Applications of bioluminescence-mediated photodynamic therapy (BL-PDT) for the treatment of deep-seated tumours

Abstract

Bioluminescence, the light generated through biochemical reactions involving luciferases in living organisms, has been the focus of extensive research due to its diverse applications. It is especially noteworthy as an internal light source for theranostic applications because of its safety and efficiency, which helps to overcome the challenges associated with the poor penetration of traditional external light sources. Recent progress in protein engineering and delivery systems has broadened the scope of bioluminescence in numerous applications, including photodynamic therapy. This review outlines the essential principles of bioluminescence and examines its recent applications in photodynamic therapy for the treatment of cancers. Additionally, it offers insights into future research avenues based on the current studies in bioluminescent systems to further enhance their potential.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-03413-2.

Introduction

Cancer remains a leading cause of mortality worldwide, highlighting the need for novel and effective treatment options [24]. Conventional modalities, such as chemotherapy, surgery, immunotherapy, radiation therapy, and molecular targeted therapy, have proven to be effective to a considerable extent, however, they are often associated with serious side effects, lack of selectivity, and difficulties in addressing deep-seated metastatic tumours. Although promising advancements have been made in cancer treatment, there remains an increasing necessity for ongoing innovation to develop more effective therapeutic strategies [28].

Photodynamic therapy (PDT) is a treatment modality that relies on the synergistic effects of light, a photosensitizer (PS), and molecular oxygen [4]. These PS molecules are usually made up of organic chromophores and cause cell damage in malignant cells [38]. Activation of PSs by light of a certain wavelength in the presence of oxygen (O₂) generates cytotoxic reactive oxygen species (ROS) that target and eliminate tumour cells [20]. In comparison to conventional treatments (such as surgery), PDT offers several benefits: (i) it is minimally invasive, (ii) it typically has few side effects, (iii) it allows for precise control over timing and location [21, 58] and (iv) it does not generally lead to significant drug resistance [58]. In addition, numerous research efforts related to PDT have investigated the creation of PS drugs either individually and in conjugation with nanoscale particles and various active targeting ligands [21].

Despite its advantages in the treatment of cancer, the clinical application of conventional PDT is constrained by the limited penetration depth of external light sources in deep tissues [18, 44]. The application of optical techniques in clinical settings has been restricted to superficial tissues [25]. To address this challenge, the use of internal light sources presents a viable solution. Bioluminescence (BL), in particular, stands out as a compelling option that eliminates the need for external light sources, which have limited tissue accessibility [21, 25].

BL refers to the natural ability of living organisms to produce and emit light. This phenomenon occurs through a chemical reaction that transforms chemical energy into light energy. The process involves a substrate commonly called luciferin, along with oxygen and an enzyme known as luciferase [22]. This phenomenon has evolved primarily in marine life, certain types of bacteria, fungi, and land-dwelling insects for various roles, including catching prey, deterring predators, and attracting mates [46]. The oldest mentions of BL are thought to originate from the texts of ancient Eastern civilizations, which talked about both fireflies and glow-worms [27]. In 1677, Robert Boyle, documented that air was needed for luminescence. This was the first scientific recording of BL [27, 46]. The exploration of bioluminescence has historically involved a variety of disciplines, including chemistry, biology, ecology, and physics, and remains a thriving field of research. This area not only enhances our understanding of key biological and chemical processes but also drives technological advancements. In the twenty-first century, bioluminescent imaging technologies have become essential for medical research, enabling non-invasive monitoring of disease progression. Additionally, ecological studies have employed bioluminescence to track species and analyse intricate ocean food chains. The blend of interdisciplinary collaboration and technological progress has transformed the study of bioluminescence from a captivating natural phenomenon into a significant scientific resource [40].

BL differs from phosphorescence and fluorescence because it does not require the initial absorption of sunlight or other types of electromagnetic radiation by a molecule or pigment to produce light. This characteristic helps to mitigate issues related to surface quenching, scattering, and sample heating. Luciferin is valuable due to its non-toxic nature, availability in biological systems, high spatial resolution, sensitivity, and remarkable quantum efficiency, making it useful in various analytical methods in chemistry, biochemistry, and microbiology [12]. These methods include both in vitro and in vivo approaches biological detection and optical imaging [12, 55]. Recently, scientists have begun to explore its therapeutic benefits. This natural self-emitting phenomenon allows for the activation of PS molecules within tumours [55].

BL-PDT leverages bioluminescent systems, such as bioluminescent bacteria or luciferase-luciferin reactions to activate PSs from within the tumour microenvironment (TME) [37]. Over 40 different bioluminescence pathways, which include specific luciferins and luciferases, have been identified, with several currently utilized in biomedical fields [22]. The first documented use of BL-PDT was described in a 2015 study by Kim and colleagues. This study presented an innovative method of PDT by employing bioluminescence resonance energy transfer (BRET) to activate photosensitizers located in deep tissues, addressing the common issue of light penetration associated with standard PDT. The findings showed that this technique successfully triggered cytotoxic effects in cancer cells and minimized metastasis in animal models [25].

This review aims to examine recent advancements in a novel area of research: using BL systems as intracellular excitation sources for PDT in cancer treatment. This subject holds significant importance, and breakthroughs in this domain could transform PDT and promote the exploration of additional applications for BL. To provide a clearer understanding, the document will also cover the fundamental mechanisms of BL and BL systems that have been utilized in PDT thus far. Table 1 below shows the differences between conventional PDT systems and BL-PDT.

Table 1.

Comparison between conventional PDT and BL-PDT

Feature	PDT	BL-PDT
Light source	External (lasers, LEDs)	Internal (BL, proteins, bacteria)
Depth penetration	Limited (superficial)	Deep-tissue targeting
Selectivity	Requires light delivery	Tumour-selective
Clinical status	Approved for superficial cancers	Preclinical

Bioluminescent systems and mechanisms

BL has evolved over time in nature, featuring unique luciferases and luciferins [12]. The bioluminescent process involves two main components: (1) a substrate that is oxidized by molecular oxygen, and (2) an enzyme, typically luciferase, that facilitates the reaction. This reaction produces a nonreactive product while releasing photons. The oxidation of the substrate is a highly energetic process, generating enough energy to form an excited singlet state intermediate (p). This intermediate subsequently emits a visible photon (hν) as it relaxes back to its ground state [35, 41].

Luciferases (Fig. 1) are a class of heterogeneous oxidative proteins that act as enzymes and, when adenosine triphosphate (ATP), oxygen, and the suitable substrate (luciferin) are present, facilitate the oxidation of the substrate [33, 45]. The BL generated by the interaction of luciferase and luciferin can pass through skin and tissues. This allows for the monitoring of physiological processes and offers valuable information related to the clinical physiology and pathology of tumours [29].

Fig. 1.

Chemical structures of known and commonly used insect and marine luciferases in bioluminescent systems [14]

d-luciferin dependent system

This system is observed in various types of beetles, such as fireflies, click beetles, and railroad worms. The light produced includes shades of yellow, orange, and occasionally red. The process involves d-luciferin, a stable and non-toxic substance with a molecular weight of about 60 kDa, requires ATP and magnesium ions (Mg²⁺) to facilitate the reaction (Fig. 2). Since the intensity of light emission depends on ATP concentration, this system is widely used in research on cancer metabolism and in analysing water that may be contaminated with bacteria. Many diagnostic tests are based on blood-related components, which are also affected by ATP levels [12].

Fig. 2.

Enzymatic reaction mechanism of d-luciferin dependent system. Beetle luciferin (d-luciferin) undergoes ATP-dependent oxidation in the presence of recombinant firefly luciferase and Mg²⁺, yielding oxyluciferin, AMP, CO₂, PPi, and visible light emission (540–600 nm) [12]

Firefly luciferase

The firefly luciferase (FLuc) and d-luciferin combination is one of the most commonly utilized BL systems. FLuc facilitates the oxidation of d-luciferin with the help of ATP, Mg²⁺, and oxygen. During this process, d-luciferin is activated through adenylation, resulting in the creation of an excited form of oxyluciferin, which releases light at approximately 560 nm as it returns to its ground state [22, 29, 32]. More than 40 species of fireflies use d-luciferin as a substrate, with the Photinus pyralis firefly being the most frequently isolated source in research [22].

Firefly luciferase has been widely utilized across various in vitro and in vivo systems. It aids in measuring protein–protein and protein–ligand interactions, assessing metabolites that play a role in cell communication and signalling, and identifying pathogenic bacteria and viruses [12, 14].

Click beetle luciferase

This group of luciferases that depend on d-luciferin is sourced from Pyrophorus plagiophthalamus. They produce light through four different types of luciferases, which emit colours ranging from green (540 nm) to orange-red (593 nm). Their ability to tolerate a wide range of pH levels, along with the presence of engineered variants and diverse colour outputs, makes these luciferases suitable for a variety of applications [12, 14].

Coelenterazine dependent system

Coelenterate-type luciferins, known as coelenterazines (CTZ), are derived from imidazopyrazinone. These CTZs are the most well-known marine luciferins and are present in various phyla, including coelenterates and ctenophores [35]. These organisms emit blue light (with an emission peak between 450–500 nm) through the oxidation of CTZ without requiring any cofactors apart from oxygen. Common CTZ-dependent luciferase systems include Renilla (RLuc), Gaussia (GLuc), and NanoLuc luciferases [14, 32].

Renilla luciferase

RLuc is a cytosolic protein of medium size (36 kDa) sourced from coral, which generates a consistent luminescent signal, with an emission peak ranging between 450–480 nm. It is frequently utilized in biomedical research areas, including bioimaging and drug screening. The existence of engineered variants that provide increased brightness and red-shifted wavelengths [12, 14] (Fig. 3).

Fig. 3.

Enzymatic reaction of coelenterazine catalysed by Renilla luciferase to coelenteramide. The reaction yields coelentramide, CO₂, and blue light emission (450–500 nm). The reaction requires molecular oxygen and produces no adenosine nucleotide by-products, distinguishing it from d-luciferin systems [12]

Gaussia luciferase

Gaussia luciferase (Gluc) is a small, alpha helix protein, weighing 20 kDa, is secreted by a small crustacean belonging to the Copepoda class, with an emission peak ranging between 480–485 nm. It demonstrates exceptional thermal stability and a rapid catalytic rate. However, the modification of its activity through the formation of disulfide bonds renders it inappropriate for specific heterologous systems. This system is advantageous for tracking tumour development and drug response, as the signals correlate linearly with the cell count [12, 14, 47].

NanoLuc luciferase

NanoLuc is a modified version of luciferase derived from the shrimp Oplophorus gracilirostris, weighing 19 kDa, and generates a bright signal suitable for various applications. This protein makes use of disulfide bonds and a cell-permeable analogue of coelenterazine, producing a strong signal that can be applied across a wide range of uses. When fused with fluorescent proteins, it creates highly luminous bioluminescent constructs with red-shifted spectra, making it ideal for single-cell and whole-body bioluminescent imaging in living organisms [12, 14]. The NanoLuc-furimazine reaction (Fig. 4) operates through an oxygen-dependent oxidation of its substrate furimazine, resulting in the emission of BL. This reaction is ATP-independent, producing a stable glow-type signal [36].

Fig. 4.

Oxidative reaction of furimazine catalysed by NanoLuc luciferase to furimamide. This enzyme converts furimazine to furimamide in an oxygen-dependent process, releasing light (460 nm) and CO₂ [12]

Bacterial dependent system

The bacterial luciferase (Lux) is found within the luxCDABE operon. LuxG, a type of flavin reductase, generates reduced flavin mononucleotide (FMNH₂) using nicotinamide adenine dinucleotide phosphate (NADPH). Meanwhile, LuxCDE converts fatty acids into long-chain aldehydes. The luciferase, LuxAB, facilitates the oxidation of these long-chain aldehydes, producing excited state intermediates in the presence of FMNH₂ and oxygen (Fig. 5). As these intermediates return to their ground states, they emit light at around 490 nm. Unlike other types of luciferases, the bacterial luminescence system is self-sustaining, as all necessary substrates are recycled and generated by the enzymes encoded within the operon [14, 22, 49].

Fig. 5.

NADPH-dependent reduction of FMN catalysed by FMN reductase to FMNH₂. This priming reaction provides the reduced flavin cofactor for bacterial luciferase (LuxAB), enabling the subsequent light-producing (490 nm) oxidation of long-chain aldehydes with molecular oxygen [12]

Table 2 summarises the most common BL systems that have been explored in BL-PDT applications.

Table 2.

Common BL systems used in BL-PDT

Luciferase	Substrate	Co-factors	Light emission wavelength (nm)
Firefly	d-luciferin	ATP, Mg2+, O2	560
Click beetle	d-luciferin	ATP, Mg2+, O2	540–593
Renilla	Coelenterazine	O2	450–480
Gaussia	Coelenterazine	O2	480–485
NanoLuc	Coelenterazine	O2, furimazine	460
Bacterial	Long chain aldehydes	FMNH2, O2	490

Bioluminescence-mediated photodynamic therapy

Bioluminescence resonance energy transfer

Luciferases not only produce visible-to-NIR light (400–700 nm) independently but can also be paired with fluorescent molecules to enhance energy transfer through a process called bioluminescence resonance energy transfer (BRET). During BRET (Fig. 6), light energy is transferred non-radiatively from a donor (luciferase) molecule to an acceptor molecule, eliminating the need for external light excitation. This process requires close proximity (usually within 10 nm) between the donor and acceptor and proper alignment between the two molecules. BRET efficiency is highly sensitive to distance, following Förster’s law. Thus, minor increases in separation drastically reduce energy transfer [22].

Fig. 6.

Schematic representation of BRET. In this system. A luciferase enzyme (donor) catalyses a reaction with its substrate to emit light (400–700 nm), which is non-radiatively transferred to a nearby fluorescent acceptor molecule. The acceptor emits a light at a longer wavelength, enabling deep-tissue penetration. Efficient energy transfer requires a distance of less than 10 nm between the acceptor and donor, with proper orientation for resonance [22]

BRET can be utilized for optical bioimaging and sensing purposes without the need for other sources. It is particularly valuable for deep-tissue imaging applications where conventional light emission methods face limitations. The most effective BRET systems utilize three classes of nanomaterials: near-infrared quantum dots (NIR QDs), gold nanoparticles (AuNPs), carbon dots (CDs), and plasmonic nanoparticles. These nanomaterials serve as efficient energy acceptors when paired with bioluminescent donors, such as RLuc, NLuc, and FLuc). Several conjugation strategies, such as physical adsorption, covalent modification, and metal-ion interactions can be utilized to create stable donor–acceptor pairs [19]. While BL is considerably less intense than traditional light sources such as lasers and LEDs, effective BRET from bioluminescent systems to PSs can greatly improve the effectiveness of PDT [22].

Photosensitizers for BL-PDT

BL-PDT introduces an innovative method for cancer treatment that leverages endogenous BL reactions to activate PSs within deeply located tumours, thus addressing the challenges posed by external light sources, which restrict treatment reach to superficial regions. The effectiveness of PDT largely depends on the choice of PS. It is crucial for PDT agents to be biocompatible and capable of being eliminated or degraded by the body since they need to reach the target tissue to be activated in place. This is generally less of an issue for porphyrin-based photosensitizers, but inorganic ones might face challenges. Additionally, the agent must have sufficient light conversion efficiency and/or release therapeutic substances effectively. Otherwise, extremely high light intensities would be necessary to achieve the desired medical outcomes. Lastly, the PSs activity may rely on specific light wavelengths, as certain wavelengths can penetrate tissues more effectively. In this regard, inorganic photosensitizers often have advantages since they typically exhibit higher energy conversion efficiency. To enhance the overall therapeutic effectiveness, it may be beneficial to modify the pharmacodynamics of PSs to increase their concentration at the target areas, or to combine PDT with other drugs or treatments, such as BL-PDT [59]. The therapeutic efficacy of BL-PDT relies on a tightly coupled molecular cascade (Fig. 7). The process involves three critical components: (1) luciferase enzyme expressed in target cells, (2) a luciferin substrate administered systemically, and (3) a PS that converts bioluminescent photons into cytotoxic ROS. As depicted in Fig. 7, the mechanism proceeds through four sequential steps:

1. Substrate delivery: Luciferin analogues (e.g., d-luciferin) diffuse into target tissues, with pharmacokinetics depend chemical modifications [16].

2. Light generation: Intracellular luciferase oxidizes the substrate, emitting visible-to near infrared light (400–700 nm) at intensities sufficient for PS activation [12].

3. Energy transfer: Emitted photons excite the PS through FRET, requiring close proximity and spectral overlap [53].

4. Therapeutic effect: The activated PS generates ROS, inducing cell death [12, 48].

Fig. 7.

Basic principle of BL-PDT. A luciferase-linked PS is introduced inside a deep-seated cancer cell. The injection of luciferin, which is later oxidized by the luciferin, allows for a series of reactions, including PS activation leading to ROS generation and cell death

The majority of PSs used in cancer treatment are derived from a tetrapyrrole structure, which resembles the one found in the protoporphyrin group of haemoglobin. Depending on their specific structures, effective PSs can be produced with absorbance characteristics ranging from 600 to 800 nm. Since light penetration into tissues improves as the wavelength increases, compounds with strong absorption in the deep-red region of the spectrum, such as chlorins, bacteriochlorin’s, and phthalocyanines, are generally more effective as PSs; however, various other factors also play a significant role [1].

In BL-PDT, their activation is linked to the emission wavelength of the bioluminescent system. Porphyrin-based PSs are effective in the red-light spectrum, around 600–700 nm, which allows them to penetrate deep tissue effectively. Chlorin-based PSs exhibit a high quantum yield and are effective in low-oxygen environments, making them particularly suitable for hypoxic tumours. Phthalocyanines have strong absorption properties in the near-infrared (NIR) spectrum, making them advantageous for tumours that require deeper light penetration. Zinc (II) protoporphyrin IX (ZnPP) is one of the photosensitizers that has garnered considerable attention due to its capacity to produce singlet oxygen when exposed to visible light, achieving a high quantum yield of 0.91 [2].

Mechanisms and applications of bioluminescent-mediated photodynamic therapy in cancer treatment

To provide a structured overview of BL-PDT in the preclinical and clinical stages, Table 3 summarizes selected studies that report therapeutic outcomes, tumour penetration capabilities, and immune-related effects. Initial studies, such as those combining FLuc with Rose Bengal, successfully demonstrated the intracellular feasibility of BL-PDT. More recent approaches, including RLuc-conjugated quantum dots and engineered bacterial systems, have improved photon output and addressed challenges related to tissue depth [15]. However, there remains a lack of standardized reporting metrics across studies, making direct comparisons difficult. Table 3 categorizes each study by luciferase-PS pairing, therapeutic efficacy, reported penetration depth, and observed immune activation.

Table 3.

Comparisons of various BL-PDT studies

Luciferase system	PS	Therapeutic efficacy	Penetration depth	Key findings	Limitations	Refs.
FLuc	Rose Bengal	90% cytotoxicity (in vitro)	< 1 mm (cell monolayers)	First study of BL-PDT; ATP-independent activation	Low photon flux; no in vivo data	[48]
FLuc	Not specified	No significant toxicity	Not reported	Photo insufficiency for PDT	Contradicts other FLuc studies; unclear PS	[39]
FLuc + PLGA nanoparticles	Rose Bengal	Tumour inhibition in mice	Intratumoral	Deep tumour activation	Lack of immune metrics	[55]
RLuc + QDs	Foscan	23% tumour growth inhibition (vs. 4.2% in controls)	2% mm	BRET-QD system achieved 0.6–0.8 J cm−2	QD cytotoxicity	[17]
RLuc + QDs	Ce6	300 M activation/min (vs. 40 M with laser)	Membrane-localized	High activation rate but limited to cell surface	No tumour penetration data	[25]
RLuc8	Ce6	Complete tumour penetration	Deep-seated tumours	80% activation ration	Requires Intratumoral injection	[52]
NanoLuc	SOPP3	Significant growth inhibition	Depth-independent	BRET ration = 1.12; HER2-targted liposomes	Limited immune profiling	[42]
Bacterial luciferase	Ce6	Supressed large tumours	Whole tumour illumination	Hydrogel delivery allowed uniform activation	Safety of engineered bacteria	[56]
Fluc plasmid + PEI-CaP	Hypericin	Sustained cytotoxicity (in vitro)	Not quantified	Gene delivery for continuous luciferase expression	No in vivo penetration	[13]

Firefly luciferase

In 2003, Theodossiou and colleagues made the initial effort to explore the capabilities of BL for PDT. They utilized the Fluc-luciferin BL system to provide intracellular excitation for Rose Bengal, a PS that is a water-soluble dye with a high quantum yield for singlet oxygen (approximately 0.75) [48]. This research was conducted in vitro, utilizing NIH 3T3 murine fibroblasts as a model system. In this study, the cells were transfected with a modified Fluc gene (Luc + , for cytosolic expression), and subsequently, both luciferin and the PS were introduced to the cell cultures [34, 48]. The results indicate that using firefly bioluminescence as an excitation source is effective, as pairing it with the PS led to a toxicity rate of 90%, while all control groups showed a 100% survival rate (accounting for experimental error). Additionally, the authors were able to determine that the cytotoxic effects are primarily due to the generation of this specific ROS when employing a singlet-oxygen quencher [48].

An intriguing aspect of this study is that it showed ATP is not necessary for cells to initiate Fluc-mediated PDT, as the levels of this metabolite in fibroblasts are adequate to activate BL. Additionally, the findings revealed that once taken up, both luciferin and Rose Bengal display a widespread distribution in the cytosol. This information is essential because BRET is effective in producing a sufficient PDT response only if both the bioluminescent donor and the photosensitizer are located in the same subcellular area [34].

The subsequent study by Schipper et al. [39] revealed that luciferase-expressing cells treated with both photosensitizer and d-luciferin did not exhibit a notable change in survival rates when compared to the control groups. The authors concluded that this intracellular bioluminescence system fails to emit a sufficient number of photons (< 1.03 × 10 − 4 mJ cm⁻² for 24 h of treatment) to enable photosensitizers to produce effective photodynamic toxicity [39].

As a typical example, Yang and colleagues [55] developed biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles that were infused with the PS Rose Bengal (Fig. 8A). These PLGA-RB nanoparticles were subsequently linked to luciferase, which emitted a fluorescent signal in the presence of the luciferin substrate. The natural BL functioned as a light source to activate the photosensitizer for the generation of ROS. The team evaluated cell toxicity, cellular emission imaging, and therapeutic efficacy through cellular assays, demonstrating effective destruction of cancer cells. The light emission images of PLGA-RB nanoparticles revealed a notable increase in light emission intensity as the concentration of fluorescein was raised. When administered via intratumour injection in H22 tumour-bearing mice, this formulation exhibited considerable tumour inhibition in comparison to the control group and displayed tumour growth suppression curves similar to those of the NP-illuminated group. This highlights the effectiveness of the BRET-PDT strategy and its potential to substitute external light sources for deep tumour treatment [55].

Fig. 8.

A Illustration of the ROS production process involving PLGA-RB nanoparticles. The oxidation of the substrate produces light emission (540–600 nm), activating the PS, resulting in ROS generation. Reprinted (adapted) with permission from [54].

Copyright 2018, Springer Nature. B Visual representation for BLS-PDT of CDs-PIX. FLuc oxidizes d-luciferin in the presence of ATP and Mg²⁺, emitting light (540–600 nm) that activates PIX to generate ROS. Reprinted (adapted) with permission from [55]. Copyright 2018, American Chemical Society. C Schematic illustrating gene-delivery based BL-PDT. Luciferase-containing plasmid DNA loaded is onto d-luciferin and hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate. Luciferase expression in lysosome leads to d-luciferin oxidation and hypericin activation, resulting in ROS. Reprinted (adapted) with permission from [13]. Copyright 2021, Wiley

Quantum dots, as semiconductor nanoparticles, exhibit distinct optical and electrical characteristics that facilitate the adjustment of their emission in various ways. This capability allows for enhanced PS involvement in BL-PDT. Nonetheless, their notable cytotoxicity raises considerable concerns regarding potential side effects, which poses a challenge for clinical implementation. On the other hand, CDs, a new category of carbon-based nanomaterials, have attracted considerable attention in biomedical fields due to their adjustable emission wavelengths, excellent water solubility, and low toxicity levels [5]. In 2018, Yang et al. [54] reported on the use of bioluminescence to activate photodynamic systems that were modified with carbon dots (CDs-PIX) (Fig. 8B). To enhance therapeutic effectiveness, CDs with excitation-independent photoluminescence were created using a straightforward hydrothermal method. The resulting CDs were engineered to conjugate with protoporphyrin IX (PIX) to form PDT agents (CDs-PIX). The findings revealed that the nano-carrier effect of CDs improves certain limitations associated with PIX and facilitates the excitation process between bioluminescent light sources (BLS) and PIX. The BLS-induced PDT system was capable of generating singlet oxygen and demonstrated approximately 60% therapeutic efficiency in SMMC-7721 hepatocellular carcinoma cells through a fluorescence resonance energy transfer (FRET) process. This indicates that the firefly bioluminescence system functions to some degree, although it is not yet perfected. While CDs-PIX serves as an excellent photosensitizer for photodynamic therapy applications, further research is required regarding its excitation inner light source [54].

Renilla luciferase

In addition to firefly BL, the RLuc–CLZ BL system has also been explored as a source of intracellular excitation. Hsu et al. were the pioneers in utilizing this system by linking the RLuc enzyme to carboxylate-functionalized quantum dots. These quantum dots absorb the photons released during the BL reaction with CLZ through a process known as BRET. Therefore, it is the quantum dots that activate the PS by emitting light at 655 nm, rather than the BL reaction itself. This approach is advantageous because the emission from the quantum dots can be more easily adjusted compared to that from the BL reaction, enhancing the adaptability of this setup for PDT and permitting its application alongside a wider array of PS [17].

This bioluminescent quantum dot conjugate was employed to intracellularly activate the clinically utilized PS meta-tetra(hydroxyphenyl)chlorin (m-THPC, Foscan) in mice that had been transfected with human lung adenocarcinoma epithelial cells. The findings were encouraging, as the average relative tumour volumes of the untreated animals (by day 20) were 4.5 to 6 times larger than their initial volumes. In contrast, mice treated with the Rluc–quantum-dot complex exhibited a significant delay in tumour growth. Additionally, the tumours in the PDT-treated group (Rluc/quantum dots/PS/coelenterazine) were notably smaller compared to those in the PS/coelenterazine group (with a tumour growth inhibition of 4.2%) or the Rluc/coelenterazine/quantum dots group (with a tumour growth inhibition of 23.3%). Nonetheless, it was noted that the Rluc–quantum-dot conjugate exhibited considerable cytotoxicity, which could pose challenges for clinical use due to the risk of this complex unintentionally affecting healthy cells and causing adverse effects [17].

Tumours treated with BL-PDT demonstrated a notable reduction in cell proliferation (27.1 ± 2.6%) when compared to the control groups (87.4 ± 1.6%; 75.2 ± 2%; 92.2 ± 2.6%). Additionally, the Rluc–quantum-dot complex contributed to a decrease in the tumours’ vascularization, which helps in suppressing tumour growth. A key aim of this study was to assess the effectiveness of BL-mediated PDT relative to traditional light irradiation. The findings indicated that, at the same concentration of the photosensitizer (PS), the result from BL-mediated PDT was similar to that achieved with light irradiation of 0.6–0.8 J cm⁻². This range is considered low compared to clinical PDT light doses, which exceed 1 J cm − 2. However, Lai's research shows that the efficacy of BL-mediated PDT is sufficient to produce a photodynamic effect in vivo. Furthermore, the efficiency achieved with this complex (0.6–0.8 J cm⁻²) is significantly greater than that observed with firefly BL-mediated PDT (1.03 × 10 − 4 mJ cm⁻²) [17].

In summary, this research demonstrated the potential of the Rluc–quantum-dot BL conjugate as an alternative excitation source for PDT. However, the photodynamic effect observed was not substantial enough to eliminate the need for further refinement of this system. The reduced efficiency may stem from the reliance on two energy transfer processes: the first being a BRET from the bioluminescent reaction to the quantum dots, and the second involving a FRET from the quantum dots to the PS. An increase in the number of energy-transfer steps can lead to more opportunities for reduced overall efficiency in PDT [34].

An investigation by Kim and associates, the first in vivo demonstration of BL-PDT, aimed to determine the viability of using BL as an excitation source in PDT, particularly due to its lower energy output compared to traditional PDT methods. They engineered self-illuminating conjugates of Rluc and quantum dots to activate the PS chlorin e6 (Ce6) within cells. The study evaluated the effects of BL-PDT on tumour growth in mice, targeting three specific cancer cell lines: colorectal cancer cells (CT26), melanoma cells (B16F10), and lung cancer cells [25].

Initially, the researchers assessed the efficiency of BRET, noting that it ranged from 60 to 65%. These findings reinforce a previous hypothesis suggesting that the low effectiveness of quantum-dot-based photodynamic therapy (PDT) is due to efficiency losses that occur right from the BRET initiation step. Additionally, the study revealed that the conjugates were predominantly found on the external cell surface and did not penetrate the cytoplasm [25].

A particularly noteworthy finding was that while Chlorin e6 (Ce6) molecules at a concentration of 100 µM were activated 40 million times per minute by a 660 nm, 2.2 mW laser, they were activated 300 million times per minute by the Rluc–quantum-dot complex. Consequently, the authors concluded that BRET energy around 100 µJ could induce a more potent photochemical reaction in the cellular membrane compared to laser energy of approximately 100 mJ [25].

In 2022, Yan and colleagues reported on an innovative approach to BRET-induced PDT using a novel photosensitizer that combines the clinically utilized photosensitizer Chlorin e6 (Ce6) with Renilla reniformis Luciferase 8 (RLuc8) proteins. By optimizing the ratio of RLuc8 to Ce6 at 1:25 and achieving excellent spectral alignment between the bioluminescent (BL) emission and Ce6 absorption, they were able to attain a high activation efficiency—an 80% probability of generating activated Ce6 per BL photon. The study demonstrated that fusogenic nano-liposomes serve as a highly effective delivery system for these conjugates into cancer cells. The researchers compared the therapeutic effects of their BL-PDT technique with those of conventional PDT on both 4T1 murine and MDA-MB-231 human triple-negative breast cancer (TNBC) cells, first in vitro and then in vivo using mouse models. Following a single injection of Luc-Ce6, they observed complete tumour remission and prevention of metastasis in lymph nodes and lungs. Additionally, they assessed the neo-adjuvant effects of intratumoural BL-PDT on advanced tumours [52].

NanoLuc luciferase

In an attempt to understand the principle of BRET-PDT, researchers developed a protein-based BRET pair consisting of NanoLuc luciferase, which serves as the energy donor upon the addition of the luciferase-specific substrate furimazine, and SOPP3, a phototoxic protein that acts as a PS. The study demonstrated that the hybrid protein NanoLuc-SOPP3 functions as an effective BRET pair with a BRET ratio of 1.12. The team successfully targeted the delivery of the NanoLuc-SOPP3 BRET pair using tumour-specific small liposomes (approximately 100 nm) to tumours that overexpress the HER2 receptor (human epidermal growth factor receptor 2), both in vitro and in vivo. The developed BRET-activated system significantly inhibited tumour growth in both subcutaneous tumours and deep-seated tumour models. Given the in vivo effectiveness of this proposed BRET-activated system, the researchers believe it holds great promise for depth-independent PDT, potentially broadening the clinical applications of PDT [42].

Bacterial luciferase

To enhance the delivery of luciferases within cells, various gene delivery techniques have been explored for PDT. Utilizing polymer particles or viral/non-viral vectors for gene delivery can achieve elevated levels of luciferases in cells, thereby improving the effectiveness of PDT [22]. Luciferase, a genetically encodable enzyme, can be heterogeneously expressed in bacterial systems [57].

In a recent study, researchers engineered bioluminescent bacteria by transforming an attenuated strain of Salmonella typhimurium, known as ΔppGpp, with a plasmid expressing Fluc (Luc-S.T.ΔppGpp) to serve as an internal light source for the uniform illumination of entire tumours. When embedded within tumours using an in-situ formed hydrogel, the colonized Luc-S.T.ΔppGpp, in conjunction with d-luciferin, was able to produce continuous light to activate the photosensitizer chlorin e6 (Ce6). This led to effective suppression of various tumour types, including opaque melanoma and large rabbit tumours. BL-PDT demonstrated significant advantages over traditional PDT, which relies on external (660-nm) light and operates at much higher light energy, offering only limited growth retardation in small subcutaneous tumours. Moreover, the results indicated that the enhanced PDT using Luc-S.T.ΔppGpp could also stimulate a strong antitumour immune response following treatment, helping to inhibit tumour metastasis and prevent tumour recurrence. Consequently, this study emphasizes that the bioluminescent bacteria-enhanced PDT represents a broadly effective therapeutic strategy for various cancers, regardless of their light-absorbing properties and tumour sizes, and shows promise for potential clinical application due to its favourable safety profile [56].

In another study (Fig. 8C), researchers incorporated d-luciferin and hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate (CaP) to address the identified challenge. They then loaded a plasmid DNA containing the luciferase gene onto this formulation, utilizing the high-density positive charge characteristic of PEI from the nanodrug, referred to as DHDC. Once the DHDC enters the tumour cells, it disassembles and releases the plasmid DNA, which harnesses the intracellular protein synthesis machinery to continuously and abundantly express luciferase. By utilizing endogenous ATP, Mg²⁺, and O₂ present in the cells, luciferase catalyses the oxidation of d-luciferin, resulting in luminescence. This luminescence, in turn, excites hypericin, leading to the production of reactive oxygen species (ROS) that effectively kill cancer cells [13].

Designing effective BL-PDT systems

BL-PDT relies on the efficient transfer of energy from a bioluminescent reaction to a PS molecule, ultimately generating cytotoxic ROS. The process involves three key steps: (1) luciferase enzyme generates light when substrate is introduced, (2) this light activates a PS molecule, and (3) the activated PS produces ROS that destroy tumour cells [6, 8]. An essential consideration is the spectral overlap between the emission peak of the luciferase and the absorption band of the PS, which is essential for efficient BRET. For BL-PDT to be effective, the system must be carefully designed to maximize light delivery, energy transfer, ad drug activation [8]. Table 4 below summarizes the key factors to consider for effective BL-PDT design:

1. Spectral matching: For effective BL-PDT, the colour of light produced by the luciferase must match the absorption range of the PS. FLuc emits yellow-green light (560 nm), which pairs well with Rose Bengal (absorbs at 560), leading to efficient energy transfer and 90% cancer cell death in some studies [48]. However, RLuc produces blue light (480 nm), which poorly matches common PS drugs like Foscan (absorbs 650 nm). To correct this, researchers use QDs as light converters to shift blue light to red [17].

2. Quantum yields: The effectiveness of BL-PDT depends on light, production, energy transfer, and ROS generation. FLuc converts approximately 40% of its substrate into light, while RLuc only manages at least 5%. When the PS is in close proximity to the light source, 80% of the energy transfers successfully. Lastly, good PS molecules, like Rose Bengal are able to convert 75% of absorbed light into toxic oxygen molecules, while others, likes Foscan are less efficient [52]. Combining these factors, FLuc systems achieve more than 25% efficiency, whereas RLuc-QD-Foscan systems fall below 5%.

3. Delivery strategies: Delivering BL-PDT components into cancer cells can be challenging. Nanoparticles, like PLGA-Rose Bengal, offer protection for the PS, improving delivery [55]. However, these conjugates may face challenges with tumour penetration. Engineered bacterial systems, are able to colonize tumours and provide continuous light [56]. Another approach uses gene therapy to use cancer cells as light sources by delivery luciferase DNA, ensuring light and PS are optimally positioned [13].

Table 4.

Key factors for BL-PDT design

System	Light emission wavelength (nm)	Best PS match	Delivery method	Target tumour
FLuc	560	Rose Bengal	Nanoparticles	Shallow tumours
RLuc	450–480	Foscan + QDs	QDs; bacterial delivery	Medium-depth tumours
NanoLuc	460	SOPP3	Gene therapy	High-precision targeting
Bacterial	490	Ce6	Direct tumour injection	Large, deep-seated tumours

In vivo delivery of luciferase and substrates

A critical aspect of translating BL-PDT into clinical application lies in the effective in vivo delivery of luciferases and their respective substrates. This remains a challenge for clinical translation of BL-PDT. Researchers have developed several strategies to address this challenge each with distinct advantages and limitations. Approaches range from nanoparticle delivery systems, such as liposomes, polymeric micelles, and extracellular vesicle, have been explored to assist in luciferase and luciferin delivery [50]. Polymeric nanoparticles, such as PLGA, have also emerged as promising carriers for luciferase and PS molecules, as these nanoparticles are able to accumulate in tumorous cells [51].

Alternative methods include plasmid transfection and viral vectors and the use of genetically engineered bacteria that preferentially accumulate in hypoxic TME [56]. Adeno-associated viruses (AAVs) represent a powerful gene delivery platform for sustained luciferase expression. The AAV9 serotype demonstrated particularly efficient tumour transduction, maintain detectable BL for 28 days post-injection in xenograft models [7]. However, pre-existing neutralizing antibodies in 30–50% of the 5human population may limit clinical translation [26]. Additionally, attenuated Salmonella typhimurium strains have shown remarkable tumour-targeting capability, higher luciferase concentrations in tumours compared to normal tissues [30].

Pharmacokinetics of luciferase substrates

Efficient BL in vivo not only depends on luciferase expression, but also critically depends on the pharmacokinetics properties of the substrates, which vary in stability, bioavailability, half-life, and clearance pathways [23, 43]. These small molecules must overcome multiple biological barriers to generate detectable light signal in target tissues. Once in circulation, the substrate needs distribute through the vasculature to the luciferase-expressing region. The substrates must then cross cell membranes, a process governed by their membrane permeability, to access intracellular luciferase enzymes [23].

The efficiency of light production depends on enzyme–substrate interactions, particularly the enzyme’s affinity for the substrate (quantified by Michaelis constant, Km) and its catalytic activity in the target tissue, following classic Michaelis–Menten kinetics. Importantly, the wavelength of emitted light significantly impacts detection sensitivity due to differential tissue penetration, longer red-shifted wavelengths experience less attenuation as they pass through biological tissue, enabling better signal detection from deeper structures compared to shorter wavelength emissions. This wavelength-dependent attenuation must be carefully considered when selecting luciferase-substrate pairs for applications, particular for deep tissue studies where signal penetration can be challenging [23].

Among available options, furimazine (the NanoLuc substrate) exhibits favourable systemic stability and low background noise in comparison to other substrates [43]. In contrast, coelenterazine is highly susceptible to auto-oxidation in biological fluids [22].

Recent advances in BL-based theranostics

The field of BL-PDT is undergoing remarkable transformation, driven by synergistic advances in nanotechnology, molecular engineering, and biomedical imaging. Recent breakthroughs have expanded the traditional boundaries of BL-PDT, overcoming long-standing limitations in tissue penetration, targeting precision, and therapeutic efficacy through several innovative approaches [3, 25].

Targeted delivery systems for enhanced specificity

Recent work by Ding et al. [11] introduced a novel NIR-II nanotheranostic probe designed for blood–brain barrier permeation, rabies virus targeting, and PDT, highlighting a significant leap in viral phototherapy. The core reporter, a novel polyacetylene fluorophore, was assembled onto an N2-PEG₂₀₀₀-R nanoparticle carrier through click chemistry. The surface was further modified with an aptamer binding the RABV glycoprotein, resulting in the construct of the final product, DK@RA-PEG [11].

After intravenous injection, the construct successfully traversed the blood–brain barrier in mice, targeting the rabies virus infection sites. It enabled real-time NIR-II fluorescence imaging with high signal-to-noise ration. Due to reduced tissue self-emission and longer wavelength photostability. Most notable, under light activation, DK@RA-PEG initiated ROS-mediated viral inactivation without thermal damage, showcasing its potential as a virus-specific PDT modality. This approach combines unprecedented tissue penetration, precise targeting, and photodynamic safety, positioning it as a promising candidate for future clinical intervention again t rabies [11].

Mitochondrial targeting for subcellular precision

Lui et al. [31] engineered and self-chemiluminescent iridium complex (IrL2H) designed to address the critical limitations of conventional PDT and tackle hypoxia. By integrating a luminol-based chemiluminescent system with an iridium PS, the conjugate enables external light-independent activation through tumour-specific hydrogen peroxide, overcoming both penetration depth restrictions and hypoxia therapy resistance. The system operates through an efficient chemiluminescence resonance energy transfer (CRET) mechanism, where hydrogen peroxide-activated luminol emission directly excites iridium centre to generate a dual type and oxygen-dependent type species. This unique combination ensures therapeutic efficacy across varying oxygen tension within tumours. When formulated into haemoglobin-loaded liposomal nanoparticles, the platform demonstrated remarkable tumour growth inhibition (78%) in aggressive tumour models without imaging through its intrinsic chemiluminescence [31].

Future adaptations could explore systemic delivery optimization and combination with emerging immunotherapies to further enhance treatment outcomes. This work establishes a new blueprint for developing self-sufficient, microenvironment-responsive phototherapeutic agents that transcend conventional PDT limitations [31]. This approach could be particularly impactful for treating diffuse malignancies where uniform light delivery remains challenging, effectively bridging the gap between localized BL-PDT and systemic photodynamic effects.

Micro/nanorobots assisted phototherapy

The integration of micro/nanorobots (MNRs) with phototherapy represents a significant advancement in precision medicine, providing targeted treatment options for conditions such as cancer, bacterial infections, neurological, and cardiovascular disorders, these small robotic systems enhance phototherapy by improving real-time navigation, controlled therapeutic delivery, and treatment monitoring, ensuring higher efficacy with reduced impact on healthy tissues. For instance, in cancer treatment, MNRs can be guided magnetically or with light to tumour locations, where they deliver PS molecules or produce heat upon light exposure [10]. By combining enhanced penetration, hypoxia modulation, and imaged-guided therapy, MNRs could significantly improve the precision and effectiveness of BL-PDT.

Smart PS molecules microenvironment-responsiveness

In the context of developing microenvironment-responsiveness antimicrobial strategies, recent work by Ding and co-workers (Ding et al.) introduced an innovative NIRPS (DHTPA) that capitalizes on the acidic conditions of bacterial infections. Their design incorporates three fundamental elements: (1) donor–acceptor structure enabling NIR absorption and emission, (2) pH sensitive tertiary amines, and (3) aggression-induced emission characteristics. This system demonstrates a remarkable increase in ROS generation under infection-mimicking acidic environments, compared to neutral environments, achieving significant bacterial reduction. Of particular note is its demonstrated efficacy in vivo where it accelerated wound healing while maintain excellent biocompatibility. The pH-activatable nature of DHTPA represents a significant advance in targeted antimicrobial phototherapy, as intrinsically discriminates between infected and healthy tissue, potentially overcoming the selectivity limitation of conventional PS molecules [9–11].

This work exemplifies the next generation of smart PS molecules that can significantly enhance BL-PDT by responding to multiple microenvironmental cues, potentially overcoming current limitations in selection for the treatment against resistant strains. The pH activable factor informs of selectively that could make BL-PDT systems safer and mor effective.

Advantages of BL-PDT

BL-PDT is a groundbreaking improvement over traditional PDT, as it overcomes several limitations of the conventional approach by using light generated from within the body. Unlike standard PDT, which depends on external light sources such as lasers or LEDs to activate a photosensitizer, BL-PDT utilizes chemical reactions, commonly involving a luciferase enzyme and a luciferin substrate, to produce light internally.

Various techniques for tumour-targeted drug delivery used in PDT and other molecular treatments can be utilized for BL-PDT. The agents used in BL-PDT may be paired with targeting agents, such as mAbs that target specific biomarkers or RGD peptides to facilitate targeted bioluminescence BRET-induced photoimmunotherapy. Another significant benefit of BL-PDT is its self-sustaining light activation mechanism, which removes the need for bulky external equipment, such as LEDs, thereby enhancing its accessibility for clinical use. It can also be effectively paired with immunotherapy and chemotherapy, improving overall treatment effectiveness and promoting immune responses that contribute to better long-term outcomes. Additionally, BL-PDT tackles the challenge of hypoxia-induced resistance—an issue prevalent in standard PDT—by utilizing oxygen-releasing nanocarriers or oxygen-independent strategies.

Limitations and future perspectives

BL-PDT offers an innovative method for treating cancer, but several challenges need to be resolved before it can be used in clinical practice. A significant biological challenge is the lower light intensity produced by bioluminescence compared to the laser sources that are typical in conventional PDT. This diminished intensity may limit the activation efficiency of photosensitizers, resulting in reduced production of ROS and weaker therapeutic effects. Additionally, luciferase enzymes, responsible for bioluminescence, can degrade in biological settings, leading to a shorter lifespan. Likewise, luciferin substrates might require multiple doses to sustain adequate light output. Another significant issue is the oxygen dependency of PDT; since ROS production depends on molecular oxygen, the often-hypoxic conditions present in tumours could compromise the effectiveness of BL-PDT, hindering its capacity to induce cancer cell death.

In terms of technical limitations, there are several challenges associated with BL-PDT. A crucial problem is the mismatch between the emission spectrum of bioluminescence and the absorption spectrum of commonly used photosensitizers. Many of these photosensitizers are designed to absorb red (620–750 nm) or NIR (750–2500 nm) light, while bioluminescent systems typically emit light in the blue (380–500 nm) or green (500–570 nm) range, resulting in less effective excitation. Additionally, the duration of bioluminescence is limited by the availability of luciferase substrates, which may require continuous or repeated dosages to ensure effective treatment. Efficiently delivering luciferase and its substrate to tumour tissues remains a challenge. Although methods and application of nanoparticles, gene therapy approach, and viral vectors are under investigation to enhance delivery, issues related to targeting accuracy, bioavailability, and potential toxicity. These must be resolved prior to their widespread application.

Moreover, several clinical and practical obstacles complicate the advancement of BL-PDT into mainstream therapeutic use. Most current research still resides in the preclinical phase, with only a limited amount of human clinical trial data available. There are no established standardized treatment protocols for choosing optimal photosensitizers, luciferase systems, or dosing strategies, making it difficult to incorporate this therapy into routine clinical practice. The costs associated with producing and purifying luciferase enzymes and substrates represent another hurdle. Furthermore, introducing foreign luciferase proteins into the body might provoke immune reactions, potentially diminishing treatment effectiveness or leading to inflammatory side effects.

Despite these challenges, ongoing research is discovering innovative solutions to enhance the feasibility of BL-PDT. Genetic engineering approaches are being explored to allow for stable and long-lasting luciferase expression in tumour cells, which could prolong the duration of bioluminescence. Efforts are also underway to improve the spectral overlap between luciferase and photosensitizer absorption to maximize light utilization. Additionally, new strategies aim to decrease PDT's reliance on oxygen, potentially boosting its effectiveness against hypoxic tumours. Conducting more clinical trials is vital to establish the safety, efficacy, and standardized protocols for BL-PDT, which are essential for advancing this promising technology. Overcoming these challenges will be key to unlocking the full potential of BL-PDT as a non-invasive and highly targeted cancer therapy.

The BL-PDT agents used in the studies discussed in this review do not inherently target cancer cells. However, various tumour-targeted drug delivery strategies designed for PDT and other molecular therapies can be utilized with BL-PDT. These agents could be synergized with monoclonal antibodies (mAbs) that target specific biomarkers or RGD peptides to facilitate targeted bioluminescent resonance energy transfer-induced photoimmunotherapy (PIT). More advanced approaches, such as protease-sensitive quenchers, can enhance tumour specificity. Certain BL molecules need cofactors like Ca²⁺ and adenosine triphATP), which may provide pathways to connect with particular signalling routes and metabolic processes. The efficiency of BRET, which is distance-dependent (~ r-6), can be leveraged to focus on certain molecular interactions. Since BL-PDT employs minimally toxic agents before activation—contrasting with traditional chemotherapeutics—molecular transport and pharmacokinetic strategies, such as fine-tuning the timing between luciferase and luciferin administration, can improve target specificity while reducing harm to healthy tissues. Additionally, advanced drug delivery systems could facilitate the systemic administration of BL molecules.

Conclusion

Photodynamic therapy is a minimally invasive approach that shows great potential in addressing malignant diseases. It can be utilized either prior to or following chemotherapy, radiation therapy, or surgical procedures, without hindering these treatments or its own effectiveness. Additionally, PDT offers significant advantages with minimal invasiveness, achieving similar or even better results while reducing the chances of complications and disfigurement. However, its effectiveness is restricted by the ability of light to penetrate tumours, whether directly or via more invasive methods like needle or endoscopy-guided fibre optics. Consequently, there is an interest in developing methods to deliver light to tumours located throughout the body.

Bioluminescence is mainly found in marine organisms. Some bioluminescent species are unable to produce luciferin on their own and therefore rely on symbiotic relationships to obtain it, while others can synthesize it independently. The interaction between oxygenated luciferin and luciferase leads to the formation of oxyluciferin, resulting in light production. Research in bioluminescence heavily focuses on genetic engineering to improve safety and convenience in various applications. We have explored several natural bioluminescent systems, including those dependent on different forms of d-luciferin, coelenterazine, as well as bacterial systems and their underlying processes. Gaussia and Renilla luciferases are less effective than FLuc because they emit blue bioluminescence (with a peak at 480 nm), which is highly absorbed by pigmented substances like haemoglobin and melanin and is scattered by tissues. In contrast, FLuc emits light that extends beyond 700 nm into the red spectrum, allowing it to penetrate mammalian tissues more effectively. The NLuc/coelenterazine system, although engineered and offering a higher photon output, is not available for commercial use. Additionally, certain luciferin systems are not widely used due to limited commercial availability. Reporter genes are developed to attach to other genes for functionality. There is a need to create more complementary pairs of luciferin and luciferase that possess enhanced properties for new applications. In BL-PDT, PSs such as hypericin, mTHPC, and chlorin e6 (Ce6) linked with luciferase proteins (like Rluc8) show great potential because they can be triggered by BL produced by luciferases.

BL-PDT presents several benefits over traditional PDT, making it a promising method for cancer treatment. One of the key advantages is its capability to penetrate deep tissues without the need for external light sources, which is especially beneficial for targeting tumours located in difficult areas such as the brain, pancreas, or deep within the lungs. Furthermore, BL-PDT reduces side effects by focusing on cancerous cells while preserving healthy tissues, thus lowering systemic toxicity and minimizing collateral damage.

The biocompatibility of bioluminescent compounds, which are often derived from natural enzyme systems, decreases the risks of immune rejection and enhances patient safety. Moreover, integrating BL-PDT with imaging techniques enables real-time monitoring of tumour responses, which helps refine treatment precision. Its non-invasive characteristics also expand its potential applications beyond oncology to include antimicrobial therapy, wound healing, and treatment for neurodegenerative diseases. Collectively, these benefits establish BL-PDT as a highly innovative and effective approach for the future of photodynamic therapy.

Abbreviations

BL

Bioluminescence

BL-PDT

Bioluminescence-activated photodynamic therapy

BRET

Bioluminescence resonance energy transfer

Ce6

Chlorin e6

CTZ

Coelenterazine

CRET

Chemiluminescence resonance energy transfer

FLuc

Firefly luciferase

GLuc

Gaussia

Lux

Bacterial luciferase

NIR

Near infrared

PDT

Photodynamic therapy

PS

Photosensitizer

PEI

Polyethyleneimine

PLGA

Poly(lactic-co-glycolic acid)

RLuc8

Renilla reniformis Luciferase 8

ROS

Reactive oxygen species

TME

Tumour microenvironment

Author contributions

MM and BPG conceptualized the study. MM and BPG drafted the manuscript. BPG and HA reviewed and edited the manuscript. All authors approved the last version of the manuscript.

Funding

This work is based on the research funded by the South African Research Chairs initiative of the Department of science and technology and National Research Foundation (NRF) of South Africa (Grant No. 98337), South African Medical Research Council (Grant No. SAMRC EIP007/2021), as well as grants received from the NRF Research Development Grants for Y-Rated Researchers (Grant No: 137788), University Research Committee (URC), University of Johannesburg, and the Council for Scientific Industrial Research (CSIR)-National Laser Centre (NLC).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Yes.

Competing interests

The authors declare no competing interests.