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

Abstract

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.

Figure 1

Schematic representation of the functionalized rHA-HJ biomolecular design formed by the annealing of complementary oligonucleotide modules (Q1, Q3, and Q4) to the rHA-oligonucleotide (Q2) scaffold. The green line represents the linker connecting Q2 to albumin and the white ribbon with blue sticks represent the oligonucleotide modules Q1, Q3, and Q4 functionalized with anti-EGFR nanobody (EgA1), Atto647, or Atto488 fluorophores, respectively.

Figure 2

Purification of rHA-Q2. (A) Cutout of the IEX-HPLC chromatogram showing absorbance at 260 nm indicating the oligonucleotide. The fraction number is shown below the chromatogram. (B) SYBR gold stained native PAGE gel of fraction 1–11 of the corresponding IEX-HPLC purification as shown above. # indicates a Q2 control lane. (C) Coomassie blue stained native PAGE gel of the pooled purified product consisting of the fractions free of excess free Q2 oligonucleotide (fraction 1–7 in frames (A) and (B)).

Figure 3

EgA1-UAA expression and oligonucleotide conjugation. (A) Coomassie stained SDS-PAGE gel. Lane 1: Molecular weight ladder, lanes 2, 3, and 4 are 10, 20, and 100 pmol of purified EgA1-UAA, respectively. (B) SYBR gold stained native PAGE gel. Lane 1: Unpurified Q1-EgA1 conjugate; lane 2: Gel purified Q1-EgA1 conjugate, lane 3: Q1 oligonucleotide control.

Figure 4

Formation of biomolecular assemblies visualized with native PAGE. (A) Lane 1: Q1-EgA1 control; lane 2: rHA-Q2 control; lane 3: Q3-Atto647 control, lane 4: HJ(Atto647), lane 5: HJ(EgA1, Atto647), lane 6: rHA-HJ, lane 7: rHA-HJ(Atto647), and lane 8: rHA-HJ(EgA1, Atto647). (B) Lane 1: Q1-EgA1 control, Lane 2: rHA-Q2 control, lane 3: Q3-Atto647 control, lane 4: Q4-Atto488 control, lane 5: HJ(EgA1, Atto647, Atto488), lane 6: rHA-HJ(EgA1, Atto647, Atto488). Black bands: SYBR gold signal; red bands: Atto647 signal, green bands: Atto488 signal, yellow bands: Overlap of the Atto647 and Atto488 signals. In the lane legend, “-” indicates presence of the corresponding non-functionalized strand (Q1–4) and “+” indicates functionalized strands (Q1-EgA1, rHA-Q2, Q3-Atto647, and Q4-Atto488).

Figure 5

Flow cytometric analysis showing levels of biomolecular assembly binding to EGFR negative 3T3 cells and EGFR positive 3T3-EGFR and A431 cells. (A) Representative chromatograms showing Atto647 fluorescence intensity in the three cell lines. (B) Quantitation of mean cell fluorescence relative to HJ(Atto647). N = 3 independent experiments; error bars indicate SD.

Figure 6

ELISA quantitation of FcRn-mediated cellular recycling of albumin biomolecular assemblies after incubation with FcRn-transduced human microvascular endothelial cell line-1 (HMEC-1-FcRn) cells. Buffer control is an albumin-free negative control. N = 3 independent experiments; error bars indicate standard deviation (SD). Statistics were performed using Student’s t test, **p < 0.01.

Figure 7

Circulatory half-lives of rHA-HJ(EgA1, Cy5.5) and HJ(EgA1, Cy5.5). Fluorescence intensity was measured and normalized to t₀ in blood taken at indicated time points. (A) Data in blood collected over 48 h. (B) Data in blood collected from time 0–6 h. N = 4 mice per group.