An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.