While counterion-mediated uptake relies on the formation of ion-pairs with exofacial anions, thiol-mediated uptake exploits the interaction with exofacial thiols. As previously mentioned, disulfides can be introduced in transporters to achieve the release of the cargo through glutathione-mediated reduction.
However, it was also found that the introduction of disulfides could improve the cellular uptake. Most of the examples in literature coupling the presence of
disulfides or thiols to efficient cellular uptake were overlooked,[41] until Sagan and coworkers proposed in 2009 that cell-surface thiols mediated the entry of a disulfide-engineered cell-impermeable peptide, a protein kinase C inhibitor (PKCi). Figure 7 shows how cell entry can be achieved only when a disulfide bridge is used to conjugate two molecules of PKCi. Moreover, a decrease in uptake was observed when the exofacial thiols were oxidized using DTNB (5,5-dithio-bis-(2-nitrobenzoic acid)), the so-called Ellman’s reagent.[42]
Figure 7. Cellular uptake of PKCi achieved without (left) and with (right) the presence of a disulfide bond. Image from reference.[42]
Moreover, in 2012, Gait and coworkers showed that the activity of a PNA containing a cysteine residue was enhanced compared to an analogue in which the thiol of the cysteine was alkylated (Figure 8).[43] These findings strongly suggested that the thiol was responsible for the increase in activity and led Gait to hypothesize that disulfide exchange between thiolated transporters and exofacial thiols could occur, leading to the concept of thiol-mediated uptake.[41]
Figure 8. Cellular uptake of a PNA sequence modified without (red) and with a free (blue) or an alkylated cysteine (green). Image from reference.[43]
Disulfide bridges have also been introduced in polymeric materials such as the capsules developed by Caruso and coworkers in 2011.[44] As shown in Figure 9, the capsules could be delivered into HeLa cells only when cross-linked with disulfides and not when thioethers or amides were used.
Figure 9. Structure and cellular uptake of polymeric capsules cross-linked with a) disulfides, b) thioethers and c) amides. Figure from reference.[44]
a) b) c)
Polymers containing disulfides, that is poly(disulfide)s, have been most commonly used as delivery systems to introduce bioreducible bonds to enable the release of the cargo they are carrying.[45] In particular, in the case of nucleic acid delivery, cationic poly(disulfide)s are used to form polyplexes that protect the nucleic acids, mediate their transport through mainly endocytosis and release them into the cytoplasm or the nucleus due to the high concentration of GSH that reduces the disulfide bonds, as shown in Figure 10.[46]
Figure 10. Nucleic acid delivery using cationic poly(disulfide)s. Image from reference.[46]
Poly(disulfide)s can be synthesized using Michael polyaddition of disulfide containing monomers, like commercially available bisacrylamide 12, to primary or secondary aliphatic amines to give poly(amido amine) 13 (Figure 11).[47]
Another strategy involves the use of disulfide containing crosslinkers, such as imido ester 14, on polyamines to give permanently charged amidinate salts 15.[48]
Controlled radical polymerizations, such as RAFT (reversible addition-fragmentation chain transfer) are also employed to obtain polymers with well-defined end groups containing thiols, such as 16, which can then undergo a
post-synthesis of cationic polypeptides, like 17, and then the thiols oxidized using mild oxidizing agents, such as dimethylsulfoxide.[50]
Figure 11. Common methods for the synthesis of cationic poly(disulfide)s: a) Michael polyaddition, b) disulfide crosslinking, c) RAFT polymerization and d) polypeptide synthesis. Figure adapted from reference.[46]
Matile and coworkers introduced in 2013 a different type of strategy to obtain cationic poly(disulfide)s that uses disulfide exchange. The procedure takes inspiration from the so-called self-organizing surface-initiated polymerization (SOSIP) developed in the group in 2011. As shown in Figure 12, the disulfide-containing initiator 18, which is bound to an indium tin oxide (ITO) electrode, is activated to the corresponding thiolate 19 under basic conditions.
Polymerization occurs by using derivatives of asparagusic acid 20, a strained cyclic disulfide, through disulfide exchange, generating new thiolates that propagate the reaction to build a polymeric architecture.[51]
12
Figure 12. Self-organizing surface-initiated polymerization. Figure adapted from reference.[51]
The concept of substrate-initiated disulfide exchange polymerization was then applied to obtain cationic poly(disulfide)s as novel CPP mimics, named cell-penetrating poly(disulfide)s (CPDs). As shown in Figure 13, a thiolated initiator is used to attack the disulfide-containing monomer. The monomer contains a strained cyclic disulfide and, when the new disulfide bond with the initiator is formed, the ring tension is released and a thiolate generated which will in turn attack the disulfide of a second monomer unit and, therefore, propagate the polymerization. To terminate the exchange, a thiol-alkylation reaction is performed using a iodoacetamide derivative.[52]
N N
Figure 13. Substrate-initiated disulfide exchange polymerization to obtain cell-penetrating poly(disulfide)s.
The CPP-mimic characteristic of the CPDs originates from the presence of a guanidinium group in the monomer leading, after polymerization, to a cation-rich poly(disulfide). The most commonly used initiators are thiolated fluorophores, such as 21-22, green-emitting and red-emitting respectively, in order to track the cellular uptake of the resulting CPDs. While commercially available iodoacetamide 23 is used as the terminator of the polymerization.
Among the different monomers that were tested over the years, the ones that gave CPDs with the highest activity are lipoic acid derivatives 24-25, as shown in Figure 14.
Initiator S S S S S
NH NH2 H2N
NH NH2 H2N
S S NH NH2 H2N
I N
H
O Terminator
polymerization
S S
S n
O NH NH
NH2 H2N
Figure 14. Building blocks used to synthesize cell-penetrating poly(disulfide)s:
initiators 21-22, terminator 23 and monomers 24-25.
Interestingly, it was found that small structural modifications of the lipoic acid sidechain, such as the introduction of aromatic groups, led to a loss of cellular uptake efficiency.[53] Moreover, in order to study the effect of a wide variety of functionalities, such as branching, π-acidic surfaces or binding to glycosaminoglycans using boronic acids, co-polymerization was developed, as shown in Figure 15.[54]
O
HO O
COO
NH O O
O SH I O NH2
22 O
N N
COO
NH O O
O SH
21 23
S S NH
O OO HN
H2N
NH2 S S
NH H O N NH2 H2N
24 25
Figure 15. Substrate-initiated co-polymerization.
As mentioned previously, fluorescent CPDs grown using monomers 24-25 were able to reach the cytosol, nucleus and nucleoli of HeLa cells after only 15 minutes of incubation at 37 °C using 500 nM concentration (Figure 16).
Moreover, compared to CPP reference poly-arginine 1, no toxicity could be observed up to 10 µM concentration suggesting that, similar to other poly(disulfide) scaffolds, the high concentration of GSH enables the depolymerization of the polymer and results in lack of cytotoxicity.[53]
Mechanistic investigations revealed insensitivity towards endocytosis inhibitors, such as chlorpromazine (which is a marker for clathrin-mediated), wortmannin (macropinocytosis) and methyl-β-cyclodextrin (caveolae-mediated). Interestingly, sensitivity towards exofacial thiols was observed when thiol-mediated uptake was inhibited using DTNB. Together these findings suggested that, in addition to counterion-mediated uptake due to the presence of the guanidinium groups, thiol-mediated uptake also strongly contributed to the efficient cell entry of the CPDs.[53]
S S S S S I NH2 O
polymerization
S S S S
O NH2 S
n m
n m
Figure 16. Cellular uptake of best-performing CPDs into HeLa cells: a) confocal laser microscopy (CLSM) images, b) cytotoxicity (poly-arginine ¡, CPD ¨) and c) DTNB inhibition. Figure from reference where CPDs 1, 4 bear propagators 24 and 25, respectively.[53]
Further studies on the cellular uptake of the CPDs revealed dependency of the intracellular localization on the molecular weight: longer CPDs were able to reach the nucleus and nucleoli, while shorter ones either stayed trapped in endosomes or localized preferentially in the cytosol.[55] Indeed, CPDs bind to the cell surface by counterion exchange with exofacial anions like common CPPs, but the shortest ones cannot outcompete endocytosis and therefore remain trapped in endosomes. With increasing length, however, thiol-mediated uptake becomes more important since CPDs, bearing a poly(disulfide) backbone, can also covalently bind exofacial thiols inducing a faster formation of transient micellar pores which enables them to reach the cytosol (Figure 17). Moreover, if the polymers are long enough, they can escape complete depolymerization by GSH, reaching nucleus and nucleoli. [21]
a) b) c)
Figure 17. Dual mechanism of entry of cell-penetrating poly(disulfide)s:
counterion-mediated (left) and thiol-mediated (right). Image adapted from reference.[21]
An important application of the CPDs is the delivery of proteins through the use of biotin-streptavidin technology. As shown in Figure 18, the biotinylated green-emitting polymer 26 was interfaced with streptavidin 27.[56] Streptavidin is a 53 kDa tetramer of β-barrels which is known for its high affinity with biotin (KD = 10-15 M). In this study, the four sites of the protein were labelled with both red-emitting biotinylated fluorophore 28 and CPD 26 to give complex 29. The complex reached the nucleus and nucleoli of HeLa cells and this general strategy was used for the delivery of larger cargos, such as quantum dots and nanobodies by the Matile group,[57] but also proteins, monobodies and nanoparticles by the Yao group.[58-59]
Figure 18. General strategy for protein delivery with CPDs with complex 29 obtained using biotinylated CPD 26 and streptavidin 27 labelled with biotinylated fluorophore 28. Figure adapted from reference.[56]
The contribution of thiol-mediated uptake to the efficient delivery of cargos was investigated further by Matile and coworkers also using small molecules. In 2015, they were able to demonstrate that by increasing the ring tension, in this case the C-S-S-C dihedral angle of cyclic disulfides, uptake efficiency in HeLa cells increased accordingly. In a first study, the more strained asparagusic acid 30, depicted in Figure 19, with a dihedral angle of 27° was found to have superior uptake compared to less strained lipoic acid 31 (35°).Inhibition was observed when the exofacial thiols were oxidized or alkylated while uptake was promoted when exofacial disulfides were reduced, suggesting the occurence of disulfide
[60]
entry. Moreover, the conjugation of asparagusic acid to a cell-impermeable peptide enables it to be delivered and to exercise its apoptotic function.[61]
Recently, the highest degree of ring tension was achieved with epidithiodiketopiperazine (ETP) 32 (dihedral angle of near 0°) with a 20-fold increase in the uptake efficiency of the model fluorophore to the cytosol and nucleus compared to 30 (Figure 19).[62]
Figure 19. Thiol-mediated uptake with increasing ring tension from lipoic acid 30 to asparagusic acid 31 and ETP 32.
In conclusion, this first chapter shows that there are two mechanisms that can be exploited to enable cellular uptake: countermediated, exploiting ion-pairing with exofacial anions by using positive charges like guanidiniums, and
SS
S S HN
O O NH
S 35°
S 27°
S S S S
S S
S S
SS S S
S 0°
S S N N
HN O
O O
30 31 32
Increased Uptake with Increased Strain
thiol-mediated, exploiting disulfide exchange with exofacial thiols by using disulfides. When dealing with polymers, such as the CPDs, motifs that can enable cellular uptake through these described mechanisms could be installed through, among others, modification of their sidechain. The next chapter will deal with strategies that can be adopted for polymer sidechain engineering.