Naphthalimide dyes as fluorescent probes for emerging applications in medical diagnostics and therapeutics: a review

Abstract

Naphthalimide dyes have become an important class of fluorophores since they combine structural adaptability with favorable photophysical characteristics as well as good compatibility with biological systems. These qualities make them strong candidates for use in medical diagnostics and therapeutics. Their tunable emission, photobleaching resistance, and capability of undergoing different processes like intramolecular charge transfer, twisted charge transfer, and photoinduced electron transfer have contributed to their applications in biosensing and cancer imaging, photodynamic therapy and multifunctional drug delivery. Recent advances have led to the extension of this family into near infrared and two-photon designs for deep tissue imaging, activatable probes having high selectivity, nanocarrier formulations and targeted theranostics. Work is also well underway to integrate naphthalimide probes with artificial intelligence platforms and multiplexed sensing approaches as well and opens up the door to real-time monitoring and systems-level diagnostics. Despite these improvements, there are still critical hurdles to overcome, such as optimising compatibility with the body, enhancing light penetration through tissue, controlling pharmacokinetics and traversing the regulatory pathways. This review focuses on innovations in structural design, biomedical field and clinical translation of the naphthalimide derivatives, and further presents future research directions in smart probes, precision medicine, and economics in healthcare. Overall, naphthalimide scaffolds seem to be well poised to make a contribution in the future of molecular diagnostics and personalised therapeutics.

1 Introduction

Naphthalimide derivatives have become a structurally diverse family of organic fluorophores with favourable photophysical properties, such as large Stokes shifts, high photostability and ease of structural modification. These attributes have attracted increasing interest in the biochemical sciences over the past decade in particular, where naphthalimides have been investigated for their use in targeted drug delivery, biomolecular sensor and diagnostics imaging [1,2,3]. While fluorophores based on rhodamines, BODIPYs, and cyanines are still widely used, naphthalimide scaffolds are increasingly considered to be one of the best candidates for next-generation molecular probes and theranostic agents.

The backbone structure is a naphthalimide core functionalized with an imide functional group on a naphthalene ring, which endows them with high electron affinity and efficient π-π stacking interactions, leading to photostability and synthetic tunability. More importantly the C-4 and N-positions can be modified to give predictable changes in photophysical properties by using electron-donating or -withdrawing groups on the molecule [4]. Such systematic shifts have been used to develop ratiometric fluorescent probes and have enhanced biological selectivity in applications from ion sensing to live-cell imaging [1, 3]. The structural sketch of 1,8-naphthalimide is given in Fig. 1.

Fig. 1

General structure and numbering of 1,8-naphthalimide

A key feature of naphthalimide fluorophores is that they can demonstrate very large Stokes shifts to reduce reabsorption and background effects during imaging. Yu et al. [5] reported an NBDNI derivative, which exhibited a significant polarity sensitive behavior and an extremely large Stokes shift of 241 nm in ethanol, owing to the twisted intramolecular charge transfer (TICT) character. In a related work, Prsir et al. [6] synthesized two derivatives of 1,8-naphthalimide-piperazine [NI-1 and NI-2] which showed Stokes shifts of around 200 nm along with the selective metal ion sensing capability. Together these results emphasize the strategic exploitation of modifications of the structure of the naphthalimide core at the 4-position, in order to obtain strong photophysical responses in varying environments. They also demonstrate the power of rational molecular design to reproducibly cause ultra-large Stokes shifts with important implications in both biological and chemical sensing.

Beyond such advantages of photophysics, many derivatives of naphthalimide exhibit a property of dual-emission, where fluorescence and phosphorescence co-exist with the same molecular structure. This property is of special value in multicolor imaging, because it allows to collect emission signals in different spectral windows without using several probes. Wang et al. [7] showed that fluorescence and room-temperature phosphorescence (RTP) is possible under ambient conditions with waterborne polyurethanes based on brominated 1,8-naphthalimide derivatives. The introduction of halogen atoms improved spin-orbit coupling and intersystem crossing, and thus the balance between the two emissive pathways. Such structural adaptability strengthens the potential of naphthalimide systems as versatile probes for biomedical imaging in which a single molecular platform can be adapted to a wide range of diagnostic requirements.

From a biomedical point of view, naphthalimide derivatives have been gaining popularity in their use as live cell imaging and as drug delivery vectors. De et al. [8] compared tethers of amphiphilic and nonamphiphilic derivatives of naphthalimides, and showed that the former molecules switch from aggregation-caused quenching (ACQ) in organic solvents to high aggregation-induced emission (AIE) in aqueous environments, a feature which is very useful for biological imaging applications. In a complementary study, Marullo et al. [9] characterized a core-functionalized 1,8-naphthalimide derivative which is able to self-assemble into supramolecular aggregates in an aqueous environment, highlighting the structural versatility of the family of dyes in biologically relevant environment. These aggregates exhibited good drug-binding and sensing properties, implying their potential as nano-scale drug delivery platforms. Taken together, these results demonstrate the structural engineering of amphiphilic naphthalimide scaffolds to achieve aggregation-induced emission (AIE) and aggregation-driven drug-carrier functionality under physiological conditions.

Researchers have been working towards enhancing the structure of naphthalimides to give it greater selectivity against cancer cells and decreased damage to healthy cells. Such selectivity is the key to reducing side effects and enhancing the level of safety in cancer treatment [10]. They have been shown to be effective against a very broad spectrum of cancer such as lung, breast, ovarian and colon cancer. Some well-known related naphthalimide family compounds include amonafide, mitonafide, elinafide, bisnafide and UNBS5162 (designed as an optimized bisnafide analogue with chemical modifications made to reduce toxicity but preserve anticancer activity) (Fig. 2) [11].

Fig. 2

Structures of heterocyclic naphthalimide derivatives already in clinical development trials. Permitted to use as an article under a Creative Commons license [11] Copyright (2024) by MDPI

Collectively, the optical tunability, photostability, low cytotoxicity, and modular chemical design of 1,8 naphthalimide compounds, present a useful platform upon which new biomedical tools can be designed. Their possible role in simultaneously performing the diagnostic and therapeutic functions - so-called theranostics - underlines their importance in the emerging field of precision medicine.

In view of these advances, there is a need for critical evaluation of the role of naphthalimide dyes against a backdrop of the general landscape of medical research and innovation in the clinic. This review aims to present an overview of the current situation and role of naphthalimide dyes in medical applications focusing on their basic photophysical features, biological interaction and possibility of clinical translation. By simultaneously integrating the findings in these crosscutting fields, the goal of our effort is to provide an integrated narrative that spans basic science and clinical practice. The aim is to present a balanced and critically synthesized view on this rapidly expanding area of research providing a perspective relevant to researchers, clinicians and industrial stakeholders interested in the current and future of naphthalimide-based medical technologies.

2 Chemical structure and photophysical properties

2.1 Core structure and modifications

The 1,8-naphthalimide framework is a very versatile fluorophore framework. Its electron-deficient naphthalene structure fused with an imide motif exhibit strong π-π stacking, extended conjugation and good photostability. These innate features help it to perform well on high-intensity fluorescence applications. Structural modifications such as π-expansion and positional isomer formation make it even more appropriate for its application as molecular probes, imaging agents and stimuli-responsive materials [12].

In particular, substitutions on two important sites, the C-4 position of the aromatic core and the position of the imide nitrogen (N-position), provide effective tools to fine-tune the photophysical behaviour of the dye. According to Li et al. [13], these C-4/N-positions with multifunctional substitution could cause a blue-to-green emission with a photoluminescence quantum yield of 61−85% and electron-donating functional groups such as hydroxyl and amide, was found to boost a lot higher photostability and environment sensitivity in cellular imaging. Such modifications make it possible to have a fine control of emission wavelengths, fluorescence quantum yields, environmental sensitivity and overall biofunctionality.

The C-4 atom is at the center of a delocalized π-system in 1,8-naphthalimide, and as such, it proved to be an important site to modulate frontier MO interactions. Electron-donating substituents introduced at this position, such as amino, hydroxyl or alkoxy substituents, have been found to enhance intramolecular charge transfer (ICT), stabilize the excited state and give a substantial bathochromic shift of the absorption and the emission spectra. For instance, Wang et al. [14] showed that derivatives of 4-amino-naphthalimide, which allow for dramatic red-shifts in polar media with emission ranges ranging from blue in the non-polar to yellow-orange in the polar, exhibit strong ICT behavior. Tian et al. [15] showed that the substituted methoxy group at the C-4 position allows an improved performance of photoinduced electron transfer probes with higher fluorescence quantum yield and sensitivity, mostly by decreasing the number of non-radiative decay pathways. Collectively, these results demonstrate the versatility of targeted modification to the C-4 position, which is capable of targeted control over emission wavelength, quantum yield, and environmental responsiveness. They are also indication that naphtalimide scaffolds are suitable for imaging and sensing applications.

In contrast, substitution at the N-imide position has proven particularly useful towards improved solubility, targeting capability, and biocompatibility but not significantly changing the electronic properties of the chromophore core. Introducing hydrophilic moieties or bioactive ligands at such site enables integration into biological systems and such derivatives are promising for live-cell imaging and targeted drug delivery [16]. The adaptability of the N-position further supports conjugation with biomolecules, polymers or nanoparticles and thus expands the biomedical relevance of naphthalimide-based probes.

In the solid-state environment as in crystalline forms, the packing of the molecules has a direct influence on the optical properties. Voss et al. [17] showed the hydroxyl and the carbamate groups are the key elements involved in the formation of hydrogen bonding between adjacent molecules. These interactions stabilize the system into forms of more planar arrangement locking chromophores in place and leading to lower non-radiative decay which imparts greater emission stability under prolonged excitation. Such behavior is extremely desirable, for example for applications such as confocal microscopy or continuous fluorescence monitoring. Even slight variations in orbital overlap distance caused either by the chemical environment or by variation of the structure can cause effects quite comparable to the effects caused by applied pressure. Wu et al. [18] have reported one-dimensional macroscopic perspective; perovskite analogues show a bandgap compression and emission shift upon compression which is analogous to the effect of electron donating substituents on the naphthalimide derivatives. These parallels make the relation between molecular structure and optical response close.

Altogether, these examples are illustrative of the reason why the derivatization of 1,8-naphthalimides at the position specific level has been so extensively pursued. Adjustments at either the C4 or N sites, allow for fine-tuning of parameters such as Stokes shift, quantum yield, excited state lifetime and even two photon absorption. The ability to modulate these properties has led the scaffold to becoming a powerful platform for use in sensor - imaging applications and, more recently, in theranostics development.

2.2 Photophysical characteristics

The photophysical properties of 1,8-naphthalimide derivatives are the key of their widespread application in fluorescence-based sensing, imaging and optoelectronic applications. Their behaviour is mainly determined by four parameters, which characterise also conventional dye families: fluorescence quantum yield, Stokes shift, photostability and tunable emission. Analysis of these parameters not only reveals the benefits of naphthalimides, but also serves as an input into molecular design of next generation probes.

2.2.1 Fluorescence quantum yields

Fluorescence quantum yield (QY) is the ratio of the number of emitted photons to the number of absorbed photons under excitation and it is a basic indicator of the efficiency of the light emitting systems. Naphthalimide-based compounds tend to have high QYs, and introducing multiple functionalities to them increases yields. The efficiency of this compares favourably with that of classical rhodamines and BODIPY dyes, but at the expense of much larger rigidifying frameworks. The QYs are further improved by non-planarity and rigidification strategies, e.g., incorporating heterocyclic moieties that inhibit nonradiative decay and produce dyes that are as bright as commercial dyes [19].

The electron-donating substituent at the C-4 position is particularly useful to increase quantum yields through the amplification of intramolecular charge transfer (ICT). Recent reviews indicate the benefit of 4-hydroxy-1,8-naphthalimide derivatives, being sensitive to environmental changes but also maintaining high QY [20].

2.2.2 Stokes shifts

The Stokes shift is defined as the difference between the absorption maximum and the emission maximum of a fluorophore and represents the energy gap between excitation and photon emission. Naphthalimides usually have large Stokes shifts ranging from 67 to 170 nm (even above 200 nm depending on solvent polarity) [5, 21, 22]. The spectrally non-overlapping emission and absorption bands and reduced self-absorption afforded by a sharp transition further enhance the sensitivity of detection in complex media. In biological imaging, this property is of particular importance, since it leads to a suppression of the background autofluorescence and an increase in signal-to-noise ratios.

Structural tailoring has a direct effect on the magnitude of Stokes shift. Substitution of electron-withdrawing groups with electron-donating groups increases the intramolecular charge transfer (ICT) components, which results in the larger bathochromic shifts. In addition, conformation relaxation pathways can be quenched and energy states stabilized by the presence of bulky or rigid side-groups, resulting in red-shifted emissions. For instance, in a recent study of a naphthalimide-based SO₂ probe (NG-TCF), a very large Stokes shift of ~ 207 nm (e_x: 463 nm, e_m: 670 nm) was reported to decay to ~ 87 nm on binding to sulfite ions with a correspondingly large ratiometric blue-shift in emission of ~ 120 nm [23]. In contrast, another study of a naphthalimide-Fe(II) spin crossover complex reported dual emission peaks in the range of 390–408 nm when excited at 310 nm, which corresponds to smaller Stokes shifts in the range of 80–100 nm [24].

These results show that both molecular structure of dye and surrounding environment affect the magnitude of Stokes shift. Even when small tweaks in design are made, modest to unusual shifts can be produced, giving the naphthalimides a fair bit of versatility for use in applications ranging from thermosensing to deep tissue imaging.

2.2.3 Photostability

Photostability is one of the most outstanding features in naphthalimide dyes. In contrast to fluorescein or cyanine dyes which bleach quickly, the stiff imide scaffold of Naphthalimides makes the excited state stable and suppresses the degradation pathways. This stability in the presence of light over a long period of time enables them to be especially suitable for applications such as time-lapse, monitoring of therapeutic treatment, and even high-intensity microscopy [22]. Recent work has suggested that the photostability can be further enhanced by certain structural modifications. Bulky substituents, protecting the imide core, will minimize the effects of solvent and oxygen and retards processes leading to bleaching. Solvent polarity has been shown to also play a role in the stabilization of the excited state, yet another method of fine tuning photostability [25].

Future advancement will require the creation of derivatives with a good combination of photobleaching resistance and high and consistent brightness. While structural rigidity is one of the contributors to stability, in some cases it may also reduce the quantum yield. Striking a balance between such properties will be critical to the development of a new class of probes, capable of performing reliably in challenging biomedical applications, from imaging of deep tissues to monitoring therapy over extended periods of time.

2.2.4 Tunable emission

One of the most valuable features of naphthalimides is their ability to produce tunable emissions. Through the change of substitution pattern on core structure, the emission maximum can be systematically shifted from the blue region into the near-infrared region. For instance, the incorporation of glycine ethyl ester or azetidine into the scaffold gives blue green shifts of emission, while more powerful donor acceptor systems may be extended into the red and near infrared regions [26]. This type of flexibility goes beyond mere spectral tuning. Unlike when single fluorophores, such as Fluorescein, are integrated into multiplexed imaging platforms, several emission colors can be used simultaneously, a feature particularly useful for researching complex biological environments. Notably, this tunability is achieved without sacrificing any essential photophysical characteristics, such as the quantum yield, Stokes shift or photostability.

The combination of the spectral flexibility and photophysical stability is an important feature which explains why naphthalimides are good candidates for applications that require a broad coverage in tandem with a constant performance [21].

2.2.5 Influence of substituents on photophysical properties of naphthalimides

The photophysical characteristics of 1, 8-naphthalimide (NI) derivatives are extremely versatile and can be modified by replacing various groups at the C-4 position and at the N-imide location. Such changes affect key properties like quantum yield (Φ_F), emission wavelength, Stokes shift and photostability which are all necessary for applications in fluorescence sensing, imaging and photodynamic therapy. As an illustration, the electron-donating groups such as amino or alkoxy group at the C-4 position may induce high fluorescence quantum yields and result in bathochromic shift of the emission spectrum, thus these derivatives are very useful in fluorescence-based applications [14]. Conversely, 4-hydroxy substitution allows excited-state intramolecular proton transfer (ESIPT), which yields high Stokes shifts, but tends to reduce the quantum yield due to competing non-radiative decay mechanisms [27]. The rigidity of the molecular framework is also important. Certain substituents such as aziridine rings are known to quench twisted intramolecular charge transfer (TICT) which results in brighter fluorescence and longer photostability [28]. N-imide substitution, in turn, exerts a less pronounced influence on the quantum yield yet is very advantageous towards increasing the solubility and receptor binding, which is much more useful in the biocompatibility of these fluorophores [16, 29]. Table 1 shows the effect of certain substituents. These modifications allow naphthalimide derivatives to be fine-tuned for numerous biomedical uses, including use as a fluorescence imaging and therapeutic agent in photodynamic therapy.

Table 1 Substituent effects on photophysical properties of naphthalimides

3 Mechanisms of fluorescence

3.1 Fundamental photophysical processes

Naphthalimide-based fluorophores show a wide variety of photophysical behavior, which may be attributed to their conjugated structures and strong ability for electronic delocalization. These properties permit for fine-tuned application in biomedical science and engineering.

3.1.1 Intramolecular charge transfer (ICT)

Intramolecular charge transfer (ICT) is observed in the case of introduction of electron donating groups like amino or dialkylamino substituents at C-4 position of naphthalimide core resulting in a donor-acceptor system. On excitation the electron density is being transferred from the electron donor substituent to the electron-deficient imide moiety. This ICT process is extremely sensitive to the surrounding environment; for example, polarity of solvent can strongly affect the dipole moment of the excited state and thus can change the response of emission [32]. ICT allows environment sensitive fluorescence and is highly applicable in the development of probes to detect polarity, pH and metals.

3.1.2 Twisted intramolecular charge transfer (TICT)

ICT is a specific type of ICT where a twisted geometry between donor and acceptor moieties forms on excitation which minimizes overlap of donor and acceptor π orbitals and stabilizes the charge separated state. This conformation-related effect has led to high sensitivity to viscosity and polarity. For instance, a water-soluble naphthalimide with a piperazinyl moiety exhibited TICT-induced fluorescence sensitivity to pH and viscosity, thereby demonstrating the role of TICT in the design of ratiometric sensors, as well as microenvironment-responsive sensors [33].

3.1.3 Photoinduced electron transfer (PET)

PET is a fluorescence quenching method in which an electron donor or acceptor group participates in non-radiative decays. The technique is most often applied in so-called turn-on or turn-off sensors, in which binding of an analyte to the sensor interferes with or restores PET, thereby changing fluorescence intensity. Dual PET-ICT systems have been developed to enhance the selectivity of the probes and background signal. More specifically, probes that integrate both mechanisms can be used to selectively sense analytes including H₂S by modulated electron transfer [34].

There is also the possibility of the strategic replacement of the donor site to alter fluorescence behavior. Fueyo-Gonzalez et al. [35] have shown that the replacement of a methoxy donor with other amines in the 6-position led to the reversal of the emission trend in the system, i.e., instead of ICT-like emission, the emission was quenched by PET. Compounds with intramolecular charge transfer (ICT) possess classic strong solvatochromism with the emission maximum shifting to higher energy as the polarity of the environment increases. The stabilization of ICT excited state in polar solvents results in a red shift of emission, and therefore these systems are useful for the detection of polarity gradients in biological and chemical media, including macrophage imaging (Fig. 3) [35]. Interestingly, Fueyo-Gonzalez et al. showed that conversion of conventional solvatochromic fluorophores into “reversed solvatochromic” systems which emit brighter fluorescence in polar than apolar environments, can be achieved by converting a hydroxyl group into a dimethylamino group. The polarity-dependent fluorescence response makes them useful both as pH sensors in organic solvents and as effective probes for imaging cellular environments.

Fig. 3

Emission spectra and fluorescence image of compound 1a (A) and 1b (B) upon UV lamp irradiation (λ_ex 365 nm) in CH₃CN and H₂O. Reproduced with permission from [35] Copyright (2019) by Elsevier

Likewise, Georgiev et al. [29] prepared a water-soluble naphthalimide, where a replacement donor gave an apparent PET process that enabled the sensing of pH and water contents sensitively. However, even more recently, Geraghty et al. [36] have been able to tune the 4-position donor by changing the electron-withdrawing nitro to an electron-donating amine leading to a shift towards ICT-driven fluorescence. These studies reveal that even subtle changes at the donor site can have an effect on the balance between photoinduced electron transfer (PET) and intramolecular charge transfer (ICT) and provide a useful design handle for tuning emission characteristics.

3.2 Environmental sensitivity

The fluorescence of naphthalimide derivatives is strongly influenced by their microenvironment, which gives them the adaptability to be used as probes for sensing and imaging. Solvent polarity is a major influence and derivatives which undergo ICT often show large solvatochromism and their emission maxima are shifted by the dielectric strength of the surrounding medium. The ICT excited state is stabilized by a solvent in more polar solvents, which shifts the emission to the red and makes them particularly suitable to sense polarity gradients in biological and chemical systems, as they have shown in polarity-responsive macrophage imaging probes [37].

Another important environmental parameter is viscosity. The twisted intramolecular charge transfer (TICT) naphthalimides can be used to detect viscosity based on changes in fluorescence. In media with low viscosity, the rotational relaxation is predominant and results in nonradiative decay and poor emission. On the other hand, increased viscosity will inhibit intramolecular movement, inhibit the TICT process, and increase the intensity of fluorescence. Molecular rotors have also been used for the investigation of microviscosity in aqueous systems [33]. Bakov et al. [33] described a hydrophilic analog of 1,8-naphthalimide including a rotor module and a switch-like “off-on” fluorescence effect. Introduction of a 4-methylpiperazinyl moiety at C-4 resulted in generation of TICT movement, evidenced by the photophysical studies carried out in different viscosity solvents (Fig. 4). In its TICT-off excited state, the probe had a weak yellow-green emission, and protonation inhibited TICT and increased the fluorescence. Viscosity-dependent measurements of glycol, glycerol, and their mixtures (Fig. 5) showed an increase in emission intensity with increasing viscosity, and the correlation between the fluorescence intensity and viscosity was found to be strong (R² = 0.9843), which is in agreement with TICT rotor behaviour.

Fig. 4

Switching mechanism of probe 2 as a function of pH. Permitted to use as an article under a Creative Commons license [33] Copyright (2022) by MDPI

Fig. 5

Fluorescence spectra of probe 2 (λ_ex = 400 nm) normalized to optical density: (A) recorded in glycol (black), glycol/glycerol mixtures at 3:2 (red), 1:1 (blue), and 2:3 (green) v/v ratios, and pure glycerol (orange); (B) measured in glycol and glycerol, as well as in their Tris-HCl (10 µM, pH 8) buffered counterparts. Permitted to use as an article under a Creative Commons license [33] Copyright (2022) by MDPI

The nature of protonation of functional groups on naphthalimide core also plays an important role in fluorescence as it affects the ICT and photoinduced electron transfer (PET) process. As an example, pH-sensitive systems can be used as molecular switches such that upon protonation or deprotonation of the system, PET pathways are switched on or off resulting in turn-on or turn-off of fluorescence. Dual-mode pH sensors were also developed that display complex on -off -on fluorescence patterns over changing pH conditions, further expanding the use scope of the instruments to biological pH mapping [38].

The temperature has a minor but meaningful impact on the photophysics of naphthalimide derivatives. Increase in thermal energy can allow nonradiative decay channels to be more efficient thereby diminishing the total fluorescence emission. In twisted intramolecular charge transfer (TICT) systems, high temperature can further vary the equilibrium between the locally excited and charge-separated states altering the emission efficiency significantly [39]. Likewise, thermally modulated fluorescence may also be seen with PET based probes, where the elevated temperature either facilitates or suppresses electron transfer depending on configuration of substituents.

Effects mediated by temperature have not been studied as intensively as those of polarity, viscosity, or even pH, but they are being regarded as an important consideration when designing in vivo or in situ probes where physiological and environmental temperature changes cannot be neglected.

To put the performance of naphthalimide-based probes into perspective, we compared the most important photophysical and functional characteristics of major fluorophore series namely rhodamines, BODIPYs, fluoresceins, and cyanines (Table 2). Although several of these systems provide high fluorescence efficiency, they vary significantly in their tunability (spectrally), photostability and responsiveness to their environments.

Table 2 Comparative fluorophore performance

4 Applications in cancer diagnostics and imaging

Naphthalimides are a class of compounds with significant anticancer activities, primarily attributed to their DNA intercalating properties. These compounds have been extensively studied for their cytotoxic effects across a variety of human and murine tumor cell lines, exhibiting potent inhibition of cancer cell proliferation. Among naphthalimide derivatives, drugs such as amonafide and mitonafide have progressed to clinical trials, underscoring their therapeutic potential in cancer treatment. Advances in chemical modification, particularly the development of bisintercalating agents like elinafide, have improved antitumor activity and pharmacological characteristics. Recent research has focused on systematic synthesis and structural alterations to improve DNA-binding affinity and anticancer effectiveness, yielding promising findings in a variety of cancer models, including breast and lung malignancies.

4.1 Tumor-specific targeting

Tian et al. [42] reviewed recent advances in naphthalimide-polyamine conjugates that target tumor cells by exploiting overexpressed polyamine transporters, improving selectivity and reducing systemic toxicity compared to traditional naphthalimides. These compounds exhibit multimodal anticancer actions, including DNA damage via the p53/PARP-1 pathway, autophagy disruption, and inhibition of angiogenesis and metastasis through VEGF/MMP suppression. Sulfonamide-containing naphthalimide derivatives exhibit low cytotoxicity and are efficiently taken up by tumor cells, enabling sensitive fluorescent imaging in melanoma models [43]. Fluorescent probe-based imaging has become a valuable approach in cancer research, providing a non-invasive, rapid, and highly sensitive method with excellent selectivity and the advantage of real-time visualization. These probes combine strong fluorescent properties with tumor-targeting groups that bind to proteins highly expressed in tumors, such as carbonic anhydrase IX. Additionally, naphthalimide probes conjugated with tumor-targeting ligands can selectively localize in mitochondria or lysosomes of cancer cells, allowing real-time imaging and differentiation between normal and tumor cells [44]. Such probes also show promise for applications in noninvasive cancer diagnosis and therapy monitoring, with good biocompatibility and minimal toxicity reported in preclinical studies The various mechanisms through which naphthalimide-based probes demonstrated tumor-targeting capabilities are highlighted in the later next sub-sections of this review.

4.1.1 Glucose-mediated targeting

Glucose-mediated targeting has emerged as an effective strategy to enhance the selectivity of naphthalimide-based agents. Calatrava-Pérez et al. [45] designed glycosidase-activated naphthalimide pro-probes which remain inactive until enzymatic cleavage by tumor-associated glycosidases, thereby enabling selective intracellular release while minimizing background uptake. Building on this approach, Bhattacharjee et al. [46] demonstrated that direct glucose conjugation of naphthalimide facilitated preferential accumulation in hepatocellular carcinoma, with mitochondrial localization leading to ROS-mediated apoptosis and inhibition of metastasis, consistent with GLUT-mediated uptake. In a similar work, Li et al. [47] reported that glucose-decorated glycopolymer nanoparticles incorporating a naphthalimide fluorophore achieved dual tumor targeting and sensitive imaging in hepatocellular carcinoma models, highlighting the utility of glycolytic avidity for delivery. Within the broader medicinal chemistry framework, Banerjee et al. [48] underscored that glycan decoration represents an important targeting handle for improving solubility, selectivity, and multifunctionality of naphthalimide scaffolds while retaining their DNA-intercalating pharmacophore.

The potential of naphthalimide in cancer diagnostics is further buttressed in a study which developed functional glycopolymer nanoparticles with 1,8-naphthalimide. These motifs showed high affinity to tumor cells with significantly faster staining rates compared to normal cells [49]. The dual tumor-targeting effects arise from dense glucose parts and boronic ester modifications that exploit the enhanced glucose uptake characteristic of cancer cells (Warburg effect) [50].

4.1.2 Metabolic targeting

Cancer cells rely heavily on metabolic fuels like glucose and amino acids for growth and survival. Unlike normal cells, cancer cells often exhibit altered metabolism such as increased glucose uptake, enhanced polyamine synthesis, and abnormal mitochondrial function to meet their elevated energy and biosynthesis demands. Targeting these metabolic differences allows for more precise treatment strategies aimed at suppressing tumor growth while minimizing harm to normal tissues.

Significant successes in metabolic targeting include the development of drugs that inhibit key enzymes in cancer metabolism, such as glutaminase inhibitors and agents targeting glycolysis or polyamine biosynthesis. These therapies have demonstrated potential in reducing tumor proliferation and improving patient outcomes in various cancers. However, due to metabolic heterogeneity and adaptability exhibited by tumours, metabolic targeting faces challenges. Currently, researchers are focused on identifying more specific metabolic targets and developing combination therapies to overcome resistance and improve the efficacy of metabolic targeting in cancer treatment.

Naphthalimide, their derivatives and analogue are recently developed to target metabolism in cancer. This section of the paper provides a summary of Naphthalimide in metabolic targeting of cancers, successes achieved and challenges.

4.2 Enzyme-responsive cancer detection

Several naphthalimide probes have been developed for cancer-specific enzyme detection. Naphthalimide derivatives are used as a key chemical framework for enzyme-activatable optical probes due to their excellent fluorescent properties [51]. As a well-established dye and fluorophore, 1,8-Naphthalimide is extensively utilized in analytical chemistry, materials science, and biochemistry due to its outstanding photostability, versatile structural adaptability, high fluorescence quantum yield, and significant stokes shift [21]. These derivatives function as fluorophore scaffolds that can be chemically tailored with enzyme-responsive groups, enabling fluorescence activation upon enzymatic interaction. Naphthalimide-based probes demonstrate high sensitivity and specificity, supporting real-time, high-contrast bioimaging of enzyme activity in tumors and disease sites. Naphthalimide offers two approaches for analyte detection: the conventional fluorescence ‘switch-on’ response and the modulation of the ICT excited state to produce a ratiometric fluorescence response [36]. Specific applications of naphthalimide probes in enzyme-responsive cancer detection are further discussed below.

4.2.1 hNQO1-responsive probes

Human NAD(P)H: quinone oxidoreductase 1 (hNQO1) is significantly overexpressed in various cancer types. This tumor-specific activation has inspired development of bioactivatable quinone drugs (NQO1 bioactivatable drugs, NBDs) like β-lapachone, which are currently in clinical trials [52]. Naphthalimide probes containing triphenylphosphonium salt demonstrated enhanced enzyme efficiency and rapid detection capabilities, with significant fluorescence increase at 540 nm upon hNQO1 activity [53]. Detection limits as low as 0.5 µg/mL have been achieved for hNQO1 activity monitoring. NQO1-targeted optical probes enable high-contrast tumor imaging and have potential for guided surgery and theranostic applications [52]. A nitronaphthalimide-based fluorescent probe was developed by Kumari et al. [54] which responds to nitro-reductase overexpressed in hypoxic tumors, demonstrating strong fluorescence signals and distinguishing hypoxic cancer cells in vitro with minimal toxicity. While Khan et al. [52] discussed how NQO1 mediates redox cycling of quinones to generate ROS and H₂O₂, thereby inducing selective cytotoxicity in cancer cells, Cao et al. [55] developed a novel NQO1-activated near-infrared theranostic probe (I-HCy-Q) which electively accumulates in mitochondria, enabling targeted photodynamic therapy through localized ROS generation, which disrupts mitochondrial function and triggers apoptotic cell death. In another study, Meng et al. [56] developed a ratiometric fluorescent probe which can detect MAO-A (monoamine oxidase A) in cells and zebrafish (Fig. 6). The probe exhibits a high affinity for MAO-A and the limit of detection is 1.1 ng/mL. When the MAO-A concentration is in the range of 0.5–3.0.5.0 ug/mL, the concentration of MAO-A and the ratio of the probe fluorescence intensity of the 550 to 452 nm chromophore has a good linear relationship, indicating that the probe can be used to qualitatively and quantitatively detect MAO-A in cells and in zebrafish.

Fig. 6

Synthesis of probes (3 & 4) and their reaction mechanism (R = CH₃ and H for two different probes). Reproduced with permission from [56] Copyright (2024) by Elsevier

4.2.2 Caspase-3 detection

The development of ratiometric fluorescent probes targeting caspase-3 is a major step forward for the monitoring of apoptosis and drug delivery in cancer research. One of the leading examples is the Ac-DEVD-PABC-Naph which was shown to have an excellent use in visualizing programmed cell death in cancer cells [57].

The design of the probe is based on a modular approach in which a 1,8-naphthalimide fluorophore is attached by a self-cleavable p-aminobenzyl alcohol (PABC) spacer to an N-acetylated DEVD peptide sequence. The tetrapeptide DEVD (Asp-Glu-Val-Asp) is a recognition motif for caspase-3 for the cleavage of the probe in apoptosis. The PABC linker assembles again spontaneously after cleavage, releasing the naphthalimide fluorophore to provide a unique ratiometric fluorescence response (Fig. 7) [57].

This design principle has also been successfully extended to other fluorophores including cyanines and cresyl violet which demonstrate the robustness and flexibility of DEVD-based probes for monitoring apoptosis [58,59,60].

Fig. 7

Illustration of the Ac-DEVD-PABC-Naph peptide probe (5) and its activation pathway for ratiometric detection of Caspase-3 (purple indicates the linker, blue the fluorophore) Permitted to use as an article under a Creative Commons license [57] Copyright (2024) by Frontiers

The ratiometric signal is essential for precise quantification, because the two forms of the probe (the “caged” form - fluorophore masked by the peptide-PABC group, and the “uncaged” form - fluorophore released after enzymatic cleavage) have different absorption and emission spectra. This twofold response provides an internal reference so that there is a minimization of artifacts of fluctuations in the probe concentration or fluctuations in the cellular environment. As a result, the measurement of enzyme activity and drug response are independent of the probe concentration, leading to more stable results and making these systems especially interesting as an approach for therapeutic monitoring [23, 24, 58].

In addition to probes for hNQO1 and caspase-3, naphthalimide-based fluorescent sensors have great potential for detecting other important cancer biomarkers. These biomarkers are involved in tumor progression, metastasis and epigenetic changes. One of the biggest gaps in current research is the use of naphthalimide-based probes for detecting matrix metalloproteinases (MMPs) and histone deacetylases (HDACs), both of which are often dysregulated in cancer.

4.2.3 Matrix metalloproteinases (MMPs) as tumor invasion biomarkers

MMPs are enzymes that break down the extracellular matrix (ECM), a key process that helps cancer cells invade surrounding tissue and spread to other parts of the body. MMP-2, MMP-7 and MMP-9 are frequently overexpressed in aggressive cancers, and their levels are often linked to poor prognosis and advanced tumor stage [61, 62]. These enzymes are critical for tumor invasion and angiogenesis, making them important targets for early detection. Currently, many probes designed to detect MMPs rely on other types of fluorescent scaffolds, such as quantum dots or bioluminescent proteins. However, naphthalimide-based probes present distinct advantages, including bright fluorescence and large Stokes shifts, which enhance signal clarity and reduce background interference. Despite these advantages, there is no verified, peer-reviewed study that demonstrates a naphthalimide-based probe for MMP detection, marking this as an exciting area for further research.

4.2.4 Histone deacetylases (HDACs) as epigenetic targets in cancer

HDACs are enzymes that modify histones and non-histone proteins by removing acetyl groups, regulating gene expression and cell function. Abnormal HDAC activity is commonly observed in many cancers, contributing to tumor progression and resistance to apoptosis [63]. Although HDAC inhibitors are used in cancer treatment, the development of selective fluorescent probes for HDAC activity has been slower.

Recent work has shown that naphthalimide conjugates with HDAC inhibitors, such as PCI-34,051, can be used to selectively target HDAC8 in cancer cells, showing high photostability and significant fluorescence shifts for imaging [64]. This suggests that naphthalimide-based probes could be used for monitoring HDAC activity, but more research is needed to realize their full potential in clinical diagnostics.

Although naphthalimide-based probes have found extensive use in detecting other biomarkers, their application in MMP and HDAC activity detection remains largely unexplored. This presents a significant opportunity for future research to develop activity-responsive naphthalimide probes that could aid in monitoring tumor progression and personalized treatment. Given their brightness, modular design and photostability, naphthalimide probes are ideally suited to play a pivotal role in real-time cancer diagnostics.

4.3 Quantitative performance metrics

A major problem of the use of naphthalimide-based probes as bioanalytical reagents has been their quantitative performance. Their reliability in biological systems ultimately depends on crucial performance parameters including detection limits, dynamic range and ratiometric signaling capacity, since accuracy and reproducibility is paramount.

4.3.1 Detection limits and sensitivity

In the mammalian system hydrogen sulfide (H₂S) has become the significant signaling molecule, and the levels of H₂S circulating in the bloodstream and in plasma are usually stated in the micromolar scale. Experiments show that safe dosages range between approximately 4.2 and 62.7 µM, with other evidence of normal levels up to 70–125 µM by the clinical methods used in healthy individuals [65, 66]. Any significant change of such levels above or below this physiological range can have severe consequences, including cardiovascular dysfunction, neurodegenerative changes and inflammatory diseases. The operation of naphthalimide-based probes is outstanding in this regard. The NAP-Py-N₃ probe, described by Slowinski et al. [67], reached a detection limit of 15.5 nM, highlighting its high sensitivity. This is several orders of magnitude less than normal physiological concentrations, demonstrating how such probes can uncover subtle physiological variations of H₂S signaling before the disease sets in. Collectively, these developments highlight the potential of intelligent molecular design to enable probes that are highly sensitive and highly versatile to enable biomedical applications such as controlled laboratory studies, diagnostic and environmental monitoring.

With advances in the detection of H₂S, probes based on naphthalimides have also been found useful for monitoring biothiols. The interesting features of hydroxy-1,8-naphthalimide systems in terms of high photophysical behavior and versatility have been described by Zhu et al. [20] and more recent reports have exhibited experimental demonstrations of such potential. Dvorak et al. [68], by showing that even subtle changes in the molecular structure of hydroxy-1,8-naphthalimides can change their sensitivity and selectivity towards H₂S, underlined the importance of careful molecular design. In their study, Aydin et al. [69] introduced a naphthalimide probe (NASP), which showed a distinction between cysteine, glutathione and homocysteine in living cells with a detection limit of 9.06 nM. In combination with the structural information provided by Dvorak et al. [68], the results help to illustrate how naphthalimide scaffolds can be modified to yield high sensitivity and selectivity, properties which are important to the investigation of redox processes in biological systems.

pH-responsive naphthalimide probes also show a similar quantitative reliability with many often showing linear responses over narrow but biologically important ranges (pH 6.0–8.0). This allows the precise mapping of intracellular compartments, i.e. lysosomes, and mitochondria where slight changes of pH have diagnostic significance [21].

Two-photon fluorescent probes based on 4-hydroxy-1,8-naphthalimide scaffolds have been studied for their potential to also combine high sensitivity and deep-tissue imaging capabilities. Zhou et al. [70] created a probe (Np-Sn) against hydrogen polysulfides that exhibited a fluorescence enhancement of 80-fold, a detection limit of 33 nM, and effective two-photon imaging in living cells. In a subsequent study, the NP-H₂S probe was developed by Ou et al. [71] with a detection limit of 18.8 nM and good linear response up to 10 µM. The system was also demonstrated in cultured cells as well as in inflamed mouse tissue, demonstrating its usefulness for practical biological imaging. Similar study by Zhang et al. [19] investigated in this regard with design of FM1-FM3, in which seven membered heterocycle was added to the heterocyclic core of naphthalimide. The modification resulted in a much stronger performance for the new probes than the parent compound FM0. FM0 showed good quantum yield in ethanol, but its fluorescence was almost lost in water because of aggregation. By contrast, FM1-FM3 remained fluorescent in both solvents and were more than 15 times brighter. The authors linked this to greater rigidity, which reduced nonradiative losses, and a nonplanar structure, which limited aggregation. These probes also proved stable to light exposure, insensitive to pH change and effective with high two photon excitation, traits that make them well-suited to biological imaging.

Yu et al. [5] discovered multi-stimuli responsive fluorophore, NBDNI, which is obtained by attaching a 1,8-naphthalimide derivative carrying a rotatable electron-donating N, N-dimethylaniline group at its 4-position. Such a molecular design provides NBDNI not only with the properties of the aggregate-induced emission (AIE) but also with the properties of twisted intramolecular charge transfer (TICT), which allows it to experience significant fluorescence variations in response to a range of external stimuli. It is worth noting that NBDNI is highly sensitive to solvent polarity and polymer matrices as well as being very sensitive to fluorescence response as the temperature is altered. The researchers proved the possibility of the fluorophore as a temperature-dependent fluorescence sensor by using a basic fluorescence thermometer. The experiment involved placing a chloroform solution containing NBDNI in a quartz tube with the top part being heated to 50 °C and the bottom of the tube kept at comparatively lower temperature, at 15 °C. Upon exposure to 365 nm UV-light, the solution was observed to undergo a visible transition between the weak orange fluorescence to the intense green fluorescence to stretching the length of the tube as the temperature changed (Fig. 8). Not only does this experiment prove NBDNI to be a viable technology in being used as a fluorescent temperature sensor, but also it points out to the possible applications that may arise in areas where high accuracy and strictness in temperature are essential. Combined with the earlier contributions above, these results illustrate the adaptability of naphthalimide scaffolds for applications where both sensitivity and biological relevance are required.

Fig. 8

Photograph showing the fluorescence of NBDNI (Probe 6) in CHCl₃ solution at different temperatures (15–50 °C) within a quartz tube (left). The schematic diagram illustrating the thermosensitive mechanism of the NBDNI molecule is shown on the right. Permitted to use as an article under a Creative Commons license [5] Copyright (2024) by MDPI

4.3.2 Response kinetics and time resolution

The temporal dynamics of the naphthalimide-based probes is a key element in determining their suitability for real-time biological monitoring. Most systems have fast response kinetics, generally with measurable change in fluorescence within 2 to 10 min after exposure to the analyte. Such response times are important for near real-time visualization as well as the tracking of dynamic cellular processes [19].

There are certain recognition mechanisms that allow even faster responses. Probes based on azide reduction or 2,4-dinitrobenzenesulfonyl (DNBS) cleavage tend to produce detectable signals in 30 s to 2 min and are thus valuable for time-resolved fluorescence imaging and for following short-lived biological events [72]. By contrast, probes using nucleophilic substitution usually take longer to respond, usually 5 to 10 min. For example, a 3,5-dinitropyridin-2-yl substituted naphthalimide took almost 30 min to obtain a full fluorescence response with biothiols. Although slower, such systems still work in equilibrium assays and some strong analytical performance can be achieved [73].

Ultimately, the selection of the kinetic profile, i.e. ultra-rapid or moderately paced, depends on the application for which it is intended. This adaptability enables probe design to be suited to the temporal requirement of particular biological or diagnostic situations. Most importantly, the kinetic rate constants have been instrumental in establishing a quantitative framework in order to assess the efficiency of probes. For instance, the NAP-Py-N₃ probe, which has a second-order rate constant (k₂) of 9.62 M^{− 1}s^{− 1}, allowing the half-times to be accurately determined with analyte concentrations of biologic interest [67].

4.3.3 Selectivity and specificity

One of the main advantages of properly designed naphthalimide probes is their high selectivity in spite of other biological species that may interfere.

A low level of cross-reactivity is always found in selectivity tests with a large range of common biomolecules. Indicatively, biothiols and their analogs, such as cysteine, homocysteine, and glutathione, do not generate substantial signal perturbation and most probes currently show efficiencies of discrimination with cross-reactivity less than 5% [73, 74]. Equally important, physiological metal ions like sodium, potassium, calcium, and magnesium, which are considered essential, tend to have insignificant interference and therefore a probe fidelity is preserved even with ionic biological conditions [75].

Reactive oxygen species (ROS) such as hydrogen peroxide and hypochlorite, also show low cross-reactivity in the majority of systems, and they retain the stability of probes in the presence of oxidative stress [76]. Moreover, small molecules like glucose, amino acids and other cellular metabolites are generally non-interfering, further demonstrating the high chemical specificity of naphthalimide-based probes [77].

These results support the view that naphthalimide-based probes have an unprecedented level of specificity, reliably identifying target analytes against a diverse background of structurally related or highly abundant biological species.

5 Photodynamic therapy applications

5.1 Photosensitizer development

5.1.1 Evolution beyond traditional porphyrins

Historically, photosensitizers based on porphyrins and chlorine have been used in photodynamic therapy (PDT), and these small molecule photosensitizers effectively cause adverse effects, including long-term cutaneous photosensitivity, insufficient water solubility and reduced tunability of photophysical properties. More recently, naphthalimide analogs have become the new competitors based on their structural flexibility, deep absorption, and the ability to tune the photochemical reactivity [79].

In contrast to traditional heavy-atom-containing photosensitizers, it is possible to rationally design naphthalimides to increase intersystem crossing (ISC) and formation of reactive oxygen species (ROS) without the inclusion of elements linked to long-term toxicity. This is a heavy-atom-free method to overcome one of the greatest challenges impeding PDT agents in the past [79].

5.1.2 Structural engineering strategies

Naphthalimide PDT scaffold has been optimized by a variety of molecular engineering methods.

Thionation approach: Thio-naphthalimides are generated by substituting the oxygen atom in the carbonyl group of the imide with sulfur and this leads to enhanced ISC. The replacement favors population of triplet states via n-π* → π-π* crossing channels, enhancing the output of ROS without increasing the heavy-atom content of the design. Interestingly, lysosome-targeting Thio-Naphthalimides (LSNI-S) exhibit high specificity toward subcellular localization, and their photocytotoxicity is very high, and thus would have great potential as a targeted cancer therapy agent [78].

Donor-π-Acceptor (D-π-A) engineering: Naphthalimide frameworks can be engineered by attaching electron-donating and electron-withdrawing moieties to strategic positions to increase intramolecular charge transfer and triplet yield. This method enhances the ROS production and the phototoxicity indices. For example, DPA-NI-Bu derivatives exhibited phototoxicity indices above 66, a property linked to their irradiation-mediated interference with cellular redox regulation [80].

Orthogonal dyad systems: One strategy for overcoming the low performance of PDT in low-oxygen environments has been the design of orthogonal dyads of naphthalimides and cyanines. By imposing orthogonality between the donor and acceptor systems, the triplet lifetimes are favorably extended into the hundreds of microseconds range and Type-I photochemistry (leading to formation of superoxide radicals, rather than the more oxygen-consuming singlet oxygen) is induced. Yao et al. [81] reported dyad (T-BNCy5) which showed long triplet lifetime and high Type-I reactivity that leads to the wide treatment of tumors even in oxygen deficient conditions. These results show how structural design at the molecular level can aid in the resolution of one of the long-standing barriers to photodynamic therapy.

5.1.3 Two-photon activation systems

Another limitation that is critical to conventional PDT is that visible light has a low penetration depth in tissues. Naphthalimide derivatives have been of much interest as two-photon (TP) photosensitizers that allow excitation in the near-infrared (NIR) range and propagate deeper while minimizing collateral tissue damage. More recent computational and experimental work has discovered thiophene-conjugated naphthalimides with not only high two-photon absorption cross-Sects. (1,028 − 1,384 GM) but also remarkably high triplet lifetimes (1,337 − 11,609 µs) [80]. These properties make them very effective in deep-tissue TP-PDT applications. Notably, true spatial accuracy can be achieved in two-photon activation as well, where local excitation to the focal plane is localized and minimizes off-target phototoxicity. In addition, more comprehensive two-photon research shows that these NIR-based approaches do not only increase tissue penetration but also significantly reduce the scattering and improves the efficacy of treatment in deep-seated tumors [82].

5.2 Mechanism of PDT action

5.2.1 Fundamental photophysical processes

The photodynamic mechanism works on a canonical photophysical pathway, which commences with the selective localization process of a photosensitizer to tumor tissue, then it is excited upon irradiation with a suitable wavelength. Photosensitizer first fills a singlet excited state that combines with intersystem crossing (ISC) to a longer-lived triplet state. This trimeric state is the node of cytotoxic activity because it contributes to both the processes of electron transfer and energy transfer, which result in the formation of reactive oxygen species (ROS) [83].

5.2.2 Type-I and type-II pathways

PDT results are characterized by two important photochemical pathways. In the Type-II, the triplet photosensitizer donates its energy to ground-state molecular oxygen, (^3O₂) to generate singlet oxygen (^1O₂), a highly reactive species that induces lipid peroxidation and protein oxidation. This is the classical PDT pathway and prevails under normoxic conditions [84].

In comparison, the Type-I mechanism is that of electron or hydrogen transfer reaction between the excited triplet state and the direct neighbors, to yield radical species superoxide (O₂•⁻), hydroxyl radicals (•OH), and hydrogen peroxide. The mechanism is especially beneficial in hypoxic tumor micro-environment with limited oxygen production to restrict singlet oxygen generation. Naphthalimide-based dyads and donor-acceptor constructs have been made to specifically induce Type-I ROS generation to extend PDT activity into oxygen-depleted tumor niches [85].

5.2.3 Cellular death mechanisms

The produced ROS of PDT result in various types of controlled cell death. The classical effects are apoptosis, which is mediated by caspase activation, and necrosis at the higher dosages of photosensitizer. Notably, it has also been found that naphthalimide-based and other heavy-atom-free photosensitizers can induce ferroptosis, which is an iron-dependent cell death pathway with lipid peroxidation and glutathione peroxidase 4 (GPX4) inhibition. Ferroptotic signaling cascades have been directly associated with naphthalimide-cyanine dyads of orthogonal naphthalimides that are highly selective in Type-I superoxide formation [86]. Figure 9 shows the general photophysics and photochemistry of fluorophores [87].

Fig. 9

a Illustration of the main photophysical and photochemical pathways in fluorophores that contribute to photobleaching and phototoxic effects. S0: ground state; S1: first singlet excited state; T1: first triplet excited state; EX: excitation; F: fluorescence; IC: internal conversion; ISC: intersystem crossing; P: phosphorescence; TET: triplet energy transfer; ROS: reactive oxygen species. b Representative mechanisms by which photostabilizers deactivate the triplet state of fluorophores. PET: photoinduced electron transfer; gISC: intersystem crossing within geminate radical pairs; gBET: back electron transfer in geminate pairs; ESC: dissociation of geminate radical ion pairs from the solvent cage; Redox: reduction or oxidation. Reproduced with permission from [87] Copyright (2024) by Elsevier

In addition to direct tumor cell killing, indirect mechanisms which add to the therapeutic effect of PDT also occur. Vascular injury by ROS results in the collapse of endothelium and ischemia of tumor, and the subsequent release of damage-associated molecular patterns (DAMPs) stimulates antitumor immune responses. Moreover, the mitochondrial dysfunction caused by the local formation of ROS interferes with the energy metabolism of the cell and predisposes cancer cells to oxidative stress [88].

5.3 Advantages and clinical considerations

5.3.1 Superior properties of naphthalimide photosensitizers

Minimal invasiveness, spatial localization, and relatively low systemic toxicity compared to chemotherapy and radiation, are the current significant benefits of Photodynamic Therapy. Much more value may be attached to the naphthalimide derivatives because they are designed as heavy-atom-free photosensitizers, which increases the intersystem crossing (ISC) rate without the toxicity risk regarding platinum- or palladium-based agents over a long time [79, 83]. Their structural flexibility also enables controlling absorption wavelengths, fluorescence quantum yields and ROS production so that these molecules would constitute versatile scaffolds which can be tailored to particular therapy requirements [89]. The other benefit is that they are able to enter into tissues better. Two-photon-activated naphthalimides with near-infrared absorption properties have an advantage of deeper transmission through scattering and absorption, which helps overcome one of the classical challenges of PDT [79]. Lastly, their dual role as therapeutic and imaging agents makes them translational in real-time tracking of the responses to the therapy [89]. The superior properties of naphthalimide photosensitizers are depicted in Fig. 10.

Fig. 10

Superior properties of naphthalimide photosensitizers

Within the clinical purview, the naphthalimide-based PDT systems bear a number of advantages. The procedure is least invasive and has lower side effects as compared to traditional modalities. The other strength is its repeatability; local high-spatiotemporal control of irradiation can be repeated, and there is no accumulating toxicity [90]. PDT is also selective to the neoplastic tissue that appears because healthy structures are spared by preferential accumulation of photosensitizers and targeted activation of light. Also, PDT may be used together with other modalities, immunotherapy, or chemotherapy, which have synergistic effects. Lastly, ROS can attack various biomolecular targets, causing PDT to be less prone to drug resistance, which commonly inhibits chemotherapy [89].

5.3.2 Current limitations and solutions

Despite these benefits, PDT continues to have significant challenges. One of its major weaknesses is that it relies on oxygen, as hypoxic tumor microbiomes inhibit the generation of singlet oxygen (^1O₂), the predominant species of reactive oxygen in the conventional PDT. To address this, alternate practices are being considered. Mitochondria-targeted endoperoxides are one of the methods to release the singlet oxygen into the organelle that help to sustain the production of ROS even in the conditions of low oxygen levels [91]. Mitochondria-targeted PDT that encourages GSDME-mediated pyroptosis is another good prospect. Gasdermin E (GSDME) is a protein that is usually held inactive in a cell but is destroyed by caspase-3 and formed into a pore-forming fragment as part of this pathway. This fragment creates holes in the cell membrane, making the cancer cell swell and break apart releasing signals that trigger and activate the immune system. This cell-killing effect, which is combined with immune checkpoint therapy, through which it is possible to stimulate an antitumor response by blocking the effect of inhibitory proteins (PD-1 or CTLA-4) that normally suppress the activity of T-cells, leads to a more robust and durable antitumor response [92].

Penetration depth of light is another problem, because ROS have both short lifetimes (< 40 ns) and scales of diffusion (< 20 nm). The methodologies that have been developed are two-photon activation with increased penetration, bioluminescence resonance energy transfer (BRET)-based PDT, and light-independent endoperoxide methods [80]. Lastly, photosensitizer delivery is of critical concern to clinical translation. There is a proliferation of nanocarrier formulations, antibody conjugates, and targeted delivery systems to increase tumor tissue accumulation and retention at minimum off-target effects [93]. These measures are important milestones in providing therapeutic options that will bypass drug-kinetic obstacles and guarantee reproducible therapeutic outcomes.

6 Biosensing and molecular detection

6.1 Ion detection

Naphthalimide-based sensors have been extensively developed for detecting various metal ions of biological and environmental significance.

6.1.1 Mercury detection

Mercury (Hg²⁺) is a very toxic heavy metal of biological and environmental interest, and naphthalimide-based probes have shown extraordinary sensitivity in the detection of this chemical. A 2024 study described a straightforward naphthalimide fluorophore with a high fluorescence quenching efficiency of 560 nm and a limit of detection of 19.5nM, which can be practically realized so far in water samples, in living cells, and even in zebrafish imaging [94]. Other than single-ion systems, Yu et al. [95] also designed a naphthalimide probe that has multiple capabilities enabling the simultaneous detection of Ag⁺ and Hg²⁺. Their system provides practical use in analyzing toxic metals in a complex sample by forming a pair of emission signals which identifies the two ions.

6.1.2 Copper detection

Copper (Cu²⁺) has crucial physiological functions yet it is toxic in high concentrations. Naphthalimide probes have turn-on fluorescence responses in the case of Cu²⁺ binding. As an example, bifunctional naphthalimide sensors exhibit opposite responses to Hg²⁺ and Cu²⁺, where fluorescence change was detected with an increase with Hg²⁺ and a decrease with Cu²⁺, which discriminate ratiometrically between the two ions [96]. The changes in conformations caused by coordination, triggering the emission intensity and increasing up to 70 times in fluorescence at 520 nm, make these systems practically applicable to selective copper sensing.

6.1.3 Iron detection

Iron is a key molecule in the transportation of oxygen, enzymatic catalysis, and cellular redox, yet iron misregulation is strongly correlated with various disease conditions, including neurodegeneration and cancer. Recently, Probes of naphthalimide type have been modified into a highly selective Fe³⁺ detection in the biological systems. Li et al. [97] characterized a 1,8-naphthalimide-based dual-capability probe (MNP) with a low limit of detection of 65.2 nM that is able to not just detect Fe³⁺ with high-sensitivity but also localize to lysosomes, rendering it a viable live-cell imaging probe. It is a convenient platform to investigate iron localization in cellular environments because of this combination of analytical performance and organelle specificity. Sarkar et al. [98] through another design strategy developed fluorescent organic nanoparticles that had a naphthalimide core that presented aggregation-induced emission (AIE) properties. These Fe³⁺ selective nanoparticles had a detection limit of approximately 12.5 ± 1.2 mM and could be used as diagnostic tools to detect Fe²⁺/Fe³⁺ redox transitions.

6.1.4 Zinc detection

Zinc is a biologically important metal ion that plays many roles in the biological process, among which are enzyme control, protein synthesis and gene expression. Zn²⁺ dysregulation in the neurodegenerative disease has been linked in conditions like Alzheimer disease whereby abnormal levels of zinc influence receptors of neurotransmitters and the formation of amyloid plaques. Zinc imbalance is considered as one of the causes of the pathology of these diseases, and other neurological disorders [99]. Zn²⁺ sensing and detection in biological systems is thus vital in the determination of its role in the diseases.

Recent reports have shown the efficiency of the Zn²⁺ detection using naphthalimide-based fluorescent probes, due to the photophysical characteristics of naphthalimide scaffold, exhibiting bright fluorescence and a big Stokes shift. Liu et al. [100] showed a derivative of 4-amino-1,8-naphthalimide that was Zn²⁺ selective and sensitive. The addition of the Zn²⁺ aqueous solution in the physiological pH increased the probe fluorescence five-fold with the emission at about 550 nm, and it was effectively utilized to trace Zn²⁺ in living cells.

In another study, Ahmad et al. [101] were able to synthesize a 1,8-naphthalimide-dipicolylamine conjugate with turn-on fluorescence when binded to Zn²⁺. This probe had a detection limit of 80–120 nM, and a high specificity for Zn²⁺ compared to other metal ions, and was used effectively for in vitro and in vivo imaging. In addition, rationale design approaches, including the incorporation of detection groups reacting to Zn²⁺ coordination in an exclusive manner, have facilitated the development of Zn²⁺-sensitive probes, which amplify the fluorescence response [102]. All these studies support the idea that the naphthalimide-based probes could be an excellent platform in detecting Zn²⁺ in biological systems, and are non-invasive, highly selective, and sensitive in detecting Zn²⁺ concentrations especially in the context of neurodegenerative diseases and cellular homeostasis of Zn²⁺.

6.1.5 Detection of Peroxynitrite (ONOO^–)

The peroxynitrite (ONOO⁻) is a product of reaction between nitric oxide (NO) and superoxide anion (O₂⁻) producing an extremely reactive nitrogen species. Because ONOO⁻ is able to oxidize and nitrate amino acids, lipids and nucleic acids, excessive ONOO⁻ has been implicated in oxidative stress, inflammation and cell damage. A number of pathological conditions are linked to these processes, including neurodegeneration, hepatotoxicity, cardiovascular disease and liver injury [103].

Recent studies have demonstrated the 1,8 naphthalimide scaffold to be a potent system to design ONOO⁻ detecting fluorescent probes with high sensitivity and good biological compatibility. A study by Liu et al. [104] was one of the landmark studies describing a probe named HCA-OH, where a p-aminophenol trigger is attached directly to the naphthalimide core. Oxidative N-/O diarylation is observed to release the highly fluorescent naphthalimide derivative in the presence of ONOO⁻. The probe was almost non-fluorescent in phosphate buffer (pH 7.4) and ONOO⁻ resulted in a significant signal of fluorescence (emission at 548 nm, excitation at 460 nm). Significantly, ONOO⁻ was imaged using HCA-OH in living human liver (HepG2) cells and in nematode C. elegans. The probe had low cytotoxicity and suitable photostability, which implied that it can be used in real-time in vivo studies.

Building on this success, a more recent probe was developed by Wu et al. [105]. In their design, a naphthalimide fluorophore was linked to an isatin receptor; the reaction of the receptor with ONOO⁻ causes the probe to undergo a chemical reaction to turn on fluorescence (see Fig. 11). In order to enhance solubility and prevent aggregation-induced quenching, the probe (known as HN-I) was encapsulated in PEG (DSPE-PEG/HN-I). This probe was effective in the detection of externally added OONO⁻ and endogenously produced ONOO⁻ (e.g. in the macrophage cells under inflammatory stimulation), with high selectivity over other reactive species.

Fig. 11

Schematic for use of DSPE-PEG/HN-I (Probe 7) in the detection of peroxynitrite reaction mechanism of HN-I. Reproduced from [105] with permission from the Royal Society of Chemistry

Also, Wu et al. [106] reported a dual-channel fluorescent probe that can detect peroxynitrite and ATP at the same time. It is an innovative probe based on detection of ONOO⁻ with the help of naphthalimide and ATP with rhodamine, which allows to track oxidative stress and energy metabolism in live cells. The probe returned a turn-on signal for ONOO⁻, and at the same time it measured the levels of ATP by a red emission channel. This dual-sensor method was effectively used in hepatocytes which were subjected to drug-induced stress (acetaminophen) to analyse the ONOO⁻ production and ATP consumption.

Another remarkable development is the effort of Wu et al. [107] who created a dual-channel ratiometric probe named CD–N-I. The probe is based on carbon dots (CDs) that have been conjugated with naphthalimide-isatin to detect ONOO⁻. When exposed to ONOO⁻, cleavage of the isatin moiety takes place and FRET (Fluorescence Resonance Energy Transfer) occurs between the carbon dots and the naphthalimide to generate a fluorescence signal (see Fig. 12). CD–N-I was a highly selective and sensitive probe for the detection of ONOO⁻ even in the complex biological systems, including liver cells in the process of induction of acetaminophen-induced injury. The introduction of carbon dots enhanced solubility in water and reduced quenching that allowed more precise detection of ONOO⁻ at low concentrations.

Fig. 12

Schematic diagram of the mechanism of peroxynitrite detection using CD–N-I Probe 8). Reproduced from [107] with permission from the Royal Society of Chemistry

These naphthalimide-based probes provide an effective instrument to study ONOO⁻ dynamics on a real-time basis since peroxynitrite takes part in numerous cellular stress and disease mechanisms. Their level of sensitivity, selectivity and biocompatibility implies that they may be useful in monitoring oxidative stress, investigate the damage caused by toxins and drugs and investigate disease pathogenesis of nitrosative stress or inflammation.

All these developments are important in highlighting the sensitivity, adaptability and versatility of naphthalimide scaffolds to sense ions. They are important because of their ability to detect toxic metals such as mercury, iron, zinc as well as copper in live biological systems and environmental samples. At the same time, the scaffold structure allows establishing numerous formats, including small molecules with organelle-targeting characteristics to nanoparticle-based frameworks, enlarging their usage in ion sensing and their impact in chemical, biological and diagnostic domains.

6.2 Biological molecule sensing

The high structural flexibility of the naphthalimide scaffold is one of the most appealing features that allow its usage on a wide variety of biologically relevant targets. Naphthalimide functional groups could be programmed to detect proteins, enzymes, and gasotransmitters with high selectivity and sensitivity by optimally fine-tuning their photophysical properties, including intramolecular charge transfer (ICT), twisted intramolecular charge transfer (TICT) and photoinduced electron transfer (PET). These features make them superior to regular fluorophores, as a universal scaffold in clinical research, metabolic imaging and mechanistic research in chemical biology.

6.2.1 Protein detection

Proteins are a category of biomarkers used in diagnosing and monitoring of diseases, with the generation of selective fluorescence detection of these biomarkers continuing to be a persistent challenge. Human serum albumin (HSA), the most abundant plasma protein, is widely employed as a clinical indicator of renal function, hepatic integrity and nutritional condition. Naphthalimide-based HSA probes make use of the TICT mechanism, where binding to albumin’s hydrophobic cavity restricts intramolecular motion and leads to a strong increase in fluorescence intensity.

New research has brought improvements in this direction. In a 2024 study, Dai and associates [108] made a study targeted on generating a deep-red-coloured fluorescent probe constructed on a naphthalimide scaffold and demonstrated that the probe was capable of differentiating between human serum albumin (HSA) and bovine serum albumin (BSA). The probe showed a sensitivity of nanomolar limits and high fluorescence in complex biological conditions as it surmounted the long-standing limitation of signal overlapping with proteins surrounding the probe. In addition to analytical sensitivity, the probe also demonstrated a possible usage in bioimaging as it permitted real-time interaction of HSA in live cells. The selectivity, sensitivity and the ability to capture images make this probe a potential tool in diagnostics as well as biological research.

Similar advances have been achieved by Rani et al. [109], who developed and characterized a collection of triphenylthiourene-naphthol-imides conjugate and studied their reactions with HSA. They noted the typical HSA absorption band at 280 nm (Fig. 13a) which is the result of aromatic residues that appear at Trp214, Tyr411, and Phe403. This absorption increased by the addition of their most active conjugate, TPE-naph7 and a new band appeared at 400 nm (Fig. 13b). Fluorescence spectroscopy also revealed that the process of excitation at 280 nm gave an emission 340 nm (ascribed to tryptophan), which was seamlessly de-excited with increasing concentrations of TPE-naph7. This showed conformational alteration of Trp214, which was substantiated by time-resolved fluorescence experiments that differentiated between fixed and dynamics quenching pathways (Fig. 13c). The insights not only demonstrated how naphthalimide derivatives can be customized to recognize proteins, but also emphasize the potential offered by naphthalimide derivatives to study the structural dynamics of proteins at the molecular level.

Fig. 13

Effect of the incremental addition of analogue 7 (Probe 9) on (a) UV/Vis absorption and (b) fluorescence emission (c) Fluorescence decay profile of HAS of HAS in phosphate buffer (pH7.4). Permitted to use as an article under a Creative Commons license [109] Copyright (2024) by Wiley & Sons, Inc

All these developments highlight the clinical importance of the naphthalimide-based probes in the study of hypoalbuminemia in chronic kidney disease, cirrhosis, and malnutrition. On this basis, researchers have been able to expand their application to enzyme activityg monitoring, wherein analogous design schemes enable probes not only to sense catalytic processes, but to real-time image them inside biological systems.

6.2.2 Enzyme activity monitoring

Enzymes play a key role in the metabolism of the cell and their activity in most cases is a warning of disease development or progression. Enzyme-responsive naphthalimide probes have become a significant innovation because they can provide real-time measurements of enzymatic activity of living systems. xanthine oxidase (XO), an enzyme in purine metabolism, is one of the targets that have been well studied and has been associated with gout, hyperuricemia, cardiovascular diseases, and liver damage. In one study by Sidhu et al. [110], a turn-on naphthalimide probe of XO was described with a detection limit reaching 0.7 µM and also able to measure the level of uric acid in living cells. This probe was also specific as validated by inhibition studies, and thus it has value in not only mechanistic studies but also could be used in a diagnosis.

Structural flexibility of the naphthalimide scaffold has also enabled the generation of probes of other enzyme systems, such as caspases and b-galactosidase. Indicatively, Lozano-Torres et al. [111] proposed HeckGal, a naphthalimide-styrene probe with a β-galactose trigger. When the probe was cleaved in an enzymatic reaction, it generated a fluorescent turn-on effect, which makes it possible to visualize cellular senescence in vitro and in vivo.

6.2.3 Gasotransmitter detection

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S) are members of a group of gaseous signaling molecules called gasotransmitters, and each of those has extensive physiological effects. H₂S has received specific interest among them due to its duality as at normal concentrations, the compound is protective, but with dysregulation, it is likely to cause toxicity and disease progression.

Applications of naphthalimide scaffolds in H₂S sensing Naphthalimides have been extensively used in the method of making H₂S sensors, and the most common method is the azide-to-amine reduction reaction, where an azide group (or non-fluorescent group) is selectively reduced by H₂S to result in strong fluorescence emission. Such systems have a high degree of sensitivity and quickness of reaction. In an example, NAP-Py-N₃ probe as reported by Slowinski et al. [67] had a detection limit of only 15.5 nM, rapid response kinetics (k₂ = 9.62 M^{− 1}s^{− 1}), and greater than 50-fold fluorescence increase, making it suitable to use in real-time bioimaging.

The overall concept of selective H₂S detection through the enhancement of fluorescence was first established by Guo et al. [112] ten years ago, and since then the associated probes have been improved into the newer models with high signal density and better applicability in living cell and in vivo models. The recent research has developed the design of multifunctional naphthalimide probes, which have been used to combine sensitivity and biology. As an illustration, Shang et al. [113] created a red-emitting probe (XDS), which could work well in biosystems and real-world food samples, and Dvorak et al. [68] created structural analogues with increased selectivity and signal output in cellular systems. In the same way, Dai et al. [108] have described a long-wavelength probe that can detect H₂S and thiols simultaneously with great stability in physiological conditions.

In addition to simple turn-on probes, ratiometric designs have also been considered to provide spatial and mechanistic information. The hybrids such as cyanine-naphthalimide can be localized to the mitochondria and used to track the H₂S at the subcellular level in real-time [114]. Gasotransmitter probes targeted to organelles open the possibility to directly visualize the effects of H₂S on processes including redox balance, neurodegeneration, cardiovascular activity and tumor progression. Taken together, these advances show that naphthalimide fluorophores are further being developed into useful instruments of mechanistic biology, disease diagnostics, and therapeutic monitoring beyond being proof-of-concept probes.

6.3 Subcellular targeting and localization

One of the greatest strengths of naphthalimide-based probes is that they can be sufficiently modified to target organelles and, as a result, can be used to localize biochemical pathways in specific cellular compartments. Selective targeting to mitochondria, lysosomes or nuclei can be done using structural variations, including cationic tags, morpholine units and pH-sensitive substituents. This spatial manipulation in addition to improving the imaging fidelity, improves the diagnostic and therapeutic versatility of these fluorophores.

6.3.1 Mitochondrial targeting

Mitochondria are the seat of metabolism and apoptosis hence the main target of molecular probes. Modification of naphthalimide derivatives with triphenylphosphonium (TPP+) groups have especially been successful in reaching mitochondrial localization because the lipophilic cation has a strong electrostatic affinity to the mitochondrial membrane potential. Recent ones include probes like TPP-Naph-SS-Fc which consists of a naphthalimide fluorophore conjugated with a ferrocene quencher and a disulfide. However, when reacting with glutathione (GSH), a “turn-on” reaction is obtained with PET inhibition allowing to selectively image the mitochondria and even discriminate between normal and cancer cells [115]. Likewise, mitochondria-localized probes have been used to identify reactive oxygen species as well as hypochlorous acid (HClO), which could be used to gain information about oxidative stress at the organelle level [116].

6.3.2 Lysosomal targeting

Lysosomes maintain intracellular degradation, signaling and autophagy, and lysosomal pathology has emerged as an incumbent in cancer and neurodegeneration. These Naphthalimide scaffolds modified with morpholine groups show selective deposition in the lysosomes with acidic microenvironment protonation. An example is that Li and Qin [97] determined a dual-function naphthalimide probe (MNP) that can detect Fe³⁺ ions and track lysosomes in HeLa cells, with a nanomolar detection limit (65.2 nM), and an excellent ability to imaging selectivity. Previously, Jiang et al. [117] described Lyso-NA, a morpholine-functionalized probe that is a high-specificity and high-stability photostable probe, thus enabling it to be a powerful lysosomal dynamics imaging tool. More complex systems combine lysosomal targeting with near-infrared (NIR) emission to be used in vivo in order to detect lysosomal and mitochondrial glutathione simultaneously [116].

6.3.3 Nuclear targeting

Nuclear-targeted naphthalimide systems have not been studied extensively in comparison with lysosomal and mitochondrial probes, but some studies have shown that they have potential. Fueyo-Gonzalez et al. [37] reported the DNA-intercalating naphthalimide analogs that can be utilized to perform selective nuclear staining in macrophages, which are useful in nucleus-imaging due to low toxicity. In a similar development, Wang et al. [118] described naphthalimide compounds with acylhydrazine groups that, in addition to showing strong DNA binding, could be used to increase cytotoxicity making the compounds effective in nucleus-specific imaging and use as a therapeutic. Sarkar et al. [119] also provided insight into a nuclear-localized naphthalimide probe to detect carbon monoxide in live cells by showing how the scaffolds can be adapted to nuclear signal readouts. These reports suggest that though nuclear targeting is a relatively young field in naphthalimide research analysis in comparison with other organelles, the integration of DNA intercalation, nucleolus-specific localization, and versatile functional inducers could point to obvious prospects in the study of nuclear biology and its pathology-related changes.

7 Drug delivery systems: enhancing tumor accumulation of naphthalimides via targeted delivery

Incorporating naphthalimide dyes into nanoparticle-based delivery systems can significantly enhance their clinical potential in medical diagnostics and therapeutics. Naphthalimides can be enhanced in solubility, stability and biocompatibility by polymeric nanoparticles, liposomes and other nanocarriers. The targeting of tumors through these carriers is also possible through both active and passive delivery processes, which have unique benefits compared to standard methods of free drug delivery. Nanocarriers have demonstrated great potentials on better tumor accumulation and targeted delivery thereby minimizing side effects and improving therapeutic efficacy.

7.1 Nanoparticle formulations

Nanoparticles can also be used in the effective delivery of naphthalimide-based agents to enhance their therapeutic and diagnostic properties. These formulations have been created to enhance solubility, circulation time and specificity of their payloads to the tumors. Polymeric nanoparticles and liposomal systems are under investigation as a target therapy and diagnostics in cancer.

7.1.1 Polymeric nanoparticles

One of the most researched nanocarrier systems in the delivery of drugs and diagnostic agents is polymeric nanoparticles. Such nanoparticles have a number of benefits such as being biodegradable, tunable to size, and surface charge which can be optimized to achieve better tumor accumulation. Targeting ligands can be modified on polymeric nanoparticles with ease to produce selective delivery to tumour cells. Recent reports indicate that polymeric nanoparticles with encapsulated naphthalimides have an extended release and greater accumulation of tumors because of the increased permeability and retention (EPR) effect [120, 121]. Also, naphthalimide derivatives have been encapsulated in polymeric micelles and polymeric nanoparticles to be used in fluorescence imaging, with preferential uptake in cancer cells [122, 123]. Functionalization of the surface of such nanoparticles with targeting moieties also increases their capacity to reach specific tumor locations, thereby increasing selectivity and therapeutic efficacy [124].

Polymeric nanoparticles, including PLGA (poly(lactic-co-glycolic acid)) and PCL (polycaprolactone), have been utilized successfully to entrap different chemotherapeutic agents and the naphthalimides might be an added advantage to achieve both imaging and therapy [125]. This bifunctional capability, which integrates diagnosis and therapy onto a single platform, is a vital feature of theranostic nanocarriers.

7.1.2 Liposomal systems

Another highly promising platform for the delivery of naphthalimide-based agents is liposomes, which are lipid-based nanocarriers. They are the best vectors when delivering naphthalimide dyes because of their capacity to entrap hydrophobic drugs and their biocompatibility and stability in aqueous conditions. Liposomes may be modified, such that they will deliver their cargos as a response to a particular stimulus, like pH or temperature, as are often present in the tumor microenvironment [126].

Recent developments have demonstrated that liposomal systems that encapsulate fluorescent probes like the naphthalimides can be better utilized in tumor targeting of the probes, and that their distribution of the drug can be real time imaged. Hamad et al. [127] reported about the application of the liposome-based drug delivery system in cancer treatment, whereby the liposomes were functionalized with antibodies to enhance the tumor cell targeting ability. The specificity of naphthalimide-loaded liposomes could be increased similarly using similar strategies to be used in imaging and therapy of cancer cells. The benefit of liposomal formulations is that they can entrap both hydrophilic and hydrophobic drugs, and this has a wide therapeutic and diagnostic application [128]. Specifically, liposomes could be functionalized with surface ligands (e.g., folic acid, antibodies) to bind to tumor-associated receptors, which would make sure of a high precision in the delivery of the liposomes to the tumor site.

7.2 Delivery mechanisms

The effectiveness of nanoparticle-based drug delivery system is very much dependent on the type of targeting strategies that are used. Active and passive targeting are two key approaches that are typically employed to make tumors more specific and effective in therapy.

7.2.1 Active targeting

Active targeting is the functionalization of nanoparticles with targeting ligands, which specifically bind the tumor-associated receptors. This approach enables the selective uptake of the nanoparticle by the tumor and enhance the efficacy and selectivity of the nanoparticle-based drugs. Several studies have shown how antibodies, peptides and small molecules that have been conjugated to nanoparticles can be used as receptor-mediated endocytosis [120, 128].

As an example, folate receptor-targeted nanoparticles are commonly used to actively target cancer cells because folate receptors are over-expressed in various types of tumors [122]. In the same way, RGD (Arginine-Glycine-Aspartic acid) peptides, which bind to integrins that are overexpressed on the tumor vasculature, have been effectively embedded into polymeric nanoparticles to deliver anticancer agents directly to the tumor site [121].

More recent advances in tumor targeting include cell-penetrating peptides and nanobodies, which are also able to increase cellular uptake and concentration of the therapeutic or diagnostic payload at the site of localization. In the case of naphthalimide-based systems, the inclusion of targeting molecules would enable the receptor-specific imaging of fluorescence and increased accumulation of naphthalimides at tumor sites, which would provide superior diagnostics and more effective treatments [123, 126].

7.2.2 Passive targeting

Passive targeting is based on the effect of enhanced permeability and retention (EPR) that is typical of tumors. The leaky blood vessels in tumors frequently lead to the extravasation of nanoparticles from the bloodstream into the tumor interstitial space. This process finds application especially in enhancement of the uptake of naphthalimide-loaded nanoparticles by the tumors.

The size, surface properties and surface charge of the nanoparticle are some of the factors affecting the EPR effect. Particles size of 10–100 nm is most suitable for passive targeting because they are small enough to escape the blood circulation and yet large enough to remain within the tumor tissue due to the absence of lymphatic drainage [128]. These size range of nanoparticles have been demonstrated to accumulate in tumors more favorably, where their naphthalimide payloads can be released to produce enhanced fluorescence and improved therapeutic response [120].

It has been demonstrated that the EPR effect can be utilized by polymeric nanoparticles (e.g., PLGA) to accumulate in tumors and enhance fluorescence imaging [122]. Naphthalimides are able to be effectively delivered to tumors with high specificity and low toxicity to normal tissue by optimizing the properties of nanoparticles in terms of size, surface chemistry and drug loading efficiency.

These systems are promising but further research is required to perfect them and apply them in clinical situations, especially in vivo trials to determine the pharmacokinetics, biodistribution and toxicity of the naphthalimide-loaded nanocarriers. The types of nanocarriers for drug delivery systems in cancer therapy are shown in Fig. 14 [129].

Fig. 14

Nanocarriers utilized for drug delivery systems in cancer therapy. a Polymeric micelles (b) liposomes (c) carbon nanotubes and (d) dendrimers. Reproduced from [129], distributed under the terms of the Creative Commons License (CC BY-NC-ND 4.0)

7.3 Theranostic applications

Cancer nanomedicine has been transformed by the introduction of therapeutic and diagnostic capabilities into one nanoplatform. Naphthalimide-based nanocarriers are specifically suitable to theranostics due to inherent fluorescence, structural tuning, and photophysical and photochemical reactivity. These systems offer a combination of drug delivery, real time imaging, and therapeutic control, offering one all-in-one approach to precise oncology. The main benefit of naphthalimide theranostic systems is the fact that it enables real time tracking of the distribution and accumulation of drugs. As naphthalimide scaffolds have been shown to emit bright and stable fluorescence, drug carriers based on this motif may serve as self-reporting systems to visualize biodistribution and pharmacokinetics in vivo. The internal optical feedback removes the requirement of exogenous dyes or labels, lowers complex formulation and allows translation of therapeutic release of the payload to imaging outputs [130].

The theranostic capability of naphthalimide derivatives is further confirmed by the work of Seliga et al. [131]. They synthesized a novel series of naphthalimide polyamine conjugates and evaluated them for in vitro antiproliferative activity against human leukemia (Jurkat), human breast adenocarcinoma (MCF-7), human lung adenocarcinoma (A549) and human cervical adenocarcinoma (HeLa) cell lines. The inherent fluorescence properties of the tested naphthalimide derivatives were used to confirm their penetration to the tumor cells and subcellular distribution and it was discovered that Compound I1 (Probe 10) and A3 (Probe 11) exhibited highest antiproliferative activity with the IC₅₀ values of 5.67–11.02 µmolL^{− 1}. The fluorescence of these probes in the membranes, cytoplasm and nuclei after 2 h drug exposure is shown in Fig. 15.

Fig. 15

A549 cells incubated with I1 (a) and A3 (b) (both 30 µmolL^{− 1}) for 2 h. Reproduced with permission from [131] Copyright (2013) by Springer Nature

These platforms also support evaluation of treatment efficacy in addition to distribution monitoring. The change in fluorescence intensity and spectral characteristics can be directly used as the indicator of therapeutic activity, drug delivery, or cellular response. Both increased specificity and quantitative treatment monitoring can be achieved by using stimuli-responsive theranostic systems, in which naphthalimide fluorescence is switched on when the drug is released in the tumor microenvironment [129]. Such designs reduce background signals and enable a clinician to compare therapeutic responses in near real time.

Notably, naphthalimide photosensitizers have dual functions, and this feature has allowed their use in photoactivatable theranostics. In this case, naphthalimides can be used as imaging agents and Photodynamic therapy (PDT) photosensitizers, which produce cytotoxic reactive oxygen species (ROS) when irradiated with light. Recent multifunctional liposomal and polymeric systems that co-deliver chemotherapeutics and naphthalimide-based PDT agents have been shown to have synergistic efficacy, which also provides imaging guidance and combination therapy [115].

Finally, naphthalimide-based theranostic systems align with the vision of personalized medicine. Because fluorescence responses are susceptible to tumor-specific microenvironmental conditions (e.g., pH, redox condition, enzyme activity), it can give personal results regarding drug uptake, release, and treatment response. Such flexibility provides an opportunity to shape treatment courses individually per patient to minimize the chances of either under- or overtreatment. Continuous studies propose that the process can be further optimized by pairing naphthalimide probes with artificial intelligence (AI)-driven imaging analysis that allows the predictive and adaptive treatment of cancer [132]. These theranostic uses show that naphthalimide scaffolds can be used to convert conventional drug delivery vehicles into multimodal nanomedicine platforms to integrate diagnosis, therapeutic, and monitoring into one, patient-centered solution.

8 Recent advances and emerging frontiers

The development of naphthalimide-based probes has provided a new perspective in bioimaging, diagnostics and therapeutics. In addition to their well-known use in visible-light fluorescence, there is recent work to adapt them into near-infrared (NIR) (or more broadly, multiplexed) detections, using artificial intelligence (AI) to design probes and interpret results, and building multiplexed detectors that can monitor complex biological systems. The goal of these advances is to overcome the present limitations in translational achievement and place naphthalimide fluorophores at the core of the next generation in chemical biology and nanomedicine.

8.1 Near-Infrared naphthalimides

Conventional visible-range sensors are affected by short maximum tissue penetration, high background autofluorescence, and this limits their application in vivo. Recent attempts have worked around the extension of naphthalimide emission into NIR-I (700–950 nm) and NIR-II (1000–1700 nm).

NIR-I window: Naphthalimide derivatives with this wavelength have the benefit of reduced autofluorescence, and have deeper depths of penetration up to several millimetres, and thus are applicable to deep tissue imaging and intraoperative guidance [133].

NIR-II window: The emission in the NIR-II region provides better spatial resolution, less scattering of the photon, and better signal to noise ratios, which enable imaging of a whole body of a small animal and accurate tumor localization [134].

Two-photon excitation (TPE): TPE controls photodamage and photobleaching and enhances spatial resolution of 3D in thick tissues by allowing excitation with longer wavelength NIR light. The latest naphthalimide scaffolds (with thiophene bridges or donor-acceptor functional groups) have very high two-photon cross-sections (> 1000 GM) and long triplet lifetimes, and are promising candidates in two-photon photodynamic therapy (TP-PDT) [80]. The NIR-tuned naphthalimides, collectively, have a lot of potentials as imaging, theranostics, and real-time surgical navigation in vivo.

8.2 Artificial intelligence integration

Artificial intelligence (AI)-based systems combined with the development of fluorescent probes have enormous potential in improving the prediction and optimization of naphthalimide-based dyes to imaging and diagnostics of tumors. Machine learning (ML) models based on Artificial Intelligence have been successful in predicting the photophysical characteristics of fluorescent molecules, such as fluorescence emission wavelengths and quantum yields. These developments have the potential to speed up the identification of new naphthalimide analogs that have optimal imaging characteristics such that the extensive experimental work can be significantly reduced.

8.2.1 Fluorescent property prediction

AI and machine learning (ML) algorithms have achieved a lot of progress in the prediction of the photophysical characteristics of organic fluorescent materials. In their study, Ju et al. [135] trained ML models that predict emission wavelengths and quantum yields of different fluorescent compounds using their molecular structure. Training the model on large datasets of fluorescent molecules enables the algorithm to accurately predict which molecular modifications are likely to enhance the fluorescence of a given dye. This methodology is of great usefulness to naphthalimides, in which it is critical to predict the TICT (Twisted Intramolecular Charge Transfer) behavior to obtain the best imaging performance of these dyes. These paradigms may greatly facilitate the design of naphthalimide-based probes because they allow fast screening of candidates with the best photophysical characteristics prior to experimental synthesis.

Similarly, Souza et al. [136] showed the application of machine learning to forecast wavelengths of fluorescence emission and quantum yield of a vast library of organic dyes, including aggregation-induced emission (AIE) molecules, which have comparable photophysical properties with naphthalimides. This work has demonstrated that it is possible to use ML models to screen naphthalimide derivatives and predict their fluorescence behavior in different situations, thereby providing a rational basis for the design of fluorescent probes with superior characteristics to be used in imaging applications.

Moreover, modular AI systems have been introduced to support the design of fluorophores, such as naphthalimides. Zhu et al. [137] suggested an artificial intelligence model based on a combination of ML algorithms with the use of computational chemistry, through which fluorescent materials are designed by taking into account photophysical functionality, as well as chemical stability and biocompatibility. This modular strategy may be scaled to maximize naphthalimide analogs, so that they can be used in both imaging and treatment to meet the target requirements of in vivo imaging.

8.2.2 AI-assisted imaging and fluorescence analysis

AI-driven image analysis techniques are also playing a transformative role in enhancing the efficacy of naphthalimide-based imaging systems. After the incorporation of naphthalimides into nanoparticles as tumor-targeted drug delivery, AI algorithms, including deep learning (DL) models can be used to analyze the fluorescence imaging data in real-time. This is particularly important with cancer diagnostics where the microenvironment of the tumor tends to induce low-contrast and complicated images. AI has shown its ability to enhance signal to noise ratio in fluorescence images, and increase tumor detection and tumor margin delineation [138].

Wen et al. [128] studied AI-assisted fluorescence lifetime imaging (FLIM) to enhance the diagnostic accuracy through detecting dissimilar tumor cell populations that react in varying ways to therapy. Such an application of AI is of particular significance to naphthalimide-based probes, in which case a more accurate mapping and quantification of fluorescence signals of naphthalimide-loaded nanoparticles can be tracked and quantified in real-time. Complex fluorescence datasets are also analyzed using deep learning models to enhance image segmentation and feature extraction which is significantly faster.

Also, CNNs (convolutional neural networks) have been used effectively in imaging of cancer to automatically identify and classify tumor cells using fluorescent data. Research has demonstrated that these AI methods enhance the detection precision of tumor cells in case of fluorescent dye such as naphthalimides, which do not require manual analysis and also, make diagnoses processes faster [136].

8.2.3 Future directions: AI-driven synthesis and design of naphthalimides

In the future, AI implementation in chemical synthesis of naphthalimides can make the process even more streamlined. Indicatively, Large language models (LLMs) and synthesis platforms based on AI are being employed to predict the best synthesis pathways of new compounds. The end-to-end AI platform suggested by Ruan et al. [139] automates the design and synthesis of chemical compounds and optimises the conditions of experiments and predicts the yield and purity of synthetic reaction outcomes. These platforms can be modified to produce naphthalimide based compounds, saving time and money in the drug development pipeline.

Moreover, the optimization of continuous-flow synthesis of naphthalimides, including the ones presented by Oshchepkov et al. [140], is also optimized using AI models to improve the efficiency of production and guarantee the scalability of naphthalimide-loaded nanoparticles synthesis on a larger scale with the use of AI. Such AI-assisted solutions will facilitate the creation of new fluorescent probes in large amounts in a short time, so that the naphthalimide-based imaging systems could be effectively manufactured in research and clinical trials.

8.3 Multiplexed detection systems

Biological systems are complex in nature as multiple analytes interact dynamically and as a result, emerging multiplexed naphthalimide probes are being developed to address this complexity by allowing detection of multiple targets at the same time during a single experiment. Structural engineering enables the naphthalimide derivatives to be optimized to emit and be monitored simultaneously at different spectral channels (blue to near-infrared) to measure multiple biomarkers, including naphthalimide at the same time (ions, proteins and small molecules) [70]. Coupled with this, ratiometric probe designs adopt a dual-emission approach that offers internal calibration, which can correct differences in probe concentrations or environmental changes and thus providing more quantitative and reproducible measurements [112]. Moreover, sensor arrays comprising naphthalimide analogs with different selectivities can also be engineered to give characteristic responses in a fingerprint, which when processed through pattern-recognition algorithms, allows distinction of complex biological mixtures, be it disease-specific serum biomarkers or volatile metabolites on breath-analysis [2, 36]. These multiplexed platforms together have a tremendous potential in systems biology, high-end diagnostics and personalized medicine, where comprehensive molecular profiling is becoming increasingly important.

9 Challenges and limitations

Although naphthalimide-based probes have shown a tremendous potential in the diagnostics, imaging and theranostics fields, certain obstacles in translating to clinical use exist. These are issues of biocompatibility, drug delivery, pharmacokinetics, biodistribution, technical obstacles to optical functionality and regulatory issues that need to be resolved before it can be widely adopted in medicine.

9.1 Biocompatibility concerns

9.1.1 Cytotoxicity

In spite of the fact that naphthalimide scaffolds can be regarded as rather safe at low micromolar concentrations, dose-dependent cytotoxicity has been observed in some of the derivatives, whose possession of heavy-atom substitutions (e.g., bromine, iodine) or strong electron-withdrawing groups favor intersystem crossing and the formation of reactive oxygen species (ROS) [7, 84, 141]. Although this phototoxicity is beneficial in photodynamic therapy (PDT), it poses a problem to diagnostic imaging or long-term monitoring in which minimal alteration of cellular activity is necessary. The interplay between bright and stable emission and low inherent cytotoxicity is a delicate issue and this highlights the value of systematic structure-activity relationship (SAR) studies. These endeavours are progressively emphasising strategies formed by heavy atoms-liberated schemes such as donor-π-acceptor engineering and thionation to accomplish high fluorescence quantum yields and effective generation of ROS without compromising security. These optimizations are especially significant when the probe is intended to be used in chronic imaging, in real-time or in repeated therapeutic cycles.

9.1.2 Immunogenicity

In addition to cytotoxicity, the immunogenicity has also not been fully explored and this is a significant clinical translation obstacle to naphthalimide-based nanocarriers. Polymeric nanoparticles, dendrimers and liposomes have been demonstrated to cause undesired innate and adaptive immune responses, which include: complement activation, cytokine release and rapid elimination through the reticuloendothelial system (RES) [142]. These not only compromise safety, but also limit circulation half-life, degrading the therapeutic index of the nanocarrier. Strategies to remove these risks, comprehensive preclinical immunotoxicity studies together with rational design are needed. PEGylation, zwitterionic surface modification and biomimetic coating (e.g. cell membrane cloaking) approaches have demonstrated a potential to increase systemic circulation and reduce immune activation [143,144,145]. Critically, individual strategies that take into consideration the immunological differences of the patients might be required to guarantee the adoption of uniform clinical outcomes.

9.2 Pharmacokinetic properties

The process of clinical translation of naphthalimide-based theranostic systems and probes relies on the concepts of pharmacokinetic optimization. The pharmacokinetic profiles, especially biodistribution, clearance, and stability) are one of the major obstacles. One of the problems is the fact that such fluorophores as many other small aromatic dyes and nanoparticle systems are likely to be deposited in the liver and kidneys via hepatobiliary and renal clearance. This sequestration is not only challenging to interpret an image due to the high background signal but poses safety issues, as repeated sequestrations may increase the possibility of organ-specific toxicity [146,147,148]. To address this, methods have been explored to minimize reticuloendothelial uptake, including surface PEGylation, zwitterionic coatings and biomimetic cloaking (e.g., red blood cell membranes) to minimize these effects [143,144,145]. Clarification optimization is also important: although long circulation times may increase tumor concentration due to enhanced permeability and retention (EPR) and a receptor-mediated uptake mechanism, long retention leads to potentially serious toxicity and off-target retention. New approaches include the utilization of biodegradable linkers that can be degraded in vivo and renal-clearable fluorophores (≤ 5 nm hydrodynamic size) that can be excreted in the urine rapidly retaining their optical properties [149]. Furthermore, probe stability in vivo is also an issue, since enzyme degradation, plasma binding, and oxidative stress can extinguish fluorescence or alter biodistribution. In order to preserve the signal quality, researchers are using chemical stabilization methods such as addition of electron-donating substituents to inhibit oxidative quenching or incorporating naphthalimides inside the polymeric nanoplatforms to prevent early degradation. These and other nanocarrier-based systems have shown enhanced photostability and fluorescence stability leading to better longitudinal imaging reliability in complicated biological settings [150].

9.3 Regulatory and standardization issues

Despite the fact that a great deal of success has been attained in laboratories, clinical translations of naphthalimide-based probes remain at bottlenecks because of the complexity of regulations and lack of standard practices. In contrast to established fluorophores (e.g. indocyanine green), the naphthalimides are still in the preclinical and investigational phases, and there is minimal regulatory precedent to help direct their approval.

Lack of standardized protocols: The primary obstacle is the non-standardization of synthesis, physicochemical characterization and biological validation schemes. Existing literature is frequently varied in terms of the preparation of probes, the assessment of purity and testing conditions, thus impeding both comparability and reproducibility between labs. A variety of standardization efforts, including those coordinated by the European Nanomedicine Characterization Laboratory (EU-NCL) and ASTM International, offer models that may be used to guide the creation of naphthalimide probes [151].

Regulatory approval: Fluorescent probes undergo strictly designed toxicological and pharmacological testing such as testing of metabolism, bio distribution, clearance, genotoxicity, and long-term safety. In the case of naphthalimides, this is further difficult because they are not only diagnostic but also therapeutic agents (theranostics) which must be considered within a combination product regulatory framework. These processes are both resource intensive and time consuming, and can go beyond the capabilities of academic laboratories and require industrial collaborations to produce on a Good Manufacturing Practice (GMP) scale [152].

Quality control: Batches-to-batches reproducibility is one of the most enduring issues in commercialization of nanoparticles and probes. Differences in size, surface charge, fluorophore loading and stability can change pharmacokinetics and imaging performance substantially. Regulatory authorities focus on Good Manufacturing Practice (GMP), in which process analytical technologies (PATs) and quality-by-design (QbD) strategies help maintain the quality of the product [153]. In the case of naphthalimide-based formulations this entails incorporation of strong manufacturing controls, tested stability tests, and performance metrics into the pipeline of development.

Collectively, getting over these challenges will necessitate multidisciplinary efforts of chemists, biologists, clinicians and regulatory scientists as well as the international harmonization of protocols to achieve reproducibility and pace up clinical translation.

10 Future perspectives and clinical translation

The bench-to-bedside route in regards to the naphthalimide-based probes and nanocarriers is not only promising but needs a systematic approach to overcome translational obstacles. These fluorophores have the potential to transform clinical diagnostics and therapeutics in the future by incorporating preclinical rigor, regulatory compliance, approaches to customized medicine and technological innovation.

10.1 Clinical translation pathways

10.1.1 Preclinical development

To achieve successful movement of naphthalimide-based systems beyond laboratory work into clinical practice a stringent preclinical development pipeline is essential. The initial move is an extensive toxicological profiling, which is an acute, sub-chronic and chronic exposure. These investigations provide safety margins and possible organ-specific toxicities, with hepatotoxicity and nephrotoxicity frequently being of the concern of small aromatic fluorophores and nanoparticle formulations [146].

Furthermore, pharmacodynamic (PK/PD) and pharmacokinetic analyses are essential in the interpretation of absorption, distribution, metabolism, excretion, and toxicity (ADMET) variables. This characterization assures the optimization of dosing schedules and offers predictor knowledge on human pharmacology [154]. The investigation on biodistribution is of special interests since, in most cases, naphthalimides tend to accumulate non-selectively within the clearance organs. This finding prompts a need to change the design to enhance the systemic performance of PEGylation or utilization of biomimetic cloak.

Lastly, efficacy validation has to be done in clinically relevant preclinical models. Compared to the traditional subcutaneous xenograft, the more relevant orthotopic and patient-derived xenograft (PDX) models can better mimic the tumor microenvironment and thus have greater predictive capabilities in human response. Such designs will be necessary to investigate the diagnostic and therapeutic (theranostic) potential of naphthalimide probes, particularly the imaging-guided application and photodynamic therapy [155].

10.1.2 Regulatory considerations

Regulatory alignment is also of utmost importance to biological validation to enable promising findings in the laboratory to advance to clinical adoption. The processes of manufacturing are expected to be based on the Good Manufacturing Practice (GMP) standards, ensuring the batch-to-batch reproducibility, purity, stability, and scalability. The GMP-compliance is especially problematic in terms of multifunctional nanocarrier systems, with minor changes in particle and surface chemistry or drug content being able to radically impair pharmacokinetics and efficacy [156, 157].

Naphthalimide-based systems deserve a clinical trial design adhering to the gradual evolution of systems characteristic of therapeutics. Phase I trials are aimed at safety, tolerability and dose escalation and Phases II and III look into efficacy, pharmacodynamics and long-term effects [158]. There are also special concerns with theranostic probes, whose borderline cases between drugs and diagnostic agents remain unclear. In the case of such products, regulatory bodies such as the Food and Drug administration (FDA) and the European Medicines Agency (EMA) approach them through frameworks of combination products, frequently necessitating further safety and efficacy procedures, outside of the traditional trial template [159].

Furthermore, dialogue with regulatory bodies at an early stage and throughout the development period is of significant importance to define the ways of obtaining approval, foresee the possible obstacles and position developmental plans in accordance to regulatory demands. A proposed schematic flowchart showing the translation pipeline is depicted in Fig. 16. The current trends in regulation of nanomedicine focus on standardisation of preclinical testing, the implementation of harmonised protocols and real-world evidence (RWE) integration to cut regulatory timelines. In the case of naphthalimide probes, this regulatory interaction will be the key to navigating the complicated cost-effectiveness of its dual-diagnostic-therapeutic profile.

Fig. 16

Schematic flowchart figure showing the translation pipeline (Preclinical → GMP Manufacturing → Regulatory Approval → Clinical Trials → Clinical Adoption)

10.2 Emerging applications

10.2.1 Precision medicine

The systems based on naphthalimides are highly relevant to the objectives of precision medicine, because they enable the customization of diagnostic and treatment strategies to the requirements of each specific patient. Their modular architecture allows the biomarker specific probes to be optimized to allow the sensitive detection of disease-relevant signatures. An illustrative case is the use of fluorescent probes that are designed to bind biomolecules that are specific to cancer, which can be used to help stratify the patients and identify those who may benefit most from the targeted therapies [160]. In addition to detection, the other significant benefit of naphthalimide systems is the ability to monitor treatment. They have a bright and tunable fluorescence that enables the real-time visualization of therapeutic responses that can enable clinicians to modify treatment strategies during the process. In oncology especially, this may come in handy because the response of tumors is not always homogenous, hence making it hard to decide clinically.

The importance of naphthalimides in the detection of resistance is also of interest. These systems can detect early biochemical indicators of drug resistance by taking advantage of the properties of activatable probe designs whereby fluorescence is emitted only when a specific enzyme is encountered or an oxidative environment is present. This enables prevention of clinical relapse and lowering rates of treatment failure and fits the increasing trend towards customised therapy informed by dynamic biomolecular feedback [161].

10.2.2 Combination therapies

The use of combination therapies is becoming a more common option to improve the effectiveness of treatment and minimize the shortcomings of a single-modality treatment. In a study by Jin et al. [162], a multifunctional nanohybrid was formed where a naphthalimidecyanine conjugate was placed in a ROS responsive carrier, which enabled PDT, PTT, and chemotherapy to work synergistically. The platform was found to be more efficient in tumor ablation than mono-therapy strategies thereby showing how a combination of multi-pronged therapeutic strategies can improve on tumor hypoxia and resistance. Yao and colleagues [81] have taken this concept a step further where an orthogonal naphthalimide-cyanine dyad (T-BNCy5) increases the triplet life and yields Type-I reactive oxygen species, thus allowing PDT even in oxygen-hypoxic tissues. The system could eliminate tumor cells and provide a long-term anti-tumor immune response in ablation in combination with immune checkpoint therapy, which highlight the importance of naphthalimide scaffolds as immunotherapy candidates.

Other applications of the naphthalimide platforms include the use of naphthalimide in drug-delivery carriers in addition to phototherapy. Most recent reviews bring out the adaptability of the use of PLGA nanocarriers in cancer therapy, not only in providing a controlled release of chemotherapeutics, but also in tracking of diagnostic markers. Kesharwani et al. [163] mentioned the PLGA nanoparticles of PEGylated polymer as sophisticated delivery systems that prolong the circulation duration, enhance tumor concentration, and have multifunctional capabilities. By incorporating naphthalimide probes into these nanoplatforms, it would be possible to have the nanoprobes to be addressed fluorescence-guided drug delivery as well as even in delivering therapeutic functionality, which would offer a theranostic advantage. These demonstrations indicate the potential of naphthalimides to be modified into universal hybrid platforms - such as PDT/PTT/chemo nanohybrids, immunotherapy-linked Type-I dyads, and drug-delivery nanoplatforms - and make the case that naphthalimides will remain central to the next-generation combination therapies.

10.3 Technological innovations

10.3.1 Advanced imaging modalities

The use of naphthalimides in multimodal imaging systems is a fast-emerging direction in the field of biomedical diagnostics. Combination systems with fluorescence, magnetic resonance imaging (MRI) or positron emission tomography (PET) enable researchers and clinicians to take advantage of the high sensitivity of optical probes, as well as to simultaneously provide the deep penetration and anatomical localization of established modalities. As an example, naphthalimide-gadolinium hybrids were described as dual fluorescence/MRI probes, which can be used to image the structure and perform functional diagnostics with fluorescence guidance [134].

In the meantime, super-resolution microscopy innovations have increased the applications of naphthalimide fluorophores on a nanoscale level. These derivatives are now able to map the sub cellular structures like mitochondria and lysosomes with a newly found resolution and assist in the dissection of dynamic biological processes with near-molecular detail. Also, advances in the creation of NIR-II fluorophores (1000–1700 nm) have allowed visualization of the entire body. NIR-II naphthalimides have less scattering, a higher spatial resolution, and a deeper tissue penetration than visible and NIR-I systems, and would move the area of operation closer to clinical translation [164].

10.3.2 Smart probe design

The architecture of dynamic, programmable and context sensitive naphthalimide probes represents a paradigm shift in relation to traditional, non-programmable, imaging instruments. The activatable probes are optically silent and only become active on their exposure to tumor-specific microenvironmental signals, which include acidic pH, over-expressed enzymes, or hypoxia. This provides a low-level of background noise and increased specificity of disease detection. Activatable systems of this type have demonstrated great potential in imaging the activity of enzymes in tumors, allowing real-time evaluation of the disease progression [165]. Supramolecular chemistry is applied in self-assembling probes which form nanostructures on-site. Under the influence of biological environments, these probes are able to form nanoparticles or fibrils, which promotes retention at disease sites and fluorescence persistence [166].

Programmable probes are further improved by tuning their fluorescence intensity, emission wavelength or ROS-producing output to more than two biological cues. This not only makes it possible to multiplex the detection of complex disease signatures, but also opens the door to probes that do not only report but form real-time molecular decision-makers. These technologies demonstrate how naphthalimides can serve as next generation theranostic systems, able to target accurately, monitor diagnostics, and activate therapeutically in a single molecular scaffold.

10.4 Market potential and economic impact

The market of the global fluorescence probes has been growing consistently as a testament to the increased need in accurate diagnostic instruments as well as the overall use of fluorescence imaging in medicine and investigative procedures. According to recent projections, the market which is estimated at several billion dollars in 2024 is likely to keep increasing with a compound annual growth rate of approximately 10.5% till 2033 [167]. This has been accelerated by their use in medical diagnostics, drug discovery and environmental testing where more precise and quicker detection devices are in great demand. This growth is also reflective of innovations in probe chemistry as well as the creation of tunable and multifunctional scaffolds such as naphthalimides in line with the trend to precision medicine and real-time monitoring.

In healthcare economics terms, the capability of these systems to monitor treatment at real time and identify disease at its earliest stages may help avert massive expense through preventing costly later interventions. The burden of chronic care would also be decreased by increase in the treatment outcomes and decrease of emergency or rescue therapies. These commercial potentials are broad with targeted diagnostic imaging agents in oncology, neurology, photosensitizers in photodynamic therapy (PDT) and nanomedicine pipeline in imaging, targeted delivery and combination therapy. Nevertheless, neither the complete economic nor clinical potential of these systems can be fully fulfilled until issues like regulatory approval pathways, large-scale manufacturing reproducibility and standardized quality control have been considered (as explained in Sect. 9).

11 Conclusion

Naphthalimide fluorescent probes have showed great potential in biomedical applications such as cancer imaging, biosensing and photodynamic therapy. Their unique photophysical characteristics of controllable emission, high photostability and large Stokes shifts place them as excellent candidates for incorporation into diagnostic and therapeutic systems. Additional technological improvements in both nanoparticle preparations and artificial intelligence (AI) expand their potential with AI-based predictions about photophysical characteristics and its applications in fluorescence imaging analysis.

Nevertheless, the extent of this review is mostly preclinical trials rather than clinical trials and this limits our knowledge of the complete clinical translation of these probes. Besides, the review provides qualitative information but does not provide a quantitative comparison of the different naphthalimide formulations, and although AI has been investigated in enhancement of fluorescence prediction and image analysis, a number of the AI methods remain in the early stages of proof of concept, and thus they have to be validated. These points highlight the necessity to conduct further studies, especially in the clinical field and practical implementation of AI, to maximize the clinical capabilities of naphthalimide-based probes.

Finally, although it is clear that naphthalimides have the potential to be used in precision medicine, it will be necessary to overcome these remaining challenges by further research in order to maximize their ability to improve diagnostic and therapeutic outcomes.