Molecular tools to generate reactive oxygen species in biological systems

Abstract

Reactive oxygen species (ROS) not only are by-products of aerobic respiration, but also play vital roles in metabolism regulation and signal transductions. It is important to understand the functions of ROS in biological systems. In addition, scientists have made use of ROS to kill bacteria and tumors through a process known as photodynamic therapy (PDT). This paper provides a concise review of current molecular tools that can generate ROS in biological systems via either non-genetic or genetically-encoded way. Challenges and perspectives are further discussed with the hope of broadening the applications of ROS generators in research and clinical settings.

Graphical Abstract

1. Introduction

Reactive oxygen species (ROS), including radicals and non-radical molecules, such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radical (HO•), and singlet oxygen (¹O₂), have gained interest for decades as harmful by-products of cellular metabolism.^1–4 The production of ROS occurs constantly in chloroplasts, mitochondria, peroxisomes, and the cytosol during photosynthesis and the aerobic respiration process. More importantly, recent work has uncovered ROS as vital cellular signaling molecules in maintaining homeostasis of many physiological processes, such as the cell cycle, apoptosis, autophagy, and immunity.^5,6 When the balance between generation and consumption of ROS is dysregulated, diseases may occur.⁷ Studies have shown that many cancer cells are well adapted to oxidative stress due to their flexible redox regulation systems.⁸ As such, modulating the redox status of cancer cells has long been recognized as an effective strategy for cancer treatment.⁸ Photodynamic therapy (PDT), during which a large amount of ROS are generated by exposure of photosensitizers to excitation light, has indeed become a widely used therapeutic approach for superficial cancer.^9–11 Moreover, PDT has shown promise as a new approach to combat drug-resistant micro-pathogens, including Gram-positive and Gram-negative bacteria, yeasts and fungi.¹² Photosensitizers are typically synthetic molecules or nanoparticles. In addition to these conventional ROS generators, the adventure of using fluorescent proteins and enzymes as genetic encoded ROS generators has created another important research domain. Moreover, synthetic photosensitizers have been integrated with genetically encoded elements to form hybrid ROS generators. This paper, which is not meant to be a thorough review, will convey these basic concepts through a few selected examples.

2. Photodynamic therapy (PDT)

2.1. Brief history of PDT

The history of PDT can be traced back to ~1900.¹³ Oscar Raab, a German chemist, firstly proposed such conception after observing that micro-organisms could be killed by light when co-cultured with certain dyes.^13,14 This phenomenon inspired him and his followers to investigate further and subsequently identify oxygen as an indispensable factor for antibiotic performance. The practice of PDT on skin cancer research was conducted not long after the initial attempt on antibiotic studies.⁹ However, due to World War II, further development of PDT was largely delayed between 1940s and 1960s. In 1970s, the stagnation was ended by Dr. Thomas Dougherty who introduced “hematoporphyrin derivatives” (HpDs), mixtures of water-soluble porphyrins, into PTD.¹⁵ Although HpDs had several practical problems, such as inefficient absorption in the far-red and near-infrared spectral region and low in vivo clearance rate, this seminal work has attracted intensive attention and paved the way for development of modern photosensitizers.^13,16 Since then, hundreds of photosensitizers have been chemically synthesized, thereby providing clinicians with a plethora of options for photodynamic agents. In addition to the wide use of PTD in cancer therapy, PDT has recently refocused onto its original purpose as antimicrobial methods because multidrug-resistant ‘super bacteria’ have become one of the greatest, global health challenges.¹⁷

2.2. Mechanism of PDT

The detailed photophysics of PDT is beyond the scope of this topical review and interested readers may refer to other publications.^18,19 Briefly, the ground-state photosensitizer is excited to a high-energy state after absorbing excitation photons (Figure 1). The excited molecule could then dissipate its energy by either nonradiative pathways (a.k.a. internal conversion or IC), or radiative pathways such as fluorescence emission, or intersystem crossing (ISC). Among all dissipation pathways, ISC is especially important for ROS generation as it converts excited, singlet-state molecules into a triplet state, which is a metastable electronic state and would dissipate its energy via phosphorescence or photochemical reactions. The lifetime of the triplet state is usually within microseconds, which is much longer than the singlet state (nanoseconds), and thus could set stage for efficient, energy-transferring collisions. Two hypothetical types of photochemical processes could take place to form ROS (Figure 1). In the type I process, the excited photosensitizers may directly react with substrates, such as proteins, lipids and nucleic acids, to acquire or lose a single electron to form radical anions or cations. These radicals may further react with molecular oxygen (O₂) to generate ROS. Moreover, photosensitizers in the triplet state may directly transfer one electron to nearby oxygen through collisions, resulting in O₂^•−. O₂^•− is not very reactive in biological systems and can be disproportionated by superoxide dismutase (SOD) to generate H₂O₂ and O₂. Through a cascade electron acquisition/donation process catalyzed by redox-active transition metal ions such as iron or copper, O₂^•− and H₂O₂ could be further converted into highly reactive hydroxyl radical (•OH) through a so-called “Fenton reaction”. The Type II process is mechanistically simpler than the Type I process as energy is directly transferred from the excited triplet molecule to a ground-state triplet oxygen (³O₂), resulting in highly reactive singlet oxygen (¹O₂*). To date, the type II process is considered to be more important for most photosensitizers during PDT.²⁰ It is worthwhile to note that the type I and type II classification refers to the initial electron/proton or direct energy transfer process.²¹ These highly reactive radicals and singlet oxygen are actually interconvertible. Moreover, for a given photosensitization process, type I and type II reactions may occur simultaneously.^19,21

Figure 1. Jablonski diagram and mechanisms for photosensitizer-induced ROS generation.

Photosensitizers in the ground state are excited to a high-energy singlet state, which could subsequently be converted into a triplet state via ISC. This triplet state may further undergo photochemical reactions via either a type I or a type II process to generate ROS. (Abs, absorption; IC, internal conversion; ISC, intersystem crossing; SET, single electron transfer; ET, energy transfer; SOD, superoxide dismutase)

3. Non-genetic ROS generators

3.1. Small-molecule-based photosensitizers

A large number of small-molecule-based photosensitizers have been developed and they are mostly from three categories: tetrapyrrole derivatives, heavy-atom-containing fluorescent dyes, and transition metal complexes. Below we briefly discuss the advantages and disadvantages of photosensitizers in each category. To facilitate the discussion, we provide the information on the absorbance of a few representative, small-molecular-based photosensitizers in Table 1.

Table1.

Representative examples of small-molecule-based photosensitizers.

Class	Example	Absorbance peak	Reference
	porphyrin	Soret band (~400nm) Q band (~630nm)	22
Tetrapyrrole derivatives	chlorin	Soret band (~400nm) Q band (~650nm)	23
	bacteriochlorin	Soret band (~400nm) Q band (~730nm)	24
Heavy-atom-containing fluorescent dyes	DIMPy-BODIPY	530 nm	27
	Rose-Bengal	540 nm	29
Transition metal complexes	Ruthenium(Ru) complex	450 nm	33
	Rhodium(Rh) complex	450 nm	35
	Iridium(Ir) complex	600 nm	36

3.1.1. Tetrapyrrole derivatives

Tetrapyrrole (Figure 2), which is naturally occurring in heme, chlorophyll, and bacteriochlorophyll, possesses the richest structural diversities among all types of synthetic photosensitizers. Tetrapyrrole derivatives can be further classified into three subgroups (porphyrin, chlorin, and bacteriochlorin), each featuring different numbers of C-C double bonds. As the number of conjugated double-bonds decreases from porphyrin to bacteriochlorin, the Q band absorption is red-shifted (Table 1).^22–24 A prominent advantage of tetrapyrrole derivatives is their strong absorption within the near-infrared (NIR) optical window in biological tissue (650–900 nm), enabling an efficient excitation of photosensitizers at a relatively deep depth. However, tetrapyrrole derivatives often have poor solubility, limiting their applications in biological systems. This solubility problem has recently been partially addressed by introducing the sulfonate functional group into tetrapyrrole.²⁰. Photofrin, a mixture of tetrapyrrole derivatives, has been approved by the US FDA to treat cancer since 1995.²⁵ Moreover, several second-generation agents (e.g., benzoporphyrin derivative monoacid) have been recently approved by the US FDA²⁶.

Figure 2.

Representative, small-molecule-based photosensitizers from three major categories. Tetrapyrrole (porphyrin) is shown in the red circle. A heavy atom (iodine) containing synthetic dye (Rose Bengal) is shown in the blue circle. A transition metal (ruthenium) complex is shown in the yellow circle.

3.1.2. Heavy-atom-containing fluorescent dyes

Many photosensitizers were developed by structurally modifying classic fluorescent dyes, such as BODIPY,²⁷ fluorescein,^28,29 and phenothiazinium salts³⁰ with a number of heavy atoms such as iodine and bromine. Heavy atoms can facilitate ISC and increase the yield of triplet states, thereby increasing ROS production. Some heavy-atom-incorporated fluorescent dyes, such as Rose Bengal (Figure 2), have been explored to treat various cancers or skin conditions.²⁹ A formulated Rose Bengal, known as PV-10, demonstrated good locoregional tumor control of cutaneous melanoma patients.³¹ PV-10 is currently undergoing clinical trials for melanoma, breast cancer, and liver cancer. Compared to tetrapyrrole derivatives, the blue-shifted absorbance of fluorescein and BODIPY derivatives (Table 1) inevitably decreases their efficiency for ROS generation because of strong tissue scattering and absorption of excitation photons.

3.1.3. Transition metal complexes

Organo-transition metal compounds have demonstrated their versatile roles in catalysis, synthetic chemistry, and development of organic light emitting diodes (OLED).³² Organo-transition metal compounds, such as ruthenium(II) polypyridyl complexes³³, have also been examined for light-induced ROS generation. In a recent example, a collection of 17 ruthenium(II) polypyridyl complexes with varying substituent(s), molecular symmetry, electrical charge, and counterions were characterized for absorption coefficient, ¹O₂ generation quantum yield, and their antibacterial activity in photodynamic assays using Gram-positive and Gram-negative bacteria.³⁴ Interestingly, in addition to ¹O₂ production efficiency, other parameters, especially those impacting on interaction with bacteria, were identified be key factors for killing of bacteria. In addition, there are a handful of reports on rhodium (Rh)³⁵ and iridium (Ir)³⁶ complexes serving as PDT reagents. The impressively high ¹O₂ production yields and stability of transitional metal complexes make them a promising category of photosensitizers. However, their poor water-solubility and potentially high cytotoxicity hinder their wide-scale adoption and usage in biomedicine.

3.2. Nanotechnology-enhanced photosensitizers

Nanotechnology has received significant attention in recent years because of its outstanding performance in targeted delivery and controlled release of cargos, which may overcome disadvantages of many synthetic photosensitizers.^37,38 The integration of nanotechnology with synthetic photosensitizers has resulted in significant advancement. For example, the low-solubility issue of synthetic photosensitizers has been significantly alleviated by encapsulating photosensitizers within liposomes and micelles.³⁹ Additionally, these modifications are also helpful to enhance tumor accumulation after intravenous administration. In another example, the photosensitizer phthalocyanine was assembled on gold nanoparticles covalently bound with anti-HER2 antibodies to achieve targeted delivery.⁴⁰ Indeed, the cytotoxicity of phthalocyanine drastically decreased while the drug concentration in breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor elevated significantly.

4. Genetically encoded ROS generators

Certain fluorescent proteins have been shown to be effective photosensitizers. In addition, some enzymes can effectively generate ROS via biochemical reactions (Figure 3). These protein-based ROS generators have attracted much attention, because they are excellent research tools for not only understanding the biological functions of ROS,⁴¹ but also precisely inactivating specific targets, such as proteins or protein complexes, in live cells and in vivo.⁴²

Fig 3.

Genetically encoded ROS-generating systems, including fluorescent protein-based photosensitizes and enzyme-based ROS generators. Shown in the graph are the structures of KillerRed and RgDAAO based on Protein Data Bank (PDB) entries 3A8S and 1C0I.

4.1. Fluorescent-protein-based photosensitizers

When certain fluorescent proteins are genetically fused to target proteins, excitation of these fluorescent proteins may generate ROS, which can diffuse and inactivate surrounding molecules in living cells.⁴³ By using this chromophore-assisted light inactivation (CALI) technique, one can selectively inactivate proteins within cells. Fluorescent proteins are particularly important for this application, because these genetically encoded photosensitizers can be readily fused to almost any target protein. The absorbance peaks of representative fluorescent-protein-based photosensitizers are given in Table 2.

Table 2.

Representative examples of genetically encoded photosensitizers.

Name	Absorbance peak	Reference
KillerRed	585 nm	46
SuperNova	579 nm	50
SuperNova Green	440 nm	51
KillerOrange	455 nm	52
MiniSOG	448 nm	47
SOPP	440	55
Pp2FbFP	449	58

In early studies, green fluorescent protein (GFP) was demonstrated as an effective CALI fluorophore, although its ROS generation capability is worse than synthetic dyes such as fluorescein or malachite green.^43–45 From the hydrozoan chromoprotein anm2CP (a GFP homolog), Lukyanov and co-workers developed KillerRed,⁴⁶ the first fluorescent protein that was specifically engineered for photosensitizing. This red fluorescent, dimeric protein indeed showed 1,000-fold higher phototoxicity than GFP. KillerRed is believed to mainly generate O2^•− via the type I reaction.^47–49 Recent effort has resulted in its monomeric and/or color-shifted variants, such as SuperNova,⁵⁰ SuperNova Green,⁵¹ KillerOrange,⁵² and mKillerOrange.⁵³

In addition to GFP analogs, some flavoproteins are also effective photosensitizers. Shu et al. engineered a mini singlet oxygen generator (miniSOG) from the flavin mononucleotide (FMN)-based LOV2 domain of Arabidopsis thaliana phototropin 2. This protein was shown to produce ¹O₂ upon excitation, suggesting that the photosensitization process occurs via the type II reaction.⁵⁴ Variants of miniSOG with enhanced ¹O₂ production, such as SOPP,⁵⁵ SOPP2, SOPP3,⁵⁶ and miniSOG2,⁵⁷ have also been described. In addition, a recent study analyzed eleven LOV-based flavoproteins and all were able to produce ¹O₂ and/or H₂O₂, despite that there were remarkable differences in ROS selectivity and yield.⁵⁸ In particular, two variants, Pp2FbFP and DsFbFP M49I, were demonstrated to be new tools for light-controlled killing of bacteria and studying of specific ROS-induced cell signaling.⁵⁹

In addition to CALI, genetically encoded photosensitizers have been targeted to mitochondria, chromatin, or plasma membranes to selectively kill cells in cell culture models and in vivo.^60,61 Not surprisingly, they have also been explored as phototoxic cancer therapeutic agents.^61,62

4.2. Enzymes that produce ROS without need of light

Perhaps one of the most well-known ROS-producing enzymes is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which was first characterized in mitochondria of neutrophils and is a major source for endogenous ROS in the form of either O₂^•− or H₂O₂.^63,64 In addition, xanthine oxidase (XOR) and myeloperoxidase (MPO) are effective ROS generators.⁶⁵ Furthermore, several components of the mitochondrial respiratory chain can be modulated to generate a large amount of O₂^•−.⁶⁶

The most useful ROS-generating tool in this category is D-amino acid oxidase (DAAO), a flavin adenine dinucleotide (FAD)-containing enzyme that catalyzes the conversion of D-amino acids in the presence of molecular oxygen (O₂) to alpha-keto acids, leading to enzyme- and D-amino-acid-dependent H₂O₂ production (Equation 1).^67,68

(Eq. 1)

DAAO was first identified by Krebs in 1935.⁶⁹ In particular, DAAO from the yeast Rhodotorula gracilis (RgDAAO) has been widely used due to its high enzymatic activity toward D-alanine.⁷⁰ Overexpression of RgDAAO is an effective and innovative way to induce oxidative stress in cell culture models and in vivo. For example, RgDAAO has been used to spatiotemporally control H₂O₂ concentrations in astrocytes to study the role of H₂O₂ in astrocyte-dependent neuro-protective mechanisms.⁷¹ Recently, RgDAA was expressed in heart of rats fed with D-alanine to induce chronic generation of H₂O₂.⁶⁷ The rats developed a dilated cardiomyopathy with significant systolic dysfunction. The study thus showcased a powerful chemogenetic approach to explore redox signaling and physiology in vivo.

5. Hybrid ROS generators

To precisely control the localization of synthetic photosensitizers, hybrid ROS generators have been developed by integrting synthetic photosensitizers with genetically encoded elements. For example, chemical photosensitizers could be conjugated to antibodies and this strategy actually enabled the first CALI experiment.⁷² Moreover, chemical photosensitizers could be linked to proteins via genetically encoded tags by using FlAsH, ReAsh, or HaloTag technologies.^73,74 In another example, a fluorogen-activating single-chain antibody was engineered to bind an iodine-substituted malachite green analog, resulting in ‘on-demand’ activation of the photosensitizer to produces O₂^•− and fluorescence in the presence of near-infrared (NIR) excitation light.⁷⁵ This so-called FAP-TAP (fluorogen-activating protein - targeted and activated photosensitizer) technology was validated for light-induced cell ablation in HEK293 cell culture and in live zebrafish. One advantage of these hybrid strategies is the availability of diverse, synthetic photosensitizers, including those excitable with NIR light to facilitate manipulation of proteins and cells in deep tissues of live organisms. Their main disadvantage is the dependence of the uptake of exogenous photosensitizers, whose penetration, localization, and distribution may be problematic for live cell, tissue, and/or in vivo applications.

6. Perspectives

ROS have been recognized as by-products of aerobic respiration and important signaling molecules. Tools that can generate or detect ROS are equally important for understanding biology regulated by ROS signaling and oxidative stress. This topical review discusses tools that have been adapted to control ROS production in biological systems via either photosensitization or enzyme reactions. In particular, a number of genetically encoded photosensitizers are now available for specific generation of O₂^•− or ¹O₂ via the type I or type II photosensitization mechanism. However, these current photosensitizers often require blue, green, or orange light for excitation. Further studies are urgently needed to develop red-shifted variants that can be excited in the NIR optical window. In addition, an elegant chemogenetic approach, which uses DAAO and D-amino acids to generate H₂O₂, may enable a large array of studies. D-amino acids are unfortunately essential in certain tissue types such as the brain.⁷⁶ Therefore, this DAAO system is not completely bioorthogonal. It remains an open question whether a truly bioorthogonal system can be developed to chemogenetically control ROS in cells and organisms. It is promising that a NanoLuc luciferase was recently fused to MiniSOG to generate ¹O₂ via energy transfer in a cell culture model.(77) However, it still remains a future task to expand this strategy to in vivo animal models and to the generation of other types of ROS.

Genetically encoded or hybrid photosensitizers are excellent tools for specific deactivation of proteins, a process known as CALI. In addition, photosensitizers may be utilized to selectively destroy organelles, intracellular machineries, or whole cells. When they are applied to therapy, photosensitizers are promising for the treatment of cancer, bacterial infection, and other diseases. A number of synthetic photosensitizers have been approved for uses or clinical trials in cancer patients. Further studies may pursue strategies to achieve selective delivery of photosensitizers and to enhance their tissue penetration, bioavailability, and ROS production efficiency. Moreover, because multidrug resistant pathogens have become a threat to global heath, exploration of ROS generators to combat multidrug resistant pathogens is highly worthwhile.