3 research outputs found
Toward Artificial Immunotoxins: Traceless Reversible Conjugation of RNase A with Receptor Targeting and Endosomal Escape Domains
The
specific transport of bioactive proteins into designated target
cells is an interesting and challenging perspective for the generation
of innovative biopharmaceuticals. Natural protein cytotoxins perform
this task with outstanding efficacy. They enter cells with receptor-targeted
specificity, respond to changing intracellular microenvironments,
and by various mechanisms translocate their cytotoxic protein subunit
into the cytosol. Here we imitate this toxin-based delivery strategy
in an artificial setting, by bioreversible conjugation of a cytotoxic
cargo protein (RNase A) with receptor-targeting PEG-folate and the
pH-specific endosomolytic peptide INF7 as synthetic delivery domains.
Covalent modification of the cargo protein was achieved using the
pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified
delivery modules. This linkage is supposed to enable traceless intracellular
release of the RNase A after exposure to the endosomal weakly acidic
environment. Delivery of RNase A via this polycation-free delivery
strategy resulted in high cytotoxicity against receptor-positive KB
tumor cells only when both PEG-folate and INF7 were attached
pH-Reversible Cationic RNase A Conjugates for Enhanced Cellular Delivery and Tumor Cell Killing
Intracellularly-acting
therapeutic proteins are considered promising
alternatives for the treatment of various diseases. Major limitations
of their application are low efficiency of intracellular delivery
and possible reduction of protein activity during derivatization.
Herein, we report pH-sensitive covalent modification of proteins with
a histidine-rich cationic oligomer (689) for efficient intracellular
transduction and traceless release of functional proteins. Enhanced
Green fluorescent protein (EGFP), as model for the visualization of
protein transduction, and RNase A, as therapeutic protein with antitumoral
effect, were modified with the pH-sensitive bifunctional AzMMMan linker
and varying amounts of cationic oligomer. The modification degree
showed impact on the internalization and cellular distribution of
EGFP as well as the biological effect of RNase A conjugates, which
mediated considerable toxicity against cancer cells at optimal ratio.
The presented conjugates demonstrate their qualification to achieve
efficient intracellular delivery and controlled release without protein
inactivation and potential prospective applications in protein-based
therapies
Adenoviral Vectors Coated with PAMAM Dendrimer Conjugates Allow CAR Independent Virus Uptake and Targeting to the EGF Receptor
Adenovirus type 5 (Ad) is an efficient gene vector with
high gene
transduction potential, but its efficiency depends on its native cell
receptors coxsackie- and adenovirus receptor (CAR) for cell attachment
and α<sub>v</sub>β<sub>3/5</sub> integrins for internalization.
To enable transduction of CAR negative cancer cell lines, we have
coated the negatively charged Ad by noncovalent charge interaction
with cationic PAMAM (polyamidoamine) dendrimers. The specificity for
tumor cell infection was increased by targeting the coated Ad to the
epidermal growth factor receptor using the peptide ligand GE11, which
was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles
were examined by measuring surface charge and size, the degree of
coating was determined by transmission electron microscopy. The net
positive charge of PAMAM coated Ad enhanced cellular binding and uptake
leading to increased transduction efficiency, especially in low to
medium CAR expressing cancer cell lines using enhanced green fluorescent
protein or luciferase as transgene. While PAMAM coated Ad allowed
for efficient internalization, coating with linear polyethylenimine
induced excessive particle aggregation, elevated cellular toxicity
and lowered transduction efficiency. PAMAM coating of Ad enabled successful
transduction of cells in vitro even in the presence of neutralizing
antibodies. Taken together, this study clearly proves noncovalent,
charge-based coating of Ad vectors with ligand-equipped dendrimers
as a viable strategy for efficient transduction of cells otherwise
refractory to Ad infection