Supramolecular Crafting of Self-Assembling Camptothecin Prodrugs with Enhanced Efficacy against Primary Cancer Cells

Abstract

Chemical modification of small molecule hydrophobic drugs is a clinically proven strategy to devise prodrugs with enhanced treatment efficacy. While this prodrug strategy improves the parent drug's water solubility and pharmacokinetic profile, it typically compromises the drug's potency against cancer cells due to the retarded drug release rate and reduced cellular uptake efficiency. Here we report on the supramolecular design of self-assembling prodrugs (SAPD) with much improved water solubility while maintaining high potency against cancer cells. We found that camptothecin (CPT) prodrugs created by conjugating two CPT molecules onto a hydrophilic segment can associate into filamentous nanostructures in water. Our results suggest that these SAPD exhibit much greater efficacy against primary brain cancer cells relative to that of irinotecan, a clinically used CPT prodrug. We believe these findings open a new avenue for rational design of supramolecular prodrugs for cancer treatment.

Keywords: CPT Prodrug; brain cancer; high potency; nanomedicine; peptides.; self-assembly.

Figure 1

(a) Schematic illustration of the concept for rationally designed self-assembling prodrugs (SAPD) with high potency. (b) Chemical structures of the studied three model drugs: dCPT-K₂, dCPT-OEG₅-K₂ and dCPT-Sup35-K₂. Two CPTs were conjugated to three different hydrophilic auxiliary segments through disulfanyl-ethyl carbonate linker (etcSS).

Figure 2

Molecular assembly and characterization of designed SAPDs. Representative TEM images of nanotubes formed by dCPT-K₂ with diameter of 8.2 ± 0.8 nm (a) and dCPT-OEG₅-K₂ with diameter of 7.5 ± 0.7 nm (b). (Insert) High resolution TEM images display the tubular morphology. (c) TEM image of nanofibers formed by dCPT-Sup35-K₂ with diameter of 6.9 ± 1.4 nm. (d) Normalized CD spectra of the three studied supramolecular prodrugs in water. All the samples were prepared at concentration of 100 μM in water and aged over three days before measurements. TEM samples were negatively stained with 2 wt% uranyl acetate and all scale bars = 100 nm. More TEM images can be found in the SI (Figure S4-S6).

Figure 3

Stability study of nanostructures formed by dCPT-K₂ (a), dCPT-OEG₅-K₂ (b) and dCPT-Sup35-K₂ (c) after dilution to 50 μM, 12.5 μM and 3.125 μM in water, as monitored by CD. All the diluted solutions were aged over three days before CD measurements. Corresponding CD results showed no significant changes for both dCPT-K₂ and dCPT-OEG₅-K₂. However, a slight decrease in signal of nanofibers formed by dCPT-Sup35-K₂ was observed, indicating partial dissociation of the supramolecular nanostructures into smaller aggregates and/or prodrug monomers.

Figure 4

Cumulative drug release from dCPT-K₂ (a), dCPT-OEG₅-K₂ (b) and dCPT-Sup35-K₂ (c) in PBS at 37℃ at 25 μM with (blue curve) or without (red curve) 10 mM GSH. Data were given as mean ± s.d. (n = 3). For samples with 10 mM GSH, they presented ultra-fast release profiles (100% CPT released out for dCPT-K₂ (a), dCPT-OEG₅-K₂ (b) and 81.4% CPT released out for dCPT-Sup35-K₂ (c) within 1h). For samples without GSH, around 20% CPT released out at 72 h.

Figure 5

In vitro cytotoxicity study of the SAPDs against human brain cancer cell lines U87 MG (a) and intraoperative derived human cancer cell lines (612 (b) and 965 (c)). All cancer cells were incubated with the appropriate SAPDs for 72 h and cell viability was determined by SRB assay. Data were given as mean ± s.d. (n = 3) and IC₅₀ values were calculated according to CPT concentration. All the SAPDs exhibited greater efficacy against three human brain cancer cell lines than irinotecan.