Molecular design and synthesis of self-assembling camptothecin drug amphiphiles

Abstract

The conjugation of small molecular hydrophobic anticancer drugs onto a short peptide with overall hydrophilicity to create self-assembling drug amphiphiles offers a new prodrug strategy, producing well-defined, discrete nanostructures with a high and quantitative drug loading. Here we show the detailed synthesis procedure and how the molecular structure can influence the synthesis of the self-assembling prodrugs and the physicochemical properties of their assemblies. A series of camptothecin-based drug amphiphiles were synthesized via combined solid- and solution-phase synthetic techniques, and the physicochemical properties of their self-assembled nanostructures were probed using a number of imaging and spectroscopic techniques. We found that the number of incorporated drug molecules strongly influences the rate at which the drug amphiphiles are formed, exerting a steric hindrance toward any additional drugs to be conjugated and necessitating extended reaction time. The choice of peptide sequence was found to affect the solubility of the conjugates and, by extension, the critical aggregation concentration and contour length of the filamentous nanostructures formed. In the design of self-assembling drug amphiphiles, the number of conjugated drug molecules and the choice of peptide sequence have significant effects on the nanostructures formed. These observations may allow the fine-tuning of the physicochemical properties for specific drug delivery applications, ie systemic vs local delivery.

Figure 1

Illustration of the drug amphiphile concept. A hydrophilic peptide is conjugated to a hydrophobic drug via a degradable linker. Under physiological conditions, the synthesized drug amphiphiles described self-assemble into nanofibrous structures with a core micelle structure as shown in the representative transmission electron microscopy (TEM) image of mCPT-buSS-Tau (scale bar is 100 nm).

Figure 2

ESI-MS analysis of the reaction solutions during drug amphiphile synthesis. mCPT-buSS-Tau after 1 h (A), dCPT-buSS-Tau after 3 h (B), and qCPT-buSS-Tau after 18 h (C). 1×, 2×, and 3× CPT indicate singly, doubly and triply reacted products, respectively.

Figure 3

Preparative HPLC chromatograms of mCPT-buSS-Tau (A), dCPT-buSS-Tau (B), and qCPT-buSS-Tau (C) showing the reaction products formed during the synthesis of the drug amphiphiles. 1×, 2× and 3× CPT indicate incomplete reaction products.

Figure 4

Comparison of CPT conjugates with Tau and Sup35 peptide segments. (A) Chemical structures of dCPT-buSS-Sup35 and qCPT-buSS-Sup35. Representative TEM images of dCPT-buSS-Sup35 (B), dCPT-buSS-Tau (C), qCPT-buSS-Sup35 (D) and qCPT-buSS-Tau (E). All samples are 100 μmol/L in H₂O, except for dCPT-buSS-Tau which is 200 μmol/L. Scale bars are 100 nm.

Scheme 1

Solid-phase synthesis of the cysteine-functionalized Tau precursor peptides. Fmoc-GVQIVYKK-Rink was created using an automated peptide synthesizer and further modified by manual synthesis techniques. Reaction conditions: (a) (i) 20% 4-methylpiperidine in DMF, (ii) Fmoc-Lys(Fmoc)-OH, HATU, DIEA (4:3.98:6 per amine); (b) (i) 20% 4-methylpiperidine in DMF, (ii) Fmoc-Cys(Trt)-OH, HATU, DIEA (4:3.98:6 per amine), (iii) 20% 4-methylpiperidine in DMF, (iv) 20% acetic anhydride in DMF, DIEA; (c) TFA, TIS, H₂O (95:2.5:2.5); (d) TFA, TIS, H₂O, EDT (90:5:2.5:2.5).

Scheme 2

(A) Directed disulfide formation method used to synthesize the described drug amphiphiles and (B) molecular structures of the three drug amphiphiles mCPT-buSS-Tau, dCPT-buSS-Tau and qCPT-buSS-Tau and their respective drug loadings.