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The molecular mechanisms of CPP-mediated PCI are not completely understood.

While it is clear that PS-CPP conjugates might induce endosomal lysis by generation of ROS(124), the fact that Fl-CPPs might be photolytic can seem surprising. Indeed, the fluorophores used to synthesize Fl-CPP conjugates are widely used for various cell biology applications and they typically do not damage cellular membranes(125, 126).

However, fluorophores, like photosensitizer molecules, can act generate reactive oxygen species upon light excitation. This can occur in two different ways using namely Type I and Type II photosensitization reactions respectively(127).

ROS production by photosensitization

In a typical photosensitization reaction, the photosensitizer (Sen) molecule absorbs a photon upon light irradiation and gets excited to one or more higher energy states. The excited photosensitizer molecule (Sen*) then undergoes a series of internal reactions that produce reactive oxygen species, which can result in chemical alternation of a substrate molecule commonly proteins, lipids and DNA. A simplified scheme representing a photosensitization reaction is shown in Figure 1-4.

Type I photosensitization

In a Type I reaction, the excited photosensitizer (Sen*) reacts with the substrate molecule directly and as a result produces a radical ion by a one-electron transfer reaction in both the photosensitizer molecule and the substrate. Commonly, a substrate cation (Substrate+) and a photosensitizer anion (Sen-) are formed as substrate donates an electron(128). Further, upon reaction with molecular oxygen, both the substrate and photosensitizer radicals produce oxygenated products or ROS. In this reaction, the photosensitizer gets oxidized and cannot be recovered. Alternatively, however, the

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photosensitizer anion (Sen-) can transfer its extra electron directly to molecular oxygen to produce a superoxide radical (O²-). Here, the photosensitizer molecule is regenerated(128, 127).

Type II photosensitization

In a Type II reaction, the excited photosensitizer (Sen*) transfers its excess energy to the ground state molecular oxygen, in turn exciting oxygen to a singlet state.

This results in formation of singlet oxygen radicals(128, 127). Here the photosensitizer (Sen) returns to ground state and is regenerated. Singlet oxygen is an excited state radical that can promptly react with substrates to generate oxidized products(129).

Figure 1-4. Scheme for the production of ROS by a photosensitizer.Reprinted with permission from (128), “Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform” by Dai, T., Fuchs, B. B., Coleman, J. J., Prates, R. A., Astrakas, C., et al., 2012.Front Microbiol 3, 120, Copyright 2014 by Frontiers journal

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Types of reactive oxygen species- Superoxides, Hydroxyl and Singlet oxygen As a result of Type I photoreactions, products containing uneven number of electrons are formed which are typically highly reactive in nature. Superoxides and hydroxyl radicals are produced by the Type I reactions(127). These radicals can cause damage to substrates by reacting with them. Malachite Green, for instance is a known photosensitizer molecule that causes damage to biological molecules by production of hydroxyl radicals(130). The type I reactions are favored by high concentration of substrates as they involve a direct interaction between the excited photosensitizer (Sen*) and the substrate. These reactions are common at low oxygen concentrations since oxygen might compete with the substrate molecule for interaction with photosensitizer(127).

Excited singlet oxygen radical is formed as a result of transfer of energy from an excited photosensitizer to molecular oxygen in a type II photoreaction(128). Most excited photosensitizers undergo a process called intersystem crossing whereby they transition to a slightly lower energy level called the triplet state(131). Excited photosensitizers tend to have a longer lifetime and are likely to undergo an energy transfer reaction at this stage. Singlet oxygen radicals typically have a very low lifetime of 3-4.5 µs in water and in most biological environments(132). Singlet oxygen radicals can readily react with biological molecules such as lipids and proteins and thus, they have low diffusion ability unlike molecular oxygen(129). Hence type II reactions occur mostly within short distances from the place of formation of the singlet oxygen. This distance is estimated to be around 0.02 µm(132) and Type II photoreactions are likely to occur under conditions of high oxygen concentration and low substrate concentrations.

Interestingly, the small diffusion distances of the singlet oxygen and hydroxyl radical suggest that localization of the photosensitizer molecule plays an important role in the type of damage caused upon light irradiation. For instance, a lipophilic photosensitizer would accumulate in the membrane lipid bilayer. Singlet oxygen in the lipid bilayer has a lifetime double of that in an aqueous environment(132).

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Recent evidence suggests that the photolytic activity of the Fl-CPP TMR-TAT requires the production of reactive oxygen species such as superoxide and singlet oxygen. The singlet oxygen inhibitor crocetin has, for instance, been shown to reduce the light-induced endosomal escape of TMR-TAT inside cells(133). However, singlet oxygen quenchers such as α-tocopherol inhibit photolysis of RBCs(133). Also, lytic damage by TMR-TAT is inhibited in the presence of tiron, a superoxide quencher.

Together, these results support the notion that Fl-CPPs produce singlet oxygen and superoxide radicals inside endosomes upon light irradiation(134, 135). Using liposomes as membrane model systems, it has been shown that Fl-CPPs such as TMR-TAT bind to negatively charged phospholipids on the membranes. This binding is suggested to be necessary for the photolytic activity shown by TMR-TAT(135). Also, ROS produced by TMR-TAT can cause oxidation of lipids. Interestingly, arginine-rich CPPs showed more binding and lytic activity than lysine-rich CPPs, suggesting that the arginine residues of the CPPs are responsible for the lytic activity of TMR-TAT(134). The production of ROS by TMR-TAT and modulation of lytic activity shown by alteration of CPP sequences implies that in addition to ROS generation, the CPP moiety of Fl-CPPs contribute directly to Fl-CPP mediated photo-destruction of membranes(134, 135).

Studies show that a synergy between ROS produced by fluorophores and the CPP exists during photo-induced lysis by Fl-CPP conjugates(134, 135).