Project

Development and optimization of cell culture protocols for studying cell-nanoparticle interactions

Duration
01 January 2013 → 31 December 2016
Funding
Regional and community funding: IWT/VLAIO
Research disciplines
  • Medical and health sciences
    • Biomarker discovery and evaluation
    • Drug discovery and development
    • Medicinal products
    • Pharmaceutics
    • Pharmacognosy and phytochemistry
    • Pharmacology
    • Pharmacotherapy
    • Toxicology and toxinology
    • Other pharmaceutical sciences
Keywords
cell-nanoparticle interactions nanotechnology
 
Project description
Nanotechnology is one of the key enabling technologies of the 21st century. Various
inorganic and organic materials show unique properties when engineered at the nanoscale,
which opens up a myriad of novel applications. In the context of medicine, organic
nanoparticles (NP) are often applied as carriers for insoluble drugs or macromolecules, such
as nucleic acids (NA). The latter require packaging into a nanocarrier to ensure
transportation to the target tissue and cellular internalization, while maintaining their
functionality. In this regard, both viral and non-viral vectors were explored in parallel. Nonviral delivery vehicles are considered relatively safe but generally fall short in terms of
transfection efficiency given the many extra- and intracellular barriers limiting NA delivery.
At the intracellular level, endosomal escape is regarded to be the major bottleneck. Even for
state-of-the-art carriers, a minor fraction of the internalized NA dose escapes to the cytosol
whereas the bulk of the NA cargo is trafficked to the lysosomal compartment for
degradation. Hence, current strategies aim to improve endosomal escape from the early or
late endosomal compartment to avoid lysosomal entrapment. In contrast to this paradigm,
we set out to specifically target the lysosomes to induce release of the accumulated NA
cargo. In particular, we aimed to improve the cytosolic delivery of small interfering (si)RNA
upon nanogel (NG)-mediated transfection using approved low molecular weight drugs,
namely cationic amphiphilic drugs (CADs), which functionally inhibit acid sphingomyelinase
(FIASMA). A general introduction into the use of siRNA to trigger the RNA interference
pathway, nanogels as non-viral nanocarriers and FIASMAs as lysosomal delivery enhancers is
provided in Chapter 1.
Since the early days of plasmid (p)DNA delivery through lipid-based nanocarriers, scientists
explored the use of small molecules to boost the delivery potential. Our literature study
(Chapter 2) revealed adjuvants can enhance tumor penetration or cellular internalization. In
turn, some adjuvants boost endosomal escape, alter intracellular trafficking or directly
promote the transgene expression of successfully delivered pDNA. Several small molecules
can furthermore improve the transfection efficiency by reducing the innate immune
response or facilitating pDNA nuclear entry. Finally, pleiotropic molecules such as
chloroquine, dexamethasone and other steroids, improve NA transfection by simultaneously
influencing various delivery-related processes. Overall, this overview underscores the
diversity small molecular adjuvant approaches to stimulate NA delivery. Of note, the activity
of several adjuvants is limited to specific NA and/or nanocarriers and the moment of the
adjuvant treatment relative to the transfection. Hence, further insights into nanocarrier
uptake, intracellular trafficking and endosomal escape should allow to rationally combine
adjuvants, NA and carriers in the future.
The data presented in Chapter 3 show that the FIASMAs induce the accumulation of
phospholipids, cholesterol and sphingomyelin. This transient phospholipidosis (PLD)
phenotype was suggested to reduce the lysosomal membrane stability, leading to enhanced
transfer of siRNA from the lysosomes into the cytosol. Of note, this assumed improved siRNA
delivery drastically enhanced the gene silencing potential of the siRNA-loaded NGs. In
addition, we showed that the lysosomes could be applied as a depot for prolonged and
controlled siRNA release, underscoring the great potential of the CAD adjuvant approach. In
Chapter 4 we explored the broader applicability of this adjuvant strategy. First, we showed
that the induction of PLD through mechanisms other than acid sphingomyelinase inhibition,
does not necessarily improve siRNA delivery. In turn, distinct adjuvants might too greatly
affect the lysosomal membrane stability wherefore the stimulated cytosolic siRNA release
coincides with significant cytotoxicity. In addition, messenger (m)RNA delivery could not to a
similar extent be improved and the delivery of siRNA by lipid-based transfection reagents
was less prone to the FIASMA adjuvant effect.
In conclusion, we showed that a sequential FIASMA treatment is able to markedly improve
the therapeutic potential of siRNA-loaded NGs. However, this effect was both carrier- and
NA-dependent since neither mRNA delivery nor transfection by lipid-based carriers could
equally be enhanced. Research on the subject could be continued by identifying more
potent adjuvants through large-scale compound screenings. A more detailed elucidation of
the cellular FIASMA effects will in turn clarify to which subgroup of NA and carriers this
method can be applied. Finally, the co-inclusion of the FIASMA and NA into the nanocarrier
or the synthesis of FIASMA-NA conjugates should be explored.
In addition to the use of organic nanocarriers as delivery vehicles for NA, inorganic NPs are
being explored in a biomedical setting, with their unique properties allowing improvements
to current detection and/or treatment strategies or the development of novel biomedical
applications. Iron oxide (IO)NPs for instance allow magnetism-guided delivery, magnetic
resonance imaging (MRI) and cancer treatment through hyperthermia. Despite extensive
proof-of-concept studies, few inorganic NPs are currently clinically applied, which can largely
be attributed to their elusive safety profiles given the inconsistent nanotoxicity data.
Chapter 5 summarizes cell-NP interactions suggested to elicit nanotoxicity, the current in
vitro approaches to study nanotoxicity and their major limitations. We discuss novel
methods and show that the complexity of the cell model influences NP uptake and toxicity,
highlighting the need for optimization and standardization of the in vitro nanotoxicity testing
paradigm.
In Chapter 6, we assessed how IONPs interact with six related neural cell types, namely
murine and human neural stem cells (NSC), neuroblastoma and immortalized neural
progenitor cells. Besides species-specific variations, the neuroblastoma cell line and NSCs
were respectively least and most affected. Of note, we obtained cell type-specific
nanotoxicity profiles in terms of the extent and nature of the effects, indicating that a single
toxicity end point will not provide sufficient information on in vitro nanotoxicity. In a followup study (Chapter 7), we reported on the influence of the cell type on the optimization of
NPs for biomedical applications. We showed that DMSA-coated IONPs outperformed PMAcoated IONPs in the same six neural cell types, although the differentially coated IONPs
again elicited cell-type specific toxicity profiles. Hence, the major cell type-dependent
variations in the observed effects warrant the use of relevant human cell models that mimic
the envisioned target cells as closely as possible.
Overall, the optimization and standardization of in vitro hazard evaluations would
significantly improve the quality of in vitro nanotoxicity research, which could boost the
clinical development of inorganic NPs. In analogy with guidelines on in vivo methods
established by regulatory bodies, a consensus should be reached on how to assess
nanotoxicity in vitro. We showed that the cell type is a critical factor that should not be
overlooked in the standardization process. Most importantly, correlations should be
established between in vitro effects and in vivo adverse events to allow extrapolation to
possible adverse events in human.