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santa`s chimney into the cell: intracellular delivery of proteins
technical journal club17.12.2019
merve avar
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introduction – why deliver proteins at all?
importance
- can be utilized as highly
specific targets to interfere
with pathways
- no genetic editing required
- lower side effects
due to its potential, it is imperative to develop methods for intracellular protein delivery
challenges
- membrane impermeability
- high molecular weight (to smallmolecules)
- hydrophilicity
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leading the way in
Miersch et. al., F1000
cargo encapsulation -> cellular internalization -> escape from endosomes -> cytosolic cargo release
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widely reported protein delivery methods
− cell-penetrating peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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endosomolytic peptides
L17E best working peptide
does not require a transporter or a receptor
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endosomolytic peptides- functionality of cargo
saporin induces cell death upon administrationwith L17E within 2 hours
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endosomolytic peptides- functionality of cargo
saporin induces cell death upon administrationwith L17E within 2 hours
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endosomolytic peptides- pros and cons
advantages- easy to use
- does not require alterations
to cargo
- reports on functionality of the
delivered product
- mechanism proposed
shortcomings
- potential immuno response?
types of cells not possible
- paper did not evaluate
versatility of the sytem
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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widely reported protein delivery methods
− cell-penetrating selective peptides
− protein resurfacing
− endosomolytic peptides
− toxin derived assemblies
− physical delivery methods
− gold particle complexes
− nanocomplexes
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newest advance –
why important?
- self assembly with cargo, does not require chemical or biological alterations to the cargo or thecarrier
- efficient in endolysosomal escape- has wide applicability to carry proteins of differing size, pI- applicable in vivo
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assembly
initial copolymerization
endcapping
E1-base polymer: amphiphilic and pH sensitive
carboxylated brancedPBAE: supports hydrogen bond formation and saltbridges
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nanocomplex delivery into different cell lines
C5 and C7 in complex with FITC-BSA is taken up by different celllines, and viability is not impaired
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investigation of route of internalization
CPZGEN
CYD
CPZ: chlorpromazineGEN: genisteinMCD: methyl-B-cyclodextrinCYD: cytochalastin D
Lipid raft mediated endocytosis
MCD
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CPZGEN
CYD
CPZ: chlorpromazineGEN: genisteinMCD: methyl-B-cyclodextrinCYD: cytochalastin D
Lipid raft mediated endocytosis
MCD
investigation of route of internalization
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grand entry and great escape
human adipose derivedmesenchymal stem cells
HEK293T cells
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the great escape: from endosomes
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robustness of the delivery method
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CRISPR through nanocomplex RNP delivery
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CRISPR through nanocomplex RNP delivery
homology directed repair
cas9 + sgRNA against CXCR4 + ssDNA repair templatec5 polymer
self-assembly strategy
edit strategy
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CRISPR through nanocomplex RNP delivery
homology directed repair
cas9 + sgRNA against CXCR4 + ssDNA repair templatec5 polymer
self-assembly strategy
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in vivo CRISPR editing in murine glioma tumors – proof of principle
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newest advance – advantages and shortcomings
advantages- nanocomplexes are easy-to-
use
- highly efficient in cargo and
delivery of a wide range of
proteins (differing in size and
pI)
- perfect for in vitro use
(storage and performance
tested in comparison to
commercially available
products)
- (potential) capability of
evading immune responses
shortcomings
- targeted delivery into certain
types of cells not possible
- optimizations of in vivo
delivery needed
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thank you for your attention and merry christmas!(and hopefully a better new year)
publications referred to in this presentation and further reading: - https://www.frontiersin.org/articles/10.3389/fphar.2018.01208/full- https://f1000research.com/articles/5-1947/v1 - https://www.sciencedirect.com/science/article/pii/S0168365914008165?via%3Dihub- https://pubs.acs.org/doi/10.1021/acs.langmuir.6b04304- https://advances.sciencemag.org/content/5/12/eaay3255.full- https://www.ncbi.nlm.nih.gov/pubmed/28781125- https://pubs.acs.org/doi/10.1021/ja907887z- https://www.nature.com/articles/nmeth.2998- https://www.nature.com/articles/nature19764- https://www.nature.com/articles/nchem.2779.pdf