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Bioconjugate Chemistry (likely; based on formatting and page range)2019ReviewNon-viral Gene Delivery

The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery

Samuel A. Smith, Laura I. Selby, Angus P. R. Johnston, And Georgina K. Such

Summary

Many emerging therapies require delivery of biological cargo into the cytosol, but nanoparticle delivery systems are hindered by entrapment and degradation in the endo/lysosomal pathway. Endosomal escape remains a rate-determining step for non-viral delivery. The review highlights the need for greater understanding of escape mechanisms and improved quantification methods to enable rational design of safer, more efficient polymeric delivery. Escape efficiency is low: Zerial and co-workers demonstrated that <2% of siRNA delivered using lipid nanoparticles is delivered to the cytosol. - Calcein assay: 27 kDa PDEAEMA nanoparticles showed leakage of calcein.

Keywords

Endosomal escapeNanoparticlesPolymericTransfectionsiRNADNAPoly(beta-amino ester)
Purpose: Many emerging therapies require delivery of biological cargo into the cytosol, but nanoparticle delivery systems are hindered by entrapment and degradation in the endo/lysosomal pathway. Endosomal escape remains a rate-determining step for non-viral delivery. The review highlights the need for greater understanding of escape mechanisms and improved quantification methods to enable rational design of safer, more efficient polymeric delivery systems.
Hypothesis: As a Topical Review, this work does not test a single hypothesis. Its central thesis is:

If the mechanisms governing endosomal escape are better understood and quantified—particularly for polymeric nanoparticles—then more efficient, nontoxic delivery systems can be rationally designed to deliver biological cargo into the cytosol.

Aims: Primary Aim: To review current understanding of the mechanisms by which nanoparticles escape the endosome and the emerging techniques to improve and quantify endosomal escape.
  • Secondary Aims:
  • To discuss assays for detection and quantification of endosomal escape.
  • To critically evaluate proposed mechanisms: membrane fusion, osmotic pressure/proton sponge, particle swelling, and membrane destabilization.
  • To highlight how material properties (pH-responsive polymers, architecture, cross-linking density, timing of disassembly) influence escape efficiency.
  • To identify limitations and future directions for the field.
Delivery system:

Component: Nanoparticle Types; Examples Discussed: Polymeric nanoparticles, polymer micelles, polymersomes, nanoparticle aggregate particles, nanogels, lipid–polymer hybrid particles, polymer-functionalized inorganic nanoparticles

Component: Polymer Chemistry; Examples Discussed: pH-responsive polymers: PDEAEMA, PDPAEMA, PDMAEMA, P(DMAEMA-r-PAA-r-BMA), PEG-b-PDEAEMA, PEG-b-PDPAEMA, PEG-PAsp(DET), poly(β-amino ester) (PBAE), PMPC-b-PDPAEMA, PImHeMA, PGMA, cross-linked PDMAEMA/PHEMA/TEGDMA particles

Component: Payloads; Examples Discussed: DNA, RNA, siRNA, proteins, small molecules (calcein), model antigens (ovalbumin), dextrans of varying molecular weights

Component: Targeting/Ligands; Examples Discussed: Not a major focus; some mention of transferrin-bearing fusogenic polymer-modified liposomes

Component: Key Property Focus; Examples Discussed: pH-responsive disassembly, buffering capacity, membrane destabilization, particle swelling, cross-linking density, polymer architecture (linear, hyperbranched, cross-linked)

Approach: This is a narrative Topical Review synthesizing existing literature. No primary experimental data are presented. The review:
  • Discusses in vitro assays using cell lines such as 3T3, HEK-293, DC2.4, and HeLa.
  • Covers model membrane assays (red blood cell lysis, liposome dye leakage, FRET).
  • Reviews mechanisms of endosomal escape for polymeric nanoparticles.
  • Evaluates studies on pH-responsive particles, cross-linking density, architecture, and timing of disassembly.
  • Does not include in vivo animal models as a central focus.
Key methods: As a review, the “methods” are literature synthesis and comparative analysis. Headline data cited from primary studies were generated using:
  • Calcein leakage assay: Membrane-impermeable dye taken up into vesicles; punctate fluorescence indicates entrapment, diffuse fluorescence indicates escape.
  • GFP complementation assay: GFP₁₁ fragment conjugated to material; fluorescence upon complementation with GFP₁₋₁₀ indicates cytosolic delivery.
  • Fluorescence microscopy / live-cell imaging: Punctate vs. diffuse distribution to assess escape.
  • Electron microscopy: Combined with fluorescence to quantify cytosolic delivery.
  • Red blood cell lysis assay: Hemolysis as an indirect measure of membrane destabilization.
  • Dye-loaded liposome leakage and FRET assays: Model membrane fusion/destabilization.
  • Immunofluorescence staining: Localization with lysosomal/endosomal markers (LAMP1, EEA1).
  • Transfection efficiency / gene silencing: Indirect end-point measures of escape.
Key results: Escape efficiency is low: Zerial and co-workers demonstrated that <2% of siRNA delivered using lipid nanoparticles is delivered to the cytosol. - Calcein assay: 27 kDa PDEAEMA nanoparticles showed leakage of calcein into the cytosol, indicating membrane breach; calcein-only controls showed no escape. - GFP complementation: HEK-293 cells transfected with only large or small GFP fragments were not fluorescent; both together produced diffuse fluorescence. - Particle swelling: A pH-responsive PDEAEMA-core/PAEMA-shell particle exhibited a 2.8-fold increase in diameter from ~200 nm to 550 nm as pH decreased from 7.4 to 4.9, with a sharp swelling transition between pH 7.0 and 6.8. It effectively released calcein and ovalbumin into the cytosol of DC2.4 cells. - Cross-linking density: Altering cross-linking from 3 mol% to 9 mol% had a large effect on transfection efficiency; low cross-linked particles showed higher transfection and greater swelling capacity (highly cross-linked particles had ~half the swelling capacity). - Cargo size limit: PBAE nanoparticles stabilized by PEG lipid could chaperone dextran of 3 or 10 kDa out of the endosome, but 40 kDa and 70 kDa remained trapped, suggesting incomplete membrane compromise. - Proton sponge debate: Polymers with improved buffering capacity have had limited success in enhancing endosome escape; PEI does not need to be internalized with polyplexes for transfection; intracellular pH measurements are inconsistent. These points argue against the proton sponge hypothesis as an independent mechanism.
Interpretation: The authors conclude that nanoparticles have significant potential for targeted therapeutic delivery, but realizing this potential requires greater focus on engineering particles with endo/lysosomal escape ability. Several mechanisms—membrane fusion, membrane destabilization, particle swelling, and osmotic rupture—are hypothesized to play roles. Greater emphasis on understanding these mechanisms and developing standardized, quantitative assays is needed for rational design of future nanoparticles.
Limitations: Limitations inherent to the review:
  • No primary experimental data; conclusions are synthesized from existing literature.
  • No systematic search strategy or meta-analysis.
  • Focus is primarily on polymeric nanoparticles; lipid, inorganic, and biological delivery systems are mentioned but not exhaustively covered.
  • No in vivo validation discussed in depth.

Limitations of the field highlighted by the authors:

  • Lack of standard quantification: No standard method to detect and quantify endosomal escape; difficult to compare across studies.
  • Indirect assays: Transfection efficiency is an indirect measure; additional steps (plasmid dissociation, nuclear translocation) confound escape ability.
  • Proton sponge hypothesis debated: Lack of predictive power; inconsistent pH measurements; PEI membrane destabilization may better explain escape.
  • Model membrane assays: Do not completely replicate biological membrane complexity.
  • Cargo size limit: Some systems only deliver small cargoes; larger therapeutics remain trapped.
  • Difficulty decoupling variables: Differences in DNA size, promoter strength, cell line, polymer concentration, and cross-linking density complicate conclusions.
  • Timing of disassembly matters: Particles that disassemble at different pH values show different escape efficiencies; optimal timing is not fully resolved.
  • Need for live-cell imaging: Fixation/permeabilization can immobilize stealth nanoparticles; live-cell methods are preferable but technically challenging.

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The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery | Brilliant Blue Biosciences