Polymeric PEG-based bioorthogonal triggers for prodrug activation in breast cancer

Abstract

Non-toxic prodrugs have proved of great value in medicinal chemistry programmes for cancer, due to their ability to selectively deliver toxic components at tumour sites once they are activated by a localised mechanism. Since activation of the prodrug to afford the toxic drug is a prerequisite for success of the approach, much interest has focused on the localised chemical and enzymatic mechanisms for activating the prodrugs. Bioorthogonal chemistry has positively impacted this area by providing biocompatible reactions that enable on-demand prodrug activation and active drug release. However, to be effective, it is essential that one of the components of the bioorthogonal reaction is localised at the tumour, in order to initiate the on-demand and on-target activation of the prodrug. Polymers such as poly(ethylene glycol) (PEG) are known to target solid tumours by passive targeting via the enhanced permeability and retention (EPR) effect. In this paper, the feasibility of derivatising long PEG chains to afford bioorthogonal activators (PEG-azide and PEG-tetrazine) for prodrug activation via the Staudinger ligation and the tetrazine ligation reactions, respectively, is evaluated. The molecular weight of the PEG in the activator and the type of linkage in the prodrug moiety were shown to significantly affect the rate of prodrug activation and hence the rate of drug release. In vitro cytotoxicity studies on breast cancer cells (MCF-7 and MDA-MB-231) showed ∼68-76% restoration of the parent drug's cytotoxicity for the Staudinger ligation-based prodrug activation strategy, and 100% restoration of the parent drug's cytotoxicity for the tetrazine ligation-based prodrug activation strategy. Restoration of doxorubicin's ability to intercalate with DNA upon activation of the prodrug by the PEG-activators was also demonstrated via fluorescence spectroscopy. Moreover, conjugation of the tetrazine bioorthogonal activator to a 10 kDa PEG polymer improved its serum stability in comparison with other reported tetrazine activators that completely lose their stability in serum over the same period of time. The feasibility of the combined passive targeting/bioorthogonal prodrug activation approach has therefore been demonstrated using a range of prodrugs, activation mechanisms, and in vitro assays.

Fig. 1. Schematic illustration of prodrug activation allowing release of the toxic drug specifically at the tumour cell by (i) delivery of the PEG-activator component to solid tumours through the EPR effect, (ii) activation of the prodrugs, that are purposefully designed to mask the toxicity of the active drug, using the PEG-activator through bioorthogonal Staudinger ligation and tetrazine ligation reactions.

Scheme 1. (a) Synthesis of PEG-Tz activator 3. Tz loading: 1.78% W/W, free Tz < 0.2% of total Tz. (b) Chemical structure of PEG-azide activator 4.

Scheme 2. (a) Synthesis of triphenylphosphine model ester prodrug 6. (b) Synthesis of triphenylphosphine-N-mustard prodrug 11. (c) Synthesis of triphenylphosphine-doxorubicin prodrug 13.

Fig. 2. (a) Effect of polymer molecular weight on release rate of 4-nitrophenol from model triphenylphosphine-prodrug 6via the Staudinger ligation reaction, release profile was monitored by HPLC as a function of time upon incubation of 6 with activators PEG-azide (10 kDa) or PEG-azide (20 kDa) or benzyl azide or without activators (control). (b) Release profile summary of doxorubicin 12 from triphenylphosphine-Dox prodrug 13via the Staudinger ligation reaction monitored by HPLC as a function of time upon its incubation with activator PEG-azide (10 kDa) 4 or without 4 (control). Data are presented as mean ± SEM (n = 3).

Scheme 3. Synthesis of (a) TCO-N-mustard prodrug 15 and (b) TCO-N-doxorubicin prodrug 16.

Fig. 3. (a) Release profile summary of 4-nitrophenol from model carbonate TCO-prodrug 14via the tetrazine ligation reaction monitored by HPLC as a function of time upon its incubation with activator PEG-Tz (10 kDa) 3 or without 3 (control). (b) Release profile summary of doxorubicin 12 from TCO-Dox prodrug 16via the tetrazine ligation reaction monitored by HPLC as a function of time upon its incubation with activator PEG-Tz (10 kDa) 3 or without 3 (control). Data are presented as mean ± SEM (n = 3).

Fig. 4. Statistical significance of the prodrugs IC₅₀ with or without activator. (a) Determined IC₅₀ of Doxorubicin 12 and its triphenylphosphine-prodrug 13 against MCF-7 and MDA-MB-231 cells and IC₅₀ of the prodrug 13 after activation by PEG-azide 4. (b) Determined IC₅₀ of triphenylphosphine-N-mustard prodrug 11 and the prodrug 11 after activation by PEG-azide 4 against MCF-7 and MDA-MB-231 cells. (c) Determined IC₅₀ of Doxorubicin 12 and its TCO-prodrug 16 against MCF-7 and MDA-MB-231 cells and IC₅₀ of the prodrug 16 after activation by PEG-Tz 3. (d) Determined IC₅₀ of TCO-N-mustard prodrug 15 and the prodrug 15 after activation by PEG-Tz 3 against MCF-7 and MDA-MB-231 cells. Data are presented as mean ± SEM (n = 3). ns represents no significance (p > 0.05), * indicates difference at the p < 0.05, *** indicates difference at the p < 0.001 significance level.

Fig. 5. Fluorescence spectra of DNA solution incubated with (a) Dox 12, TCO-Dox 16 and TCO-Dox 16 + PEG-Tz 3. (b) Dox 12, triphenylphosphine-Dox 13 and triphenylphosphine-Dox 13 + PEG-N₃4.

Fig. 6. Stability study of PEG-Tz 3 in 10% BSA in PBS and in 50% mouse serum/PBS at 37 °C monitored over 24 hours at λ = 520 nm. Data represented as mean ± SEM (n = 3).