Thiol-Mediated Uptake Transport Systems

The development of thiol-mediated uptake transporters started from a general observation that molecules are easier to be uptaken when a thiol reactive functionality was integrated into their structures. One of the first studies was reported by Ryser et al. in 1990.¹⁴⁵ An undegradable polylysine was covalently linked to a radioiodinated tyramine through a disulfide bridge and its cellular uptake was monitored over time. Uptake kinetic showed that the uptake of the conjugate was very rapid and efficient. The missing of the lag phase in the release of radioiodinated residue proved that disulfide cleavage occurred spontaneously at the cell surface. This observation was consistent with inhibition experiment, when the cleavage of disulfide bonds was significantly suppressed after treatment of the cells with DTNB.¹⁴⁵ Behr et al. introduced the first thiol-mediated uptake delivery system for gene transfection in 1995.¹⁴⁶ Transfecting particles were prepared by mixing lipospermine, DNA and a thiol-reactive derivative of phosphatodylethanolamine (PEA). This novel system led to several order of magnitude increasing in transfection yields. Transfection efficiency was correlated well with the reactivities of reactive fragments of PEA derivatives. The contribution of thiol-mediated uptake, in this case, was believed to be the reactions between the thiol-reactive

59 functions of the particles with the sulfhydryls to attach them to the cell surfaces. Subsequent absorptive endocytosis allowed the particles to internalize into cells.¹⁴⁶ While examined the cellular uptake of some cell-penetrating peptides (CPPs) containing disulfide bridges, Sagan et al. found that these CPPs could react with thiols of membrane proteins and form covalent bonds.¹⁴⁷ This reactivity increased the amount of peptides which bound to cell membrane and facilitated their internalization.

These early studies were followed up by more rational designs of delivery systems utilizing thiol-mediated uptake. One of the most promising thiol-mediated uptake transporters is cell-penetrating poly(disulfide)s (CPDs). CPDs like 112 could be synthesized under the mildest conditions (Figure 34).¹⁴⁸ Easily to prepare, various cargos could be attached to the polymer chain shortly before delivery. A thiol group in the structure of the cargos 113 would initiate a disulfide exchange cascade of monomer 114 to grow the disulfide polymer chain. The polymerization was terminated with a thiol blocking reagent such as 115. The structure of the polymers could be modified by simply applying modification at monomer level.^149–151 For monomer 114, a guanidinium functionality was introduced to couple CPPs uptake mechanism with thiol-mediated uptake. CPDs 112 was shown to be very efficiently uptaken by cells. The polydisulfide chain allowed 112 to be degraded and release cargos upon entering the cytoplasm due to high concentration of glutathione (GSH) inside cells. Thus, cytotoxicity was significantly lower in comparison with standard CPPs.¹⁴⁸ Using this transporter, cargos as big as streptavidin could be delivered to the cytosol with minimal endosomal capture.^149,152 CPDs were also tolerated for delivery of other advanced systems such as artificial metalloenzyme,¹⁵³ quantum dots,¹⁵⁴ nanoparticles and proteins.^155–160

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Figure 34. Polymerization of monomer 114 to form CPD 112 and CPD degradation inside the cytoplasm. Adapted from reference.¹⁴⁸

The power of thiol-mediated uptake was demonstrated through a series of small molecule transporters. Even though they are much smaller in comparison with CPDs, their uptake activities have been shown to be as good as or even superior. The attention was first focused on the correlation between ring strain in cyclic disulfide compounds and their uptake efficiency. A fluorescent probe was equipped with cyclic disulfides with different tension (Figure 35).^134,161 As expected, cellular uptake increased with increasing ring tension in the

61 disulfide rings. From dithiane ring 116 with the CSSC dihedral angle 62^o to 35^o in lipoic acid derivatives 117, almost double fluorescent intensity was recorded. Even a small increase in ring tension of AspA derivative 118 caused significant improvement in cellular uptake.¹³⁴ This trend was very consistent with Epidithiodiketopiperazine 119 (ETP) was the best transporter in this series. Having the CSSC dihedral angle close to 0^o, the ring tension in ETPs reach the maximum. Ring-opening disulfide exchange in ETPs occurs much faster and results more than 20 times increase in uptake compared with AspA transporter.¹⁶¹

Figure 35. Cyclic disulfide transporters and their CSSC dihedral angle. Adapted from reference.131,134,161

Considering faster diselenide exchange reactions compared with thiolate-disulfide, replacement of sulfur by selenium has been employed to improve the activity of existed thiol-mediated uptake transporters.¹³⁶ Even for the relaxed linear diselenides, their

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uptake activity surpassed ETPs. Not surprisingly, decrease CSeSeC dihedral angle in diselenolipoic acid (DiSeL) further improved uptake efficiency.^136,162 Tackling the ring tension from a different perspective, Wu et al. introduce CXC cyclic γ-turn peptides as potential thiol-mediated uptake transporters (Figure 36).^163,164 The 11-membered ring in cyclic γ-turn peptides 120 experienced significant Prelog strain. Thus, the linear CXC motif was more oxidation resistant to form the ring-closed disulfide in comparison with other popular Cysteine-rich peptide motifs in biology such as CXXC or CC. Because of that, upon ring-opening, cyclic CXC tended to form mixed disulfide bonds with other thiols. The doubly bridged disulfides created between transporter and transmembrane proteins could explain the superior transport activity of cyclic CXC γ-turn peptides since the attachment of transporter to cell membrane for further translocation was greatly enhanced.^163,164

Figure 36. General structure of cyclic disulfide CXC γ-turn peptides.

The dynamic nature of thiol-disulfide exchange in thiol-mediated uptake was exploited to its maximum with the introduction of benzopolysulfane (BPS) transporter.¹⁶⁵ In solution, BPS 121 created a library of BPSs having different sulfur ring sizes, with pentasulfide 121 is the most stable, followed by trisulfide 122. In the presence of thiolates and disulfides, the

63 library could be extended to the extreme with the formation of BPS oligomers together with acyclic members (Figure 37).

Figure 37. Library of transporters created by dynamic covalent exchange of BPS 121, with selection for the best transporters to be uptaken. Adapted from reference.¹³¹

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This rich dynamic covalent network allowed cells to select the best transporter within the library for uptake. Re-equilibrium of the whole system pushed for the amplification of the best transporter. This reactivity made BPS 121 the best small molecule thiol-mediated uptake transporter known to date.^131,165

Based on these small transporters, more advanced delivery systems have been developed.

For example, cell-penetrating streptavidin bearing BPSs has been introduced for delivery of multiple cargos at the same time.¹⁶⁶ The potential of using multivalency to transport giant cargos through lipid bilayer membrane was also addressed with versicles containing small transporter at their surface or oligomers of cyclic oligochalcogenides.^167,168 Being non-toxic and very versatile, thiol-mediated uptake is one of the best methods so far to cross the cellular membrane.