title: Peptide-conjugated dextran hybrids: Generation of a versatile module for cytosolic delivery of biomolecular cargoes
Creators: name: Becker, Bastian
Publisher: name: UNSPECIFIED
Year: 2021

description: Along with steadily increasing interest in biopharmaceuticals such as peptides, proteins, or nucleic acids, development of strategies for their effective cytosolic delivery gained comparable attention. Indeed, being very potent in frames of bioactivity, these macromolecules do not readily enter the cytosol. Therefore, their application field is often restricted to extracellular targets. To overcome this limitation, and to achieve the ability to address intracellular targets, different approaches have been done aiming at improved cellular uptake. Beside strategies based on delivery of macromolecules with liposomes,[28-29] polymersomes,[30] nanoparticles,[32] or pore-forming proteins,[35-36] cell-penetrating peptides have emerged as a versatile tool for intracellular delivery of different cargoes. In particular, arginine-rich CPPs and their even more potent cyclic versions have numerously been reported as efficient delivery vehicles.[50, 52] In the frame of the present work, a system for efficient cytosolic delivery of biomolecular cargo was developed, based on CPP-bearing dextran hybrids. The initial study was focused on two arginine-free peptides. The 13 amino acid antimicrobial peptide aurein1.2 has been reported[68] to enhance cytoplasmic delivery by mediating endosomal escape of endocytosed proteins. The cationic amphiphilic 25 amino acid peptide L17E, a variant of haemolytic M lycotoxin with diminished cytotoxicity, was reported[56] to promote cytosolic uptake of different functional proteins up to full-length antibodies. So far, L17E-mediated cargo delivery was induced upon co-incubation with the peptide merely, and no efforts have been made to introduce a covalent linkage between both components, peptide and cargo. In the present study, both aurein1.2 and L17E were not able to promote cellular uptake when covalently linked to model conjugate in single unit. Hence, it remained to be investigated, whether an uptake-increasing multivalency effect could be generated by linkage of the respective peptide on a suitable scaffold in multiple copies. Comprising α-(1-6) glycosidic bonds between D-glucose repeating units, biocompatible dextran polysaccharide has already been reported as a suitable hydrophilic scaffold for the generation of high-DAR antibody-drug conjugates[76] or multivalent platform for DR5-induced apoptosis.[78] The hydroxy groups of the glucose repeating units as well as the polysaccharide reducing end offered possibilities for multivalent functionalization of dextran.[61, 76, 78] Therefore, 10 kDa dextran was chosen as platform for covalent oligomerization of cell-penetrating peptides aurein1.2 and L17E. To this end, amine functionality was introduced to the polysaccharide reducing end via reductive amination and the hydroxy groups of the glucose repeating units were modified via carboxyethylation. Subsequent conversion of the introduced carboxyl moieties into either maleimide- or azide-functionalities enabled peptide and/or cargo conjugation via maleimide-thiol addition or Cu(I)-catalyzed azide-alkyne cycloaddition. Decoration of maleimide-functionalized dextran polysaccharide with multiple copies of aurein1.2, respectively L17E, and a fluorescent label gave peptide-dextran hybrids, whose intracellular distribution was analyzed upon uptake in HeLa cells. L17E-dextran hybrid showed considerable superior uptake compared to the aurein1.2 variant. HeLa cells treated with TAMRA-labeled and L17E-decorated dextran module 37 at low micromolar concentration showed effective uptake of the macromolecule with fluorescence signals spread all over the cytosol and even nucleus (Figure 37, Figure 92). Furthermore, TAMRA-labeled dextran 37, covalently decorated with on average 3.8 L17E units, showed increased uptake compared to a TAMRA-labeled dextran co-incubated with solitary L17E peptide, as reported in the literature.[56] These encouraging findings led to development of a strategy for intracellular delivery of various cargo beyond fluorophore molecules, applying L17E-dextran hybrid as delivery module. A possible cytotoxic effect of L17E-dextran module was assessed, applying dextran 42, decorated with on average 4.8 L17E peptide units. In the taken cell line, an IC50 value of approximately 10 µM (Figure 41) was determined, which was rather low and in an acceptable range when taken into consideration that L17E was derived from naturally haemotoxic lycotoxin. L17E-dextran mediated cytosolic and nuclear delivery of peptide nucleic acid (PNA) was validated by mis-splicing correction assay in appropriate HeLa cells, stably transfected with enhanced green-fluorescent protein (eGFP) gene. Therefore dextran 43 was equipped with multiple copies of both L17E and PNA. The L17E-dextran module was able to deliver bioactive PNA into the nucleus of HeLa cells, whereupon the cargo PNA induced mis-splicing correction, leading to enhanced GFP fluorescence (Figure 45). This validated not only cytosolic, but even nuclear translocation of L17E-dextran module. Furthermore, the potential of dextran, equipped with multiple L17E peptides, was demonstrated to mediate intracellular uptake of PNA, that in general lacks cellular permeability and is not efficiently taken up by cells. Finally, L17E-dextran module showed its ability to promote intracellular delivery of covalently conjugated protein cargo. To this end, a construct was designed, combining eGFP as model protein with L17E-decorated dextran serving as cytosolic uptake-promoting module. Synthesis was performed via iEDDA conjugation of a norbornene-functionalized dextran, bearing multiple orthogonal azide moieties, to a methyltetrazine-modified eGFP protein, followed by CuAAC “click” with alkyne-modified L17E peptide. At low micromolar concentration, yielded L17E-dextran-eGFP 44 was efficiently taken up by HeLa cells, visualized by widely spread and homogenic eGFP fluorescence inside the cytosol (Figure 52). In conclusion, decoration of dextran polysaccharide with multiple copies of L17E peptides resulted in peptide-dextran hybrid, acting as a versatile uptake-mediating module. L17E-dextran promoted cytoplasmic delivery of covalently conjugated biomolecular cargo at low micromolar concentration without largely compromising cell viability. L17E has already been reported to promote cytosolic uptake of proteins, up to full-length antibodies, upon simple coincubation of solitary L17E peptide with the cargo.[56] However, uptake-mediating ability was improved via combining the oligomerization of L17E peptide on polysaccharide scaffold with the introduction of covalent connections between all counterparts: peptide, cargo, and dextran. In this way, a modular delivery system was established, which enabled intracellular delivery of various cargo, from PNA to proteins. Dependent on type and nature of the cargo, the multifunctionalized dextran backbone could be decorated following two strategies (Figure 59): One possibility included decoration of glucose repeating units with multiple copies of L17E and with multiple copies of cargo molecules simultaneously, while a further conjugation site at the reducing end remained accessible for conjugation of a potential second type of cargo. This option seemed to be beneficial for delivery of cargo such as peptides or PNA. The second possibility was based on decoration of the glucose repeating units with multiple L17E only, while the cargo was conjugated to the polysaccharide reducing end. The latter option seemed to be most suitable for more bulky cargo, such as proteins. In addition to the encouraging results of this proof-of-concept studies, several issues remain to be investigated. For solitary L17E co-incubation-mediated cytoplasmic cargo delivery an uptake mechanism was proposed based on transient membrane permeabilization. L17E was thought to induce actin rearrangement upon interaction with the cell membrane, leading to membrane ruffling followed by macropinocytosis. Before macropinosome formation was accomplished, L17E assumedly ruptured the ruffled membrane, thus paving the way of direct cargo entry into the cytoplasm.[70] It remains to be ascertained, whether the same mechanism is appropriate for L17E in its dextran-conjugated state. Moreover, regarding L17E-dextran mediated delivery of covalently conjugated protein cargo, it remains to substitute eGFP model protein with a functional biomolecule of interest. A broad range of intracellular targets exist, whose specific binding or functional blocking could be of interest in a therapeutic context. Single-domain antibodies, also called nanobodies, could be ideal candidates for addressing intracellular targets. Therefore, cytosolic delivery of these macromolecules would be of great importance and in the future, potentially could be realized via site-specific conjugation to L17E-dextran delivery module.