Boronate-mediated biologic delivery

Abstract

Inefficient cellular delivery limits the landscape of macromolecular drugs. Boronic acids readily form boronate esters with the 1,2- and 1,3-diols of saccharides, such as those that coat the surface of mammalian cells. Here pendant boronic acids are shown to enhance the cytosolic delivery of a protein toxin. Thus, boronates are a noncationic carrier that can deliver a polar macromolecule into mammalian cells.

Figure 1

Boronation of RNase A and its putative mechanism for expediting cellular delivery. The location of each carboxyl group of RNase A is depicted in the ribbon diagram (PDB entry 7rsa(16)).

Figure 2

Elution profile of a mixture of unmodified RNase A (eluting in region A) and boronated RNase A (eluting in region B) from a column of immobilized heparin in the absence (solid lines) or presence (dashed lines) of fructose (0.10 M). Black lines, A_280 nm; gray lines, conductivity.

Figure 3

Fluorescence polarization assay of ribonucleases binding ganglioside-labeled liposomes in the presence or absence of 10 mM fructose. Data were normalized to the polarization of each ribonuclease incubated with nonextruded DOPC lipids. Each data point represents the mean ± SD for triplicate experiments. Asterisks indicate values with p < 0.05.

Figure 4

Internalization of unmodified and boronated RNase A into Pro-5 and Lec-2 cells in the absence or presence of fructose (0.25 M). Flow cytometry data were normalized to the internalization of unmodified RNase A into Pro-5 cells. Error bars represent SDs. Inset: Confocal microscopy image of live Pro-5 cells incubated for 4 h with boronated RNase A (5 μM) that had been labeled covalently with a green fluorophore. Nuclei were stained blue with Hoechst 33322 (2 μg/mL). Scale bar: 10 μm.

Figure 5

Inhibition of K-562 cell proliferation by unmodified and boronated RNase A: (●) unmodified RNase A (IC₅₀ >50 μM); (◆) boronated RNase A (IC₅₀ 4.1 ± 0.4 μM); (◇) boronated RNase A in the presence of fructose (50 mM) (IC₅₀ 9 ± 1 μM); (□) boronated RNase A alkylated with 2-bromoacetate (IC₅₀ >50 μM). The proliferation of K-562 cells was measured by the incorporation of [methyl-³H]thymidine. Each data point represents the mean ± standard error of the mean for three separate experiments performed in triplicate.