Molecular Therapy2018ResearchNon-viral Gene Delivery

The Effect of Size and Shape of RNA Nanoparticles on Biodistribution

Daniel L. Jasinski, Hui Li, Peixuan Guo29 September 2026DOI 10.1016/j.ymthe.2017.12.018

Summary

Drugs with ideal pharmacokinetic profiles require long half-lives but little organ accumulation. Generally, these factors are contradictory: smaller particles lead to faster excretion and shorter half-lives, while larger particles lead to longer circulation but stronger organ accumulation and potential toxicity. The effect of nanoparticle size on biodistribution has been inconsistent in the literature due to the confounding effect of shape. ### Nanoparticle Size Characterization (DLS) | Nanoparticle | Designed Size | Measured DLS (nm) | |------------------|-------------------|----------------------| | 5 nm square | 5 nm | 5.60 ± 1.01 | | 10 nm square | 10.

Keywords

NanoparticlesBiodistributionDrug deliveryNanocarriersGene deliveryImmune cellsTransfection

Purpose: Drugs with ideal pharmacokinetic profiles require long half-lives but little organ accumulation. Generally, these factors are contradictory: smaller particles lead to faster excretion and shorter half-lives, while larger particles lead to longer circulation but stronger organ accumulation and potential toxicity. The effect of nanoparticle size on biodistribution has been inconsistent in the literature due to the confounding effect of shape. Using RNA nanotechnology, where size can be tuned without changing shape, a true size comparison can be achieved.

Hypothesis: RNA nanoparticles constructed from the pRNA 3WJ motif—with precisely controlled size (5-25 nm) and shape (triangle, square, pentagon)—will exhibit size-dependent circulation times and biodistribution profiles. Larger RNA nanoparticles will show increased circulation time and tumor accumulation via the EPR effect, while smaller particles will be cleared more rapidly through the kidneys. Shape will have a secondary but measurable effect on biodistribution patterns.

Aims: 1. Design and assemble RNA polygons (triangles, squares, pentagons) with variable size (5, 10, 20 nm) but identical shape, and variable shape but identical size 2. Characterize nanoparticle size, assembly fidelity, and stability using gel electrophoresis and dynamic light scattering (DLS) 3. Evaluate in vivo biodistribution of size-variant and shape-variant RNA nanoparticles in KB tumor-bearing nude mice using fluorescence imaging 4. Assess serum stability and serum protein binding of RNA nanoparticles of different sizes and shapes 5. Determine the effect of size and shape on circulation time and organ accumulation

Delivery system:

Component: Nanoparticle Scaffold; Description: pRNA three-way junction (3WJ) motif from bacteriophage phi29 packaging RNA — highly thermostable, fast kinetic assembly

Component: Nanoparticle Design; Description: RNA polygons (triangles, squares, pentagons) constructed from 3WJ modules as vertices connected by RNA duplex edges

Component: Size Variants; Description: • 5 nm square (small): designed edge length 5 nm; DLS: 5.60 ± 1.01 nm • 10 nm square (medium): designed edge length 10 nm; DLS: 9.42 ± 1.80 nm • 20 nm square (large): designed edge length 20 nm; DLS: 17.51 ± 3.64 nm

Component: Shape Variants; Description: • Triangle: 10 nm edge length; DLS: 9.91 ± 1.86 nm • Square: 10 nm edge length; DLS: 9.42 ± 1.80 nm • Pentagon: 10 nm edge length; DLS: 10.90 ± 2.09 nm

Component: RNA Modifications; Description: 2′-fluorine (2′F) modifications at cytosine and uracil — confers nuclease resistance and thermodynamic stability

Component: Fluorescent Labeling; Description: AlexaFluor 647 conjugated to one strand per nanoparticle (single fluorophore labeling ensures comparable signal across particles)

Component: Assembly Method; Description: Equimolar mixing of RNA strands in PBS + 1 mM MgCl₂; thermal denaturation at 85°C for 5 min; slow cooling to 4°C over 1 h

Component: Cell Line/Tumor Model; Description: KB cells (human nasopharyngeal carcinoma); subcutaneous xenograft in male nude mice (NU/NU)

Component: Injection Dose; Description: 100 μL of 15 μM AlexaFluor647-labeled 2′F RNA nanoparticles via intravenous tail vein injection

Approach:

Parameter: Nanoparticle Assembly; Details: Agarose gel electrophoresis (2% agarose, TAE buffer, 90 V, 1 h) to confirm assembly

Parameter: Size Characterization; Details: DLS (Zetasizer nano-ZS, Malvern) at 5 μM in PBS; Gaussian distribution fitting

Parameter: In Vivo Biodistribution; Details: • KB tumor-bearing nude mice (n=2 per particle, per time point) • IV injection via tail vein • IVIS imaging at 0.5, 1, 2, 4, 8, 12, 24 h • Organ harvest at 12 and 24 h: liver, spleen, kidneys, heart, lung, tumor

Parameter: Serum Stability; Details: 0.5 μM fluorescent nanoparticles in 10% FBS at 37°C; time points 0-32 h; 2% agarose gel; ImageJ band intensity quantification; exponential decay fitting for half-life

Parameter: Serum Binding Assay; Details: 500 nM nanoparticles incubated in 0-50% FBS (5 min, 37°C); 2% agarose gel; EtBr signal quantification; equilibrium serum concentration (50% bound) determined

Parameter: Controls; Details: PBS (blank control); single-stranded RNA (rapid kidney clearance confirmation)

Parameter: Replicates; Details: n=2 mice per particle per time point; serum assays repeated twice

Key methods:

Analysis Category: Nanoparticle Characterization; Methods: • 2% agarose gel electrophoresis (assembly confirmation) • DLS (Zetasizer nano-ZS): hydrodynamic diameter • Gaussian distribution fitting for size determination

Analysis Category: In Vivo Imaging; Methods: • IVIS imager (Caliper Life Sciences) • Whole body fluorescence at 0.5-24 h • Organ fluorescence at 12 and 24 h post-injection

Analysis Category: Serum Stability; Methods: • 10% FBS incubation at 37°C • 2% agarose gel electrophoresis • AlexaFluor 647 fluorescence scanning • ImageJ band intensity integration • Exponential decay fitting (OriginPro)

Analysis Category: Serum Binding; Methods: • 0-50% FBS incubation (5 min, 37°C) • 2% agarose gel electrophoresis • EtBr signal quantification (ImageJ) • Equilibrium serum concentration calculation (50% bound)

Key results: ### Nanoparticle Size Characterization (DLS)

Nanoparticle: 5 nm square; Designed Size: 5 nm; Measured DLS (nm): 5.60 ± 1.01

Nanoparticle: 10 nm square; Designed Size: 10 nm; Measured DLS (nm): 9.42 ± 1.80

Nanoparticle: 20 nm square; Designed Size: 20 nm; Measured DLS (nm): 17.51 ± 3.64

Nanoparticle: Triangle; Designed Size: 10 nm edge; Measured DLS (nm): 9.91 ± 1.86

Nanoparticle: Pentagon; Designed Size: 10 nm edge; Measured DLS (nm): 10.90 ± 2.09

Serum Stability Half-Lives (2′F RNA Nanoparticles):

Nanoparticle: 5 nm square; Half-Life (T₁/₂): 10.6 h

Nanoparticle: 10 nm square; Half-Life (T₁/₂): 22.9 h

Nanoparticle: 20 nm square; Half-Life (T₁/₂): >100 h

Serum Binding Equilibrium Concentrations:

Nanoparticle: 5 nm square; % Serum for 50% Binding: ~20%

Nanoparticle: 10 nm square; % Serum for 50% Binding: ~25%

Nanoparticle: 20 nm square; % Serum for 50% Binding: ~30%

Nanoparticle: Triangle; % Serum for 50% Binding: ~20%

Nanoparticle: Pentagon; % Serum for 50% Binding: ~30%

Nanoparticle: Interpretation; % Serum for 50% Binding: Larger particles bind more serum proteins; shape affects binding

In Vivo Biodistribution: Size Effect (5, 10, 20 nm Squares):

Time Point: 0.5-4 h; 5 nm Square: Rapidly cleared; kidney signal; 10 nm Square: Circulation maintained; 20 nm Square: Strongest whole-body signal

Time Point: 4-12 h; 5 nm Square: Tumor only (no organ accumulation); 10 nm Square: Moderate organ signal; 20 nm Square: Strong organ and tumor signal

Time Point: 12 h organs; 5 nm Square: Tumor only; 10 nm Square: Kidneys signal; 20 nm Square: Kidneys and liver signal

Time Point: 24 h organs; 5 nm Square: Tumor only (undetectable in organs); 10 nm Square: Tumor only; 20 nm Square: Weak liver/kidney signal + tumor

Time Point: Circulation time; 5 nm Square: Shortest; 10 nm Square: Intermediate; 20 nm Square: Longest

In Vivo Biodistribution: Shape Effect (10 nm Triangle, Square, Pentagon):

Time Point: 12 h organs; Triangle: Low kidney signal; Square: High kidney signal; Pentagon: Low kidney signal; spleen signal

Time Point: 24 h organs; Triangle: Tumor only; Square: Tumor only; Pentagon: Tumor only

Time Point: Elimination rate; Triangle: Fastest; Square: Intermediate; Pentagon: Slowest

Time Point: Shape effect; Triangle: Less pronounced than size; affects early elimination pathways; Square: -; Pentagon: -

Organ Accumulation Summary:

Organ: Tumor; 5 nm Square: Strongest at all times; 10 nm Square: Strong; 20 nm Square: Strong

Organ: Kidneys; 5 nm Square: Rapid clearance signal; 10 nm Square: Moderate at 12 h; 20 nm Square: Weak at 12 h

Organ: Liver; 5 nm Square: Undetectable; 10 nm Square: Weak at 12 h; 20 nm Square: Weak at 24 h

Organ: Spleen; 5 nm Square: Undetectable; 10 nm Square: Low; 20 nm Square: Low

Organ: Heart/Lung; 5 nm Square: Undetectable; 10 nm Square: Undetectable; 20 nm Square: Undetectable

Organ: Accumulation; 5 nm Square: None in healthy organs; 10 nm Square: Minimal; 20 nm Square: Minimal

Interpretation: The authors conclude that "the size of RNA nanoparticles have a large effect on circulation time and biodistribution in vivo," with "a strong correlation between increased size and increased circulation time" demonstrated by "the increased duration of fluorescent signal in whole body images of tumor bearing nude mice over 24 h." They note that "despite increased size from 5 to 25 nm, there was still minimal fluorescent signal in healthy organs such as the liver and kidneys after 24 h compared to tumors." The authors state: "This study demonstrates a unique property of RNA nanoparticles that allows them to evade accumulation in healthy organs while still accumulating in tumor tissue by the EPR effect." They emphasize that "circulation time is an important factor in drug delivery, as sometimes a short-lived particle is preferred to reduce toxicity, and other times a long circulating particle could be beneficial for slow acting therapies."

10. Limitations (Explicitly Stated or Evident):

1. Small sample size per time point: Only n=2 mice were used per particle per time point; while consistent trends were observed, larger sample sizes would improve statistical power.

2. No therapeutic cargo tested: The study used fluorescently labeled nanoparticles without therapeutic payloads; the effect of cargo on biodistribution was not assessed.

3. No active targeting: Nanoparticles lacked targeting ligands; accumulation in tumors relied solely on the EPR effect, which may not translate to all tumor types.

4. Short observation window: Biodistribution was tracked for only 24 h; longer-term clearance and potential late accumulation were not assessed.

5. Moderate size effect correlation: The 20 nm square (DLS 17.5 nm) showed slightly smaller size than intended; size differences between 10 and 20 nm were less than the 2× design ratio.

6. Shape effect conclusions limited: The authors note that "shape was seen to have less effect on the biodistribution of RNA nanoparticles," but only three shapes (triangle, square, pentagon) were tested at a single size.

7. No immune response assessment: While RNA nanoparticles have been shown to trigger low immune responses, immunogenicity was not specifically evaluated in this study.

8. Serum stability in vitro vs. in vivo: Serum half-lives (10.6 to >100 h) were far greater than apparent in vivo fluorescent half-lives, suggesting other clearance mechanisms (e.g., renal filtration, macrophage uptake) dominate in vivo.

9. Single tumor model: Only KB xenografts in nude mice were used; results may differ in other tumor types or immunocompetent models.

10. No quantitative organ accumulation: Organ images were qualitative; quantitative fluorescence measurements (e.g., % injected dose per gram tissue) were not provided.

11. Potential for fluorophore quenching/degradation: Fluorescent signal loss could result from dye degradation or quenching, not only nanoparticle clearance.

12. 2′F modification effects: While 2′F modifications confer nuclease resistance, they may also affect protein corona formation or receptor interactions compared to unmodified RNA.

Report prepared based on the accepted manuscript in Molecular Therapy. For full experimental details, supplementary information, and complete references, please refer to the original publication.

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The Effect of Size and Shape of RNA Nanoparticles on Biodistribution

Summary

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