Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery

Abstract

The design, synthesis, and biological evaluation of a new family of highly effective cell-penetrating molecular transporters, guanidinium-rich oligophosphoesters, are described. These unique transporters are synthesized in two steps, irrespective of oligomer length, by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers followed by oligomer deprotection. Varying the initiating alcohol results in a wide variety of cargo attachment strategies for releasable or nonreleasable transporter applications. Initiation of oligomerization with a fluorescent probe produces, upon deprotection, a transporter-probe conjugate that is shown to readily enter multiple cell lines in a dose-dependent manner. These new transporters are superior in cell uptake to previously studied guanidinium-rich oligocarbonates and oligoarginines, showing over 2-fold higher uptake than the former and 6-fold higher uptake than the latter. Initiation with a protected thiol gives, upon deprotection, thiol-terminated transporters which can be thiol-click conjugated to a variety of probes, drugs and other cargos as exemplified by the conjugation and delivery of the model probe fluorescein-maleimide and the medicinal agent paclitaxel (PTX) into cells. Of particular significance given that drug resistance is a major cause of chemotherapy failure, the PTX-transporter conjugate, designed to evade Pgp export and release free PTX after cell entry, shows efficacy against PTX-resistant ovarian cancer cells. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters.

Figure 1

Comparison of select oligomeric scaffolds for drug delivery to the oligophosphoesters described in this work, specifically highlighting ease of synthesis, backbone hydrophilicity, structural diversity, and aqueous stability.

Figure 2

Overview of synthetic methodologies employed to access guanidinium-rich oligophosphoester transporters. (a) OROP of a cyclic phospholane monomer for two-step access to guanidinium-functionalized oligophosphoesters for drug/probe delivery. (b) Methods of conjugation of drugs or probe molecules to form cell-penetrating oligomeric conjugates. Strategy 1: Initiation of oligomerization by drugs or probes containing a primary alcohol, such as the dansyl initiator (4). Strategy 2: Initiation of oligomers by trityl-mercaptohexanol (5) to produce, upon deprotection, oligomers containing a free thiol which can be conjugated to a variety of thiol-reactive drugs/probes, or attached through a redox-cleavable disulfide bond to form releasable drug conjugates.

Figure 3

Uptake of Dansyl-HexPhos oligomers compared to previously studied transporters. (a) Length dependence of uptake of Dansyl-HexPhos oligomers 6a–e in HeLa cells compared to Dansyl-Arg8 (8) and Dansyl-MTC-G8 (9). Cells were treated at 10 μM for 10 min. Fluorescence values are normalized to background fluorescence of untreated cells. (b) Cell line dependence of uptake of HexPhos8 in HeLa cells (blue), Jurkat cells (red), OVCA429 cells (green), and mouse 4T1 cells (purple). (c) Structures of previously reported transporter systems Dansyl-Arg8 (8) and Dansyl-MTC-G8 (9).

Figure 4

(a) Uptake of FL-maleimide to HeLa cells by click-coupling to thiol-initiated HexPhos oligomer 8 determined by flow cytometry. Maleimide control and HexPhos8 conjugate (11 and 12 respectively) were formed by reaction of 10 with the corresponding thiol. Fluorescence values are normalized to background fluorescence of untreated cells. (b) Representative flow cytometry histogram showing a complete shift in population fluorescence for cells treated with FL-HexPhos conjugate 12. (c) Structures of compounds used for FL-maleimide delivery.

Figure 5

Confocal microscopy images of HeLa cells treated with FL-HexPhos8 conjugate 12 (10 μM) for 10 min. Cell nuclei were counterstained with Hoechst 33342 and mitochondria stained with MitoTracker prior to imaging. Images were taken 10 min and 16 h following treatment. Scale bars represent 25 μm.

Scheme 1

Synthesis of Protected Guanidinylated Cyclic Phospholane Monomer 2-(6-bis-Boc-guanidino-hexyloxy)-1,3,2-dioxaphospholane-2-oxide (HexPhos, 3)

Scheme 2

Synthesis of PTX-HexPhos Conjugate 16 by Disulfide Exchange of Activated PTX-Disulfide 15 and Thiol-Initiated HexPhos Oligomer 7