Balancing Chemical and Supramolecular Stability in OEGylated Supramolecular Polymers for Systemic Drug Delivery

Abstract

The chemical conjugation of poly(ethylene glycol) (PEG) to therapeutic agents, known as PEGylation, is a well-established strategy for enhancing drug solubility, chemical stability, and pharmacokinetics. Here, we report on a class of supramolecular polymeric prodrugs by utilizing oligo(ethylene glycol) (OEG) to modify the hydrophobic anticancer drug camptothecin (CPT). These OEGylated prodrugs, despite their low molecular weight, spontaneously self-assemble into therapeutic supramolecular polymers (SPs) with a tubular morphology, featuring a dense OEG coating on the surface. By designing biodegradable linkers with varying chemical stabilities, we investigated how the release kinetics of CPT influence the in vitro and in vivo performance of these SPs. Our findings demonstrate that self-assembling prodrugs (SAPDs) with a self-immolative disulfanyl-ethyl carbonate (etcSS) linker exhibit a faster drug release rate than those with a reducible disulfanyl butyrate (buSS) linker, leading to higher potency and significantly improved antitumor efficacy. Notably, two stable tubular SPs, Tubustecan (TT) 1E and TT 7E, outperformed irinotecan─a clinically approved CPT prodrug─in a colon cancer model, achieving enhanced tumor growth inhibition and prolonged animal survival. These results highlight the potential of supramolecular OEGylation as an important strategy for engineering drug-based supramolecular polymers and underscore the critical role of chemical stability vs supramolecular stability in optimizing supramolecular prodrug design.

Figure 1.

Monomer design and supramolecular assembly of OEGylated tubustecan 1 (TT 1) with different chemical stability. (A) Chemical structure of TT 1 with an etcSS linker (TT 1E) and a buSS linker (TT 1B), respectively. (B) Schematic illustration of self-assembly of TT 1 into tubular supramolecular polymers with the corresponding etcSS (E) or buSS (B) chemical linkers. Representative cryo-TEM images of SP TT 1E (C) and SP TT 1B (D) at the concentration of 2 mM. (E) Circular dichroism (CD) spectra of assembled TT 1E and TT 1B in water at the concentration of 200 μM.

Figure 2.

In vitro GSH-induced drug release, cytotoxicity and supramolecular stability of TT 1. (A) Cumulative release plot of the free CPT from TT 1E and TT 1B solutions (200 μM) in PBS containing 10 mM GSH at 37°C (n = 3). Representative HPLC curves of GSH-treated TT 1E (B) and TT 1B (C) solutions at different time points, showing the formation of intermediate species. Comparison of cytotoxicities of TT 1E and TT 1B in inhibiting HCT-116 (D) and HT-29 (E) colon cancer cells after 72 h incubation, respectively. (F) Critical micellization concentration (CMC) study of TT 1B using a Nile Red assay. TT 1B exhibits a CMC of ~3.2 μM, similar to that of TT 1E.

Figure 3.

Proposed drug release mechanisms of conjugated designed with different CPT-etcSS and CPT-buSS linkers. (A) Schematic representation of how carbonate-based etcSS and ester-based buSS linkers influence the release of free CPT (3). TT 1E directly degrades into free CPT and the peptide auxiliary, whereas TT 1B undergoes stepwise degradation into CPT-SS-CPT (1), CPT-SH (2), free CPT (3), and the peptide moiety. (B) Prodrugs with the self-immolative etcSS linker rapidly convert into free CPT through an intramolecular degradation pathway. In contrast, those with the buSS linker initially degrade into CPT-SH, which subsequently dimerizes into CPT-SS-CPT. The final release of free CPT requires additional hydrolysis of CPT-SH and CPT-SS-CPT. (C) Illustration of the proposed nanostructure-promoted disulfide dimer formation occurring in close proximity to supramolecular assemblies, which may hinder further conversion into free CPT.

Figure 4.

Comparison of in vivo antitumor efficacy of TT 1E and TT 1B in nude mice bearing HT-29 tumors. Mice were intravenously injected with PBS (control), irinotecan (66.7 mg/kg), TT 1E (12 mg/kg, CPT equivalent), or TT 1B (12 and 36 mg/kg, CPT equivalent) on days 1, 5, and 9 (black arrows) (n = 5 per group). Tumor volume (A), body weight (B), and cumulative survival (C) were monitored and plotted.

Figure 5.

Design and self-assembly of OEGylated TT 7. (A) Chemical structure and cartoon illustration of TT 7. (B) Schematic illustration of self-assembly of TT 7 into short tubular supramolecular polymers. Representative cryo-TEM images of assembled TT 7E (C) and TT 7B (D) at the concentration of 1 mM. Short tubular supramolecular polymers were formed with average length around 50 nm (n > 100) and diameter around 9 nm (n > 50) for both TT 7E and 7B.

Figure 6.

In vitro and in vivo study of TT 7. Comparison of cell cytotoxicities of TT 7E and TT 7B against HT-29 colon cancer cells (A), A549 non-small cell lung cancer cells (B), and HepG2 liver cancer cells (C) with free CPT as control (n ≥ 3, 72 h incubation). Antitumor efficacy study of TT 7E in nude mice bearing HT-29 tumors (D – F) and HCT-116 tumors (G – I). TT 7E (10 mg/kg, CPT equivalent, n = 7 for HT-29 and n = 5 for HCT-116) was i.v. injected on days 1, 5 and 9 with blank PBS (n = 8 for HT-29 and n = 5 for HCT-116) and irinotecan (66.7 mg/kg, n = 7 for HT-29 and n = 5 for HCT-116) as controls. Tumor volume (D and G), body weight (E and H) and cumulative survival (F and I) plots of mice. Loss of mice is a result of treatment-related death or killing after predetermined end point was reached.

Scheme 1.

Summary of synthesized OEGylated self-assembling prodrugs