Viral
and synthetic vectors for delivery of nucleic acids impacted
genetic nanomedicine by aiding the rapid development of the extraordinarily
efficient Covid-19 vaccines. Access to targeted delivery of nucleic
acids is expected to expand the field of nanomedicine beyond most
expectations. Both viral and synthetic vectors have advantages and
disadvantages. The major advantage of the synthetic vectors is their
unlimited synthetic capability. The four-component lipid nanoparticles
(LNPs) are the leading nonviral vector for mRNA used by Pfizer and
Moderna in Covid-19 vaccines. Their synthetic capacity inspired us
to develop a one-component multifunctional sequence-defined ionizable
amphiphilic Janus dendrimer (IAJD) delivery system for mRNA. The first
experiments on IAJDs provided, through a rational-library design combined
with orthogonal-modular accelerated synthesis and sequence control
in their hydrophilic part, some of the most active synthetic vectors
for the delivery of mRNA to lung. The second experiments employed
a similar strategy, generating, by a less complex hydrophilic structure,
a library of IAJDs targeting spleen, liver, and lung. Here, we report
preliminary studies designing the hydrophobic region of IAJDs by using
dissimilar alkyl lengths and demonstrate the unexpectedly important
role of the primary structure of the hydrophobic part of IAJDs by
increasing up to 90.2-fold the activity of targeted delivery of mRNA
to spleen, lymph nodes, liver, and lung. The principles of the design
strategy reported here and in previous publications indicate that
IAJDs could have a profound impact on the future of genetic nanomedicine.
Targeted
and efficient delivery of nucleic acids with viral and
synthetic vectors is the key step of genetic nanomedicine. The four-component
lipid nanoparticle synthetic delivery systems consisting of ionizable
lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled
by microfluidic or T-tube technology, have been extraordinarily successful
for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported
a one-component multifunctional sequence-defined ionizable amphiphilic
Janus dendrimer (IAJD) synthetic delivery system for mRNA relying
on amphiphilic Janus dendrimers and glycodendrimers developed in our
laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic
dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs
with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple
injection in acetate buffer, rather than by microfluidic devices,
and provides a very efficient system for delivery of mRNA to lung.
Here we report the replacement of most of the hydrophilic fragment
of the dendron from IAJDs, maintaining only its ionizable amine, while
changing its interconnecting group to the hydrophobic dendron from
amide to ester. The resulting IAJDs demonstrated that protonated ionizable
amines play dual roles of hydrophilic fragment and binding ligand
for mRNA, changing delivery from lung to spleen and/or liver. Replacing
the interconnecting ester with the amide switched the delivery back
to lung. Delivery predominantly to liver is favored by pairs of odd
and even alkyl groups in the hydrophobic dendron. This simple structural
change transformed the targeted delivery of mRNA mediated with IAJDs,
from lung to liver and spleen, and expands the utility of DNPs from
therapeutics to vaccines.
Viral and synthetic vectors to deliver nucleic acids were key to the rapid development of extraordinarily efficient COVID-19 vaccines. The four-component lipid nanoparticles (LNPs), containing phospholipids, PEG-conjugated lipids, cholesterol, and ionizable lipids, co-assembled with mRNA via a microfluidic technology, are the leading nonviral delivery vector used by BioNTech/Pfizer and Moderna to access COVID-19 mRNA vaccines. LNPs exhibit a statistical distribution of their four components when delivering mRNA. Here, we report a methodology that involves screening libraries to discover the molecular design principles required to realize organ-targeted mRNA delivery and mediate activity with a one-component ionizable multifunctional amphiphilic Janus dendrimer (IAJD) derived from plant phenolic acids. IAJDs co-assemble with mRNA into monodisperse dendrimersome nanoparticles (DNPs) with predictable dimensions, via the simple injection of their ethanol solution in a buffer. The precise location of the functional groups in one-component IAJDs demonstrated that the targeted organs, including the liver, spleen, lymph nodes, and lung, are selected based on the hydrophilic region, while activity is associated with the hydrophobic domain of IAJDs. These principles, and a mechanistic hypothesis to explain activity, simplify the synthesis of IAJDs, the assembly of DNPs, handling, and storage of vaccines, and reduce price, despite employing renewable plant starting materials. Using simple molecular design principles will lead to increased accessibility to a large diversity of mRNA-based vaccines and nanotherapeutics.
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