomal escape Cellular uptake

Purpose: Four-component lipid nanoparticles (LNPs) represent the leading non-viral vectors for mRNA delivery, but they have limitations including: (1) segregation of the neutral ionizable lipid as droplets in the LNP core, reducing transfection efficiency to ~1-2%; (2) the "PEG dilemma" where PEGylation increases circulation time but decreases cellular uptake and endosomal escape; and (3) instability at temperatures above -70°C. A one-component system that overcomes these limitations while enabling simple assembly (injection rather than microfluidics) and storage at 5°C is needed.

Hypothesis: Sequence-defined ionizable amphiphilic Janus dendrimers (IAJDs) containing ionizable amines organized in precise sequences can coassemble with mRNA into dendrimersome nanoparticles (DNPs) by simple injection. The sequence and concentration of ionizable amines will determine transfection efficiency, with lower amine concentrations in specific sequences exhibiting higher activity—similar to the "low sugar density = higher bioactivity" principle previously demonstrated for glycodendrimersomes. These one-component DNPs will overcome the limitations of four-component LNPs and demonstrate organ-specific mRNA delivery.

Aims: 1. Design and synthesize six libraries containing 54 sequence-defined IAJDs using an accelerated modular-orthogonal methodology with ionizable amines (DMBA, DMPA, DMA, PIP, MPRZ) and benzyl ether spacers 2. Characterize self-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) by simple injection (not microfluidics) and assess size, polydispersity, and stability at 5°C 3. Screen in vitro transfection of luciferase-mRNA DNPs in HEK293T cells and compare to positive controls (MC3-based LNPs, TransIT) 4. Evaluate in vivo mRNA delivery in mice by IVIS imaging and assess organ distribution (lung, liver, spleen) 5. Investigate structure-activity relationships between ionizable amine sequence/concentration and DNP activity 6. Assess DNP stability over time at 5°C and the effect of PEG incorporation on activity

Delivery system:

Component: Delivery Vehicle; Description: One-component ionizable amphiphilic Janus dendrimers (IAJDs) — sequence-defined macromolecules with hydrophilic and hydrophobic dendrons

Component: Hydrophilic Ionizable Groups; Description: • DMBA (dimethylaminobutanoate) • DMPA (dimethylaminopropanoate) • DMA (dimethylaminoacetate) • PIP (piperidinebutanoate) • MPRZ (methylpiperazinebutanoate)

Component: Hydrophobic Groups; Description: Linear and branched alkyl groups of different lengths (modules C, D, E)

Component: Key Structural Feature; Description: Benzyl ether groups (red in schematics) between ionizable amines; cation-π interactions may modulate pKₐ and facilitate mRNA interactions

Component: Libraries; Description: 6 libraries, 54 IAJDs total: • Library 1: 9 single−single IAJDs • Library 2: 7 single−single • Library 3: 6 single−single • Library 4: 3 single−single • Library 5: Twin−twin (from IAJD1-9 + IAJD33) • Library 6: 19 hybrid twin-mix

Component: Assembly Method; Description: Simple injection of IAJD ethanol solution into acidic buffer (pH 3-5.2) containing mRNA (NOT microfluidic T-tube)

Component: Nanoparticle Type; Description: Dendrimersome nanoparticles (DNPs) — vesicular structures encapsulating mRNA

Component: DNP Size; Description: Variable (~75-100+ nm; size tolerance for activity observed)

Component: Cargo; Description: Luciferase-mRNA (Luc-mRNA)

Component: Stability; Description: 19/40 DNPs stable at 5°C for up to 135 days (unoptimized)

Component: Positive Controls; Description: MC3-based LNPs (four-component, FDA-approved); TransIT

Approach:

Parameter: IAJD Synthesis; Details: Accelerated modular-orthogonal methodology; two orthogonal protecting groups (4-methoxybenzyl ether, benzyl ether); 54 IAJDs synthesized

Parameter: DNP Assembly; Details: IAJD dissolved in ethanol → injected into acidic buffer (pH 3-5.2) containing mRNA → DNPs form; pH of resulting solution ranges 4.5-7.3

Parameter: In Vitro Transfection; Details: HEK293T cells; Luc-mRNA DNPs; luciferase expression measured; 44/54 (81%) showed activity; compared to MC3 LNP and TransIT

Parameter: In Vivo Delivery; Details: Mice (6-8 weeks, female or male); 10 μg Luc-mRNA in 100 μL; IVIS imaging at 4-7 h post-injection; organs harvested for ex vivo imaging

Parameter: DNP Characterization; Details: DLS (size, PDI); Cryo-TEM (vesicle morphology); pKₐ measurements

Parameter: Stability Studies; Details: DNP size monitored at 5°C over time (up to 135 days)

Parameter: PEG Dilemma Study; Details: IAJD32 (PEG DP=45) hybrid tested alone and as 2% additive with active IAJD33

Parameter: Replicates; Details: Most active DNPs tested up to 6 times; DNP9 showed poor reproducibility in vivo

Parameter: Controls; Details: MC3-based LNP; TransIT; JD without ionizable amines; no treatment

Key methods:

Analysis Category: IAJD Synthesis; Methods: Modular-orthogonal methodology; two orthogonal protective groups; Schemes S1-S12 (Supporting Information)

Analysis Category: Nanoparticle Characterization; Methods: DLS (hydrodynamic diameter, PDI); Cryo-TEM (vesicle morphology); pKₐ measurements

Analysis Category: In Vitro Transfection; Methods: Luciferase assay in HEK293T cells; luminescence quantification; dose-response curves for selected IAJDs (9, 22, 33, 34)

Analysis Category: In Vivo Imaging; Methods: IVIS bioluminescence imaging; D-luciferin injection (15 mg/mL, 10 μL/g); exposure 15 s to 1 min; organ harvest and ex vivo imaging

Analysis Category: Stability Assessment; Methods: DLS monitoring at 5°C over time; 40 DNPs assessed; 19 stable for up to 135 days

Analysis Category: pKₐ Measurement; Methods: pH instrumentation; correlation with activity

Analysis Category: PEG Dilemma Assessment; Methods: IAJD32 (PEG DP=45) alone and as 2% additive; effect on DNP stability and activity

Key results: ### IAJD Libraries & DNP Formation

Parameter: Total IAJDs synthesized; Result: 54 (6 libraries)

Parameter: In vitro active DNPs; Result: 44/54 (81%)

Parameter: In vivo active DNPs; Result: 31/54 (57%)

Parameter: DNPs outperforming MC3 in vitro; Result: 4 (IAJD8, 9, 21, 22)

Parameter: DNPs with lung activity > MC3 control; Result: 2 (IAJD33, 34)

Parameter: Stable at 5°C for ≥135 days; Result: 19/40 DNPs

In Vitro Transfection (HEK293T):

IAJD: IAJD9; Activity vs. MC3 LNP: Higher; Notes: DNP9; stable

IAJD: IAJD22; Activity vs. MC3 LNP: Higher; Notes: DNP22; stable

IAJD: IAJD8; Activity vs. MC3 LNP: Higher; Notes: -

IAJD: IAJD21; Activity vs. MC3 LNP: Higher; Notes: -

IAJD: IAJD33; Activity vs. MC3 LNP: Comparable/high; Notes: DNP33; lung targeting

IAJD: IAJD34; Activity vs. MC3 LNP: Comparable/high; Notes: DNP34; lung targeting

In Vivo Organ Targeting (Luciferase Signal):

Organ: Lung; Highest Activity (IAJD): IAJD33, IAJD34; Signal Level: 10⁸; vs. MC3 Control: Higher than MC3

Organ: Lung; Highest Activity (IAJD): IAJD31, IAJD46, IAJD27; Signal Level: 10⁷; vs. MC3 Control: -

Organ: Liver; Highest Activity (IAJD): IAJD31, IAJD34; Signal Level: 10⁶; vs. MC3 Control: Lower than MC3 (10⁸)

Organ: Liver; Highest Activity (IAJD): IAJD30, IAJD33, IAJD46; Signal Level: 10⁵; vs. MC3 Control: Lower than MC3

Organ: Spleen; Highest Activity (IAJD): IAJD29, IAJD30, IAJD37; Signal Level: 10⁵; vs. MC3 Control: Lower than MC3 (10⁷)

Organ: Spleen; Highest Activity (IAJD): IAJD27; Signal Level: 10⁴; vs. MC3 Control: -

DNP Stability & PEG Dilemma:

DNP: DNP9 (IAJD9); Stability (5°C): Excellent; In Vivo Activity: Good (but poor reproducibility)

DNP: DNP22 (IAJD22); Stability (5°C): Excellent; In Vivo Activity: High

DNP: DNP33 (IAJD33); Stability (5°C): Excellent; In Vivo Activity: Very high (lung)

DNP: DNP34 (IAJD34); Stability (5°C): Excellent; In Vivo Activity: Very high (lung)

DNP: DNP32 (IAJD32, PEG DP=45); Stability (5°C): Excellent; In Vivo Activity: Completely inactive

DNP: DNP33 + 2% IAJD32; Stability (5°C): Excellent; In Vivo Activity: Dramatically reduced (PEG dilemma confirmed)

DNP: DNP46 (twin-twin of IAJD33); Stability (5°C): Excellent; In Vivo Activity: ~50% of DNP33 activity

DNP: DNP47 (hybrid of IAJD33); Stability (5°C): -; In Vivo Activity: Much lower than DNP33 and DNP46

Structure-Activity Relationship:

Observation: Ionizable amine concentration; Finding: Lower concentration = higher activity (in specific sequences)

Observation: Sequence dependence; Finding: Activity change > sugar-binding activity change in glycodendrimersomes

Observation: pKₐ correlation; Finding: Most active IAJDs (33, 34, 31) do NOT have lowest pKₐ values

| DNP size tolerance | >100 nm DN

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One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA

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