Green-Synthesised Nanoconjugates: Advancing Targeted Photodynamic Therapy for Lung Cancer.

1
Introduction
Cancer is a complex disease characterised by mutations in the DNA genome that cause the unrestrained proliferation of abnormal cells in certain parts of the body [1]. According to GLOBOCAN, lung cancer ranks as the top cause of cancer morbidity and deaths around the world [2]. The characteristics of cancer include persistent proliferative signalling, avoidance of growth suppression, resistance to cell death, potential for replicative immortality, promotion of angiogenesis and initiation of invasion and metastasis [3]. In the case of lung cancer, these mutations often occur due to factors such as tobacco smoke, environmental carcinogens or genetic predisposition, leading to the uncontrolled growth of abnormal lung tissue [4]. Generally, lung cancer can be differentiated into two major subtypes, namely, small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC). The SCLC consist of malignant cells with neuroendocrine characteristics, making up 15% of lung cancer cases. Conversely, NSCLC, which accounts for 85% of the cases, can be further categorised into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [5].
The global prevalence of cancer continues to rise, both in terms of new cases and deaths. Overall, cancer has a higher global mortality rate than diseases like tuberculosis, malaria and AIDS [4]. The annual number of new cancer cases is expected to reach 35 million by 2050. The survival rate for lung cancer significantly varies based on the stage of diagnosis. Unfortunately, over 57% of lung cancer cases are diagnosed after metastasis has already occurred. The 5‐year survival rate for lung cancer in most countries is below 20% [6]. The most common interventions for treating lung cancer include surgery, radiotherapy and chemotherapy. However, these treatments have limited success in effectively improving patient outcomes due to factors such as adverse reactions, difficulty in targeting specific cancer cells, multi‐drug resistance development and recurrence over the years [7].
Lung cancer stem cells (LCSCs) are a subpopulation of lung cancer cells with self‐renewal, differentiation, and heightened tumour‐initiating properties. Research has identified this subpopulation of self‐renewing tumour cells in human lung cancer, which plays a key role in cancer progression and recurrence [8].
Photodynamic therapy (PDT) is an innovative cancer therapeutic model offering great benefits in basic and clinical research [9]. Since the late 1970s, PDT has progressed significantly as a non‐invasive therapy, evolving from treating early skin infections and epidermal tumours to more recent applications in solid tumour treatments [10]. In recent years, the U.S. Food and Drug Administration (FDA), Europe, the Middle East and Africa (EMEA) have granted approval for various PDT agents for different types of cancers [9, 11]. The advantages of PDT include its effectiveness in targeting and destroying cancer cells by increasing the generation of reactive oxygen species (ROS) when exposed to light at a specific wavelength. This has the added benefit of not causing tumour resistance. In addition, when PDT is combined with photosensitisers (PS), it can induce apoptosis through ROS generation in response to controlled light exposure, thereby reducing the toxicity of the PS in the body. PDT can also prevent small surgery‐related lesions, which improves prognosis and reduces the risk of recurrence. It can be administered multiple times at the same tumour site and can also be used in combination with other therapies, such as chemotherapy, radiotherapy, immunotherapy and gene therapy [9, 10].
Nanotechnology refers to the science and development of materials at a nanoscale of between 1 and 100 nm [12]. Over the years, scientists have endeavoured to use nanotechnology methods for cancer therapy and diagnosis. The use of nanotechnology has helped in overcoming some of the limitations of PDT for enhanced therapeutic effects [13]. One such material is the nanoparticles (NPs) used in PDT for site‐selective delivery due to their excellent biodistribution and bioavailability, high organic solubility, enhanced permeability and retention (EPR) effect, amphiphilicity, large surface‐to‐volume ratios, and reduced toxicity [14, 15]. There are many types of NPs used in PDT depending on their morphology, size, surface chemistry, light absorption and emission characteristics [16]. The types of NPs utilised in PDT can be categorised into biodegradable NPs (e.g., polyester and polyacrylamide‐based, liposomal, dendrimer‐based, natural macromolecule‐based NPs) and nonbiodegradable NPs (e.g., silica, gold, silver and magnetic NPs) [15]. In recent years, the development of green‐synthesised silver NPs (AgNPs) has intrigued researchers because of their synergistic effects in combating lung cancer and offering a sustainable, eco‐friendly therapy. The term ‘Green synthesis’ or ‘Green Chemistry’ refers to the harnessing of plant extracts, microorganisms or enzymes to act as reducing and stabilising or capping agents for the preparation of the NPs. The biosynthesised AgNPs in combination with PDT carry potent anticancer properties, which have increased cytotoxic efficacy to both lung cancer cells and their stem cells [17, 18].
This review discusses the principles and mechanisms of PDT, the development of PSs, AgNP physicochemical properties, the synthesis and conjugation of AgNPs and PSs, and the challenges, limitations and future perspectives.

2
Principle and Mechanism of PDT
Essentially, PDT consists of three main elements: a light source to supply the energy for the photodynamic reactions, PS that absorbs the light energy and triggers the photochemical reaction, and molecular oxygen to initiate reactive species (ROS) generation that causes cell death [9]. During the process of PDT, light is emitted at a specific wavelength, and the PS absorbs the light energy or photons. This excites the PS from a ground state to an excited state. As the excited PS returns to the ground state, some energy is transferred to the surrounding oxygen dissolved in the cells, which can also react with non‐oxygen substrates. This Type I reaction transfers the protons to generate free radicals and further interacts with ground‐state oxygen to generate ROS, like a single oxygen singlet (1O2). Under normal conditions, ROS plays an important role in vivo, such as in phagocytosis, regulation of cell growth, intercellular signalling and so forth. However, increased ROS production causes cellular damage [19, 20]. The Type II reaction (most common reaction) occurs when the PSs react directly with the ground‐state oxygen to increase the generation of ROS, leading to cell death through apoptosis, necrosis and autophagy [9, 21]. Apoptosis is generally viewed as the major cell death pathway; however, determinants for the type of cell death mechanism that prevails are the subcellular localisation of the PS in the different organelles (e.g., mitochondria, lysosome and endoplasmic reticulum), the PDT dosage and the drug‐light interval [22].

3
PSs used in Lung Cancer Treatment
PSs are classified as agents that convert light energy into cytotoxic and vasculotoxic reactions through photochemical reactions [23]. Most of them (i.e., porphyrins, chlorins, etc.) typically feature tetrapyrrole structures similar to natural biomolecules like chlorophyll or haem, and some can also be termed theranostic PSs (i.e., Rose Bengal) as they have a dual function of diagnostic imaging and targeted therapy [24, 25]. An ideal PS requires a strong absorption peak in the red to near‐infrared (NIR) spectrum, which enables tissue penetration, high triplet quantum yield for efficient ROS generation, no dark toxicity, rapid clearance from healthy tissues, tumour‐targeting affinity through balanced hydrophobicity/hydrophilicity and good stability [22, 26].
3.1
First Generation PSs
The first‐generation PSs, such as haematoporphyrin derivative (HpD) and its modified form Photofrin (i.e., porfimer sodium), were clinically approved by the FDA in 1998 for early‐stage NSCLC [27]. Tests demonstrate the feasibility and safety of treating peripheral lung tumours using porfimer sodium with a laser light of 630 nm at 200 J/cm2 [28]. Numerous clinical trials (e.g., NCT03678350, NCT04836429) are currently investigating the safety and efficacy of Photofrin. Previous clinical trials (i.e., NCT03344861) have shown that most patients achieved complete responses, with mild to moderate photosensitivity being a common adverse effect [29]. Studies highlight limitations to Photofrin, like complex composition, poor chemical purity, low water solubility, weak tissue penetration due to suboptimal absorption spectra and prolonged skin photosensitivity [30, 31]. These shortcomings spurred the development of second‐generation PSs.

3.2
Second‐Generation PSs
The second‐generation PSs include chlorins (i.e., chlorin e6), phthalocyanines (aluminium phthalocyanine chloride) and porphycenes. These have enhanced chemical purity, absorption and tumour selectivity, but still struggle with solubility for intravenous use and nonspecific accumulation at tumour sites [31, 32]. Talaporfin sodium (talaporfin) is a Ce6 derivative, which demonstrated promising results in clinical trials for c‐stage IA peripheral lung cancer and central early‐stage lung cancer, with less skin photosensitivity compared to Photofrin [29]. Laserphyrin is another Ce6 derivative that was approved in 2004 in Japan for early‐stage lung cancer. The study showed improvement in lung cancer symptoms and survival rates for patients after treatment with PDT using a 664 nm laser wavelength [29, 33].

3.3
Third‐Generation PSs
Third‐generation PSs aim to address the gaps by conjugating second‐generation PSs with tumour‐targeting ligands (e.g., antibodies, NPs) or hydrophilic moieties (e.g., PEG, peptides) to improve specificity, solubility, and increase 1O2 yield while enabling deeper tissue activation and reduced skin photosensitivity [30, 34]. One example of this is the combination of aluminium phthalocyanine chloride (AlPcS4Cl), which has demonstrated excellent photostability and amphipathic properties, has been combined with gold (Au) and antibody (anti‐CD133) for enhanced PDT effects, leading to increased cytotoxicity, apoptosis and reduced proliferation and viability of LCSCs, compared to a standalone AlPcS4Cl [29, 35]. In another study, a novel PS was formulated through combining titanium dioxide NPs (TiO2NPs), aluminium phthalocyanine (Pc), and folic acid (FA). This resulted in superior efficacy in one and two‐photon PDT, 4.5 times more ROS production than free Pc and 1.7‐fold higher two‐photon absorption than free Pc, which enables deeper tissue penetration for PDT [36].

3.4
Fourth‐Generation PSs
The fourth‐generation PSs represent the latest advancements in PDT, focusing on overcoming the limitations of previous generations and enhancing therapeutic efficacy (Table 1). Examples of these include mesoporous silica and meta–organic frameworks (MOFs), which are advanced porous nanostructures [37]. MOFs can integrate PSs as organic linkers (e.g., porphyrin‐based frameworks) or encapsulate them within pores [38]. This offers biodegradability and controlled release, which will be triggered by specific tumour conditions like pH or hydrogen peroxide (H2O2) [37, 39]. Furthermore, they also address the long‐standing issue associated with PDT, which is hypoxia, by catalysing the decomposition of H2O2 into O2 (as seen in Ti‐TBP MOFs and PCN‐224‐Pt) or by acting as ‘oxygen shuttles’ [37, 40].

4
AgNPs and Their Physicochemical Properties
AgNPs are a promising nanoproduct created by transforming bulk silver ions into nanoscale materials through nanotechnology [44]. AgNPs have gained great significance due to their low cost relative to Au, high conductivity, oxidation resistance and biocidal characteristics [45]. The diverse array of properties of AgNPs is defined through the chosen synthesis methods [46]. Physicochemical properties such as surface area, shape, surface charge, optical properties, agglomeration and dissolution rate will influence its biomedical applications [47].
4.1
Size of AgNPs
The surface area or size of the AgNPs can affect their surface‐area‐to‐volume ratio, optical properties and conductivity. Due to the small size, this increases the effectiveness of AgNPs in applications such as catalysis, sensing, drug delivery and imaging. However, studies have shown that very small AgNPs could result in high penetration efficiency, causing increased toxicity [46]. The Trojan Horse effect hypothesises that AgNPs smaller than 40 nm will enter the cells to continuously liberate silver ions (Ag+) in the cell. This results in lipid peroxidation and potentially damaging non‐target organelles like mitochondria and DNA. Although normal cells can mediate this by secreting the reductase enzyme to reduce the effects of the Ag+, the increased accumulation of the AgNPs and Ag+ in cells will initiate cytotoxicity in the normal cells [44]. A study by Jeyaraj et al. synthesised AgNPs sized 22 nm for human breast cancer [48], while the average size reported for AgNPs synthesised for lung cancer was 21.9 nm [49]. This suggests that AgNPs are not toxic to normal cells at low doses but lose selective cytotoxicity in healthy cells at high doses. In intravenous applications, NPs larger than 500 nm are quickly removed from circulation, while those smaller than 200 nm are ideal for treating solid tumours due to their ability to navigate the tumour vasculature [50].

4.2
Shape of AgNPs
The shape of the AgNPs can affect their catalytic, optical, and electrical properties. The shapes can vary from nanospheres, nanocubes, nanoprisms, nanotriangles, nanowires, nanostars, nanodisks and nanorice to octahedron‐shaped NPs [45]. However, many studies demonstrate that a spherical shape is the most common shape assumed by AgNPs after synthesis for anticancer PDT [46, 49, 51, 52]. The spherical shape offers uniformity and facile synthesis while releasing the Ag+ more efficiently into cell membranes due to its higher surface‐area‐to‐volume ratio compared to most other shapes [46]. It seems cells take up spherical NPs between 20 and 50 nm at the highest rates [53]. Variations in the shape and size of AgNPs are due to the chosen reducing agent, capping agent and synthesis method [54].

4.3
Surface Charge of AgNPs
The surface charge (neutral, negative or positive) plays a crucial role in NP imaging and drug delivery. The versatility of NPs for cellular selectivity, uptake and therapeutic effect is due to the manipulation of surface charge. Therefore, modulating the AgNP surface charge can affect its stability, aggregation, agglomeration, solubility and reactivity [46]. First, charged NPs have been linked to increased cytotoxicity compared to neutral forms; while cationic NPs exhibit a greater plasma membrane integrity disruption, more pronounced damage to mitochondria and lysosomes and increased autophagosomes than anionic NPs. Likewise, with cellular uptake, cationic NPs generally are taken up more efficiently in non‐phagocytic cells due to their stronger interaction with the negatively charged cell membrane, facilitating their internalisation [53]. The electrical conductivity of AgNPs is significantly influenced by their zeta potential, which measures the electrical potential difference between the surface of the NPs and the surrounding medium. Zeta potential is also an important indicator of stability, as higher values (both positive and negative) indicate strong electrostatic repulsion between particles, which helps prevent agglomeration or aggregation [46, 55].

4.4
Optical Properties
The main light interactions that influence the optical properties of AgNPs are absorption and scattering (extinction) properties [56]. This phenomenon is known as localised surface plasmon resonance (LSPR), whereby incident light interacts with the NPs, causing collective oscillations of electrons on the NP surface [57]. This leads to localised electromagnetic field enhancement and increased absorption and scattering of light, as seen in Figure 1, particularly in the visible (390–700 nm) region [46, 58]. When the electromagnetic field interacts with the AgNPs, the scattering light is affected by several factors. These include the particle's size, shape, and composition, the refractive index of the NP, the refractive index of the surrounding medium, and the wavelength of the incident light [56, 59, 60]. The plasmon extinction of spherical AgNPs peaks at 400 nm, although other morphologies can result in greater plasmonic spectra redshifts [56]. Also, there seems to be a direct correlation between the size of the NPs and an increase in the LSPR peak in the absorption (redshift) spectra [61]. A study by Zhang et al. demonstrated a threefold increase in plasmon‐assisted, metal‐enhanced 1O2 generation (ME1O2) compared to the control sample [58]. This increased 1O2 yield was due to Rose Bengal near Silver Island Films. This corresponds with the review by George et al., which states that the practicability of synthesis and functionalisation of metallic NPs allows for the adjustment of their excitation wavelengths to the NIR region [61]. This significantly enhances tissue penetration and improves the overall in vivo PDT potential.

5
Mechanisms of AgNPs–PS Conjugates in PDT
To effectively utilise the mentioned AgNP properties in PDT, it is crucial to employ a deliberate and controlled process for synthesis and conjugation. This includes selecting the correct synthesis method and strategically attaching the appropriate PS drug to the AgNP carrier [62]. These two steps help transform the separate components into a synergic targeted therapeutic agent.
5.1
Synthesis of AgNPs
The various methods for synthesising AgNPs can be divided into two major categories: top‐down and bottom‐up protocols. The top‐down method involves reducing the size of bulk material to the appropriate nanoscale structure. In contrast, the bottom‐up method builds molecular, atomic or ionic components by combining them to create a more complex nanostructure. These can be further categorised into physical (i.e., sputtering, physical vapour deposition, laser ablation, arc discharge and spark discharge), chemical (i.e., sol–gel, hydrothermal, electrochemical, microemulsion, and chemical reduction) or biological methods (i.e. plant‐mediated, microbial, bio‐polymer‐mediated, enzyme‐assisted and green sols) [63, 64]. However, the appropriate synthesis method must be chosen to achieve the desired properties of the NPs [65]. This is particularly important regarding oncological treatment, as the synthesised AgNPs must exhibit multiple benefits such as eco‐friendliness, lower toxicity and economic cheapness [63]. A study by Mukherjee et al. reported that the biosynthesised AgNPs (b‐AgNPs) exhibited significant dose‐dependent inhibition of cancer cell proliferation of A549 lung cancer cells compared to the chemically synthesised AgNPs (c‐AgNPs) [66]. This makes the biological methods more attractive as they also do not need specialised conditions such as sophisticated instruments, vacuum conditions, catalysis, high energy requirements, templates, etc, which are associated with chemical or physical processes [66].
5.1.1
Green Synthesis
The biosynthesis approach or green chemistry for AgNPs has become a focal point of intensive metal‐based NP research due to its capability to reduce the Ag+ ions while preventing agglomeration and minimising toxicity [67]. This method leverages the biological entities of various microorganisms, plant parts, and enzymes. However, using plant extracts appears to have the added advantage of faster synthesis of NPs compared to microorganisms [68, 69]. This is due to their high concentration of natural reducing agents such as flavonoids, alkaloids, terpenoids, saponins, amino acids, vitamins, proteins, polysaccharides, organic acids, aromatic dicarboxylic acids and polyphenols. Also, some functional groups are found in these agents, such as hydroxyl, amidogen and carbonyl [67]. These phytochemicals and biomolecules can also act as capping agents to provide stability to the AgNPs, so they can be biocompatible for anticancer activities [70]. The biocompounds found in plants such as Descurainia sophia (e.g., proteins, phenols, alkaloids) can donate hydrogen from their ─OH groups to free radicals, resulting in the formation of stable phenoxyl radicals [71]. These phenoxyl radicals act as key indicators of cancer cell death via ROS‐driven oxidative stress, mitochondrial damage and antioxidant depletion. Phenol‐ring polyphenolics show therapeutic potential as selective prooxidants in cancer treatment, due to the vulnerability and sensitivity of cancer cells to ROS‐inducing therapy [72].

5.1.2
Synthesis of AgNPs Using Plant Materials
Before synthesis, the plant of interest must be thoroughly washed with tap water and then ‘shed’ dried to preserve the bioactive compounds in the plant. Upon shed drying, using the maceration method demonstrated in Figure 2, the plant part (i.e., leaves, root, flower, etc.) must be ground into a fine powder and extracted using water and organic solvents, such as methanol (MeOH) or chloroform (typically a 70% ethanol). This process is a long operation which may take up to 72 h; however, as it occurs at room temperature (20°C–25°C), it is friendly to the heat‐sensitive reducing agents. After extraction, the compound will be collected through evaporation of the drying methods, prepared by desalination in solvents (distilled water or phosphate‐buffered saline), and filtered using the appropriate sterile filter [73]. Another common method employed to extract compounds is the Soxhlet extraction, which utilises a Soxhlet apparatus [74]. In a method described by Chota et al., the coarse plant powder (about 10 g) is extracted using a mixture of 70% chloroform, ethyl acetate and MeOH for 36 h at a temperature of 50°C (not suitable for thermolabile compounds). The extracts are then dried with a rotary evaporator. To prepare the stock solution, the dried plant extract is dissolved in distilled water, vortexed and filtered before being stored for analysis and further use [75].
To ascertain which phytochemicals the plant part contains, several qualitative and quantitative analysis techniques can be utilised, such as the Fourier transform infrared (FTIR) spectroscopy analysis, high‐performance thin layer chromatography (HPTLC), ultra‐high‐performance liquid chromatography coupled to electrospray ionisation quadrupole time‐of‐flight mass spectrometry (UHLPC–qTOF–MS2) and so forth [74, 76, 77].

5.1.3
Application of Plant Extracts for AgNP Synthesis
In 2019, Erdogan et al. successfully synthesised AgNPs from silver nitrate (AgNO3) from Cynara scolymus leaf extracts for cancer treatment. Scanning electron microscopy (SEM) showed spherical AgNPs [78]. At the same time, the zeta potential revealed that the structure of the bio‐synthesised NPs was stable and able to preserve the spherical structure over a long period during cellular uptake. Several studies have highlighted the presence of bioactive compounds such as flavonoids, alkaloids, terpenoids, saponins and many others, for their reduction and capping action [79, 80, 81]. In a particular study, the Alternanthera tenella leaf extract was used in the phytosynthesis of the AgNPs. What was interesting to note is that although the leaves possess multiple phytochemicals (e.g., alkaloids, reducing sugars, saponins, flavonoids, tannins, etc.), the flavonoids were identified to be the main phytochemicals responsible for the NP synthesis. Flavonoids consist of hydroxyl groups, which bind to silver ions and act as reducing agents [78]. In an extensive study by Pradeep et al., it was concluded that flavonoids and phenolic acids were involved in the reduction of Ag+ ions [82]. Meanwhile, phloroglucinols and xanthones served as capping agents, while naphthodianthrones acted as both reducing and capping agents. The phytochemicals' reduction and capping actions, depicted in Figure 3, highlight the complexity and variability of plant‐mediated synthesis, as plant extracts contain various bioactive compounds.
Recently, the Dicoma anomala root extract was used in some in vitro studies for b‐AgNPs for anti‐proliferative activity on the human lung cancer cell line (A549) and human hormone‐dependent breast cancer cell line (MCF‐7) [49]. In a study by Sreekanth et al., the b‐AgNPs from the Panax ginseng root extract demonstrated a cell‐inhibitory effect on cervical cancer (HeLa) cells, but no inhibition on the normal renal (MDCK) cells with the same concentration [83]. This corresponds with another study, which utilised the leaf extract of the same plant to demonstrate the tumour cell inhibitory action of the b‐AgNPs [84]. In a study by Muthu et al., Moringa oleifera‐derived AgNPs reduced A549 cell viability to 50.75% while sparing HEK293 normal cells, which retained 97% viability, highlighting selective cytotoxicity. Gene expression studies indicated impaired tumour vascularisation and halted proliferation due to a downregulation of cancer‐related factors: VEGF and cyclin‐D1. Within the same study, Gymnema sylvestre AgNPs downregulated VEGF expression by 63.94%, thereby disrupting angiogenesis [85]. Table 2 provides an overview of some plants and their phytochemicals that have been used to synthesise AgNPs for lung cancer PDT.

5.1.4
Factors Affecting the Synthesis of AgNPs
The synthesis of AgNPs is influenced by several factors, including pH, plant extract concentration, silver salt or Ag+ ion concentration, reaction time and temperature.
The pH of the reaction mixture will significantly affect the electrical charges of biomolecules, thereby altering their reducing and capping abilities. Neutral pH results in rapid NP production, while a high pH yields stable, small‐sized NPs [99]. A study by Anigol et al. reported on the effect of pH on the synthesis and characteristics of the AgNPs, demonstrating that electrostatic attraction (pH 2) enhances adsorption and degradation, while repulsion (pH 7–9) reduces efficiency [100].

Concentration of plant extract plays a crucial role in AgNP synthesis, as its bioactive molecules (i.e., phenols and polysaccharides) contribute to the reduction of Ag+ ions and the formation of stable AgNPs. However, high concentrations of the plant extract can lead to the formation of larger NPs due to increased nucleation [101].

The Ag+ ion concentration also impacts synthesis, as higher concentrations can lead to incomplete reduction and aggregation, while optimal concentrations ensure efficient NP formation [102]. In a study by Kah et al., the average particle size of free b‐AgNPs was smaller than that of both PEG–BpAgNP and Cum–PEG–BpAgNP conjugates. This difference in size can be attributed to the synthesis conditions, particularly the concentration of plant extract and Ag+ ions used during the process [18].

The reaction time is critical for the complete consumption of Ag+ ions, as optimal durations will help control the size and shape of NPs. A study by Ansari et al. demonstrated that longer reaction times of up to 48 h yielded smaller, more uniform AgNPs. Longer reaction time may not alter the NP concentration once all ions are reduced [101].

Temperature accelerates the reaction, with higher temperatures promoting rapid reduction and smaller NP sizes, although excessively high temperatures can degrade bioactive metabolites in plant extracts, hindering synthesis [99, 102]. All these factors must be carefully optimised to achieve the desired properties. Al‐Musawi and Al‐Saadi considered the effect of boiling time (10, 15 and 20 min) of plant leaves and the exact volumes (3, 5 and 7 mL) of plant extract on the AgNP synthesis [95]. The study found that the optimal boiling time for the synthesis of AgNPs was 10 min, while longer boiling times did not improve the synthesis efficiency. The optimal volume of the plant extract was 3 mL when using the Dodonaea viscosa leaf extract at a peak absorbance of 463 nm [95].

5.2
Conjugation of AgNPs and PSs
The conjugation of AgNPs with PSs demonstrates a significant advancement in enhancing the efficacy of PDT for lung cancer (Table 3). This approach leverages the unique properties of AgNPs (i.e., their high surface area, biocompatibility and ability to generate ROS) combined with the light‐absorbing capabilities of PSs to create a synergistic therapeutic effect [21].
A study by Mota et al. demonstrated that pluronic‐based nanohybrids allow for precise tuning of AgNP–PS interactions, enhancing PDT outcomes [106]. By adjusting polymer chain length and selecting appropriate PS, researchers can optimise 1O2 generation and fluorescence for targeted therapies. This approach provides a scalable strategy for designing next‐generation, light‐responsive nanoconjugates with potential applications in oncology treatments.
PEGylation or coating the surface of NPs with polyethylene glycol (PEG) is noted for reducing toxicity, improving the stability of AgNPs, while showing no aggregation, opsonisation and phagocytosis, thereby prolonging circulation time in physiological conditions [107]. In a method described by Kah et al., the polymer (thiol‐PEG‐amine) was coated with the AgNPs via sonication and 24‐h incubation to form the PEG–AgNPs conjugate [18]. The PS (curcumin) was loaded under specific conditions (i.e., sonication, incubation and centrifugation) and purified through multiple centrifugations to form the Cum–PEG–BpAgNPs complex, then stored for up to a week for analysis.
Surface functionalisation with plant‐derived ligands (e.g., neem alkaloids) could enhance specificity by targeting overexpressed receptors like EGFR [108].
5.2.1
Conjugation Methods and Enhanced Properties
The conjugation process can involve attaching the PS molecules to AgNPs via both covalent and non‐covalent bonding. The method chosen for grafting PS to nanocarriers and the type of PS used are crucial for ensuring the stability and reliability of the nanoconjugate.
5.2.1.1
Covalent Bonding
Most PSs are hydrophobic and therefore poorly soluble in water. As such, AgNPs can help create a more efficient delivery through covalent bonding to the proposed PS [109]. Covalent bonding, such as NP‐SH linkages via thiol groups, amine groups (─NH2), carboxyl groups (─COOH) and phosphine groups (─PR3), can aid in the stability of the nanoconjugate while determining the final size and shape [110]. The cationic chlorins form stable complexes with the non‐ionic surfactants, where the PS molecules are located at the periphery of the micelles next to the hydrophilic head groups. This interaction helps to enhance the stability and effectiveness of the PS in the aqueous environment of the body [111]. The carbonyl groups (C═O) from the amino acid residues and peptides of proteins have a strong affinity to bind metals. So that protein can act as an encapsulating agent and thus protect the NPs from agglomeration [112]. It is suggested that Ag can interact with the PS, curcumin, via the C═O group [113].

5.2.1.2
Non‐Covalent Bonding

Electrostatic interactions are effective for cationic PS, such as porphyrins, as they interact with the negatively charged NPs (i.e., AgNPs) to enhance photodynamic efficiency [114]. In 2020, Tavakkoli Yaraki et al. reported the formation of nanohybrids using 4‐mercaptobenzoic acid‐capped AgNPs and a red‐emissive AIE‐PS via electrostatic interactions, which resulted in a significant enhancement in SOG and improved photostability [115]. Also, the electrostatic assembly of cationic PS with AgNPs facilitates better cellular uptake and targeting of cancer cells, which is essential for effective PDT. This is seen through efficient endocytosis and enhanced PDT effects [116, 117]. In addition, the conjugation of the cationic PS with AgNPs increases ROS generation due to improved light absorption and energy transfer facilitated by the NPs [114].

Hydrogen bonding plays a significant role in the stabilisation and synthesis of AgNPs. Short ligands like cysteine and glycine can stabilise AgNPs through hydrogen bonds, which also enhances the interaction with PSs [118].
The interaction between AgNPs and PSs can also be influenced by Van der Waals forces [119]. The study by Kah et al. observed the encapsulation of AgNPs by the non‐functionalised PEG, potentially due to the positive surface charge of the AgNPs [18]. This encapsulation also stabilises the PEG–AgNPs complex through induced dipoles between the molecules, which is driven by the van der Waals interactions between the negatively charged oxygen groups in PEG's structure and the positively charged groups on the inert AgNPs' surface, so that the PS (curcumin) encapsulates the AgNPs [18].

5.2.2
Synergistic Mechanisms and Targeting
Most AgNP–PS conjugates enter the cells via endocytosis (clathrin‐dependent or clathrin‐independent), while some may be taken up through diffusion or lipid peroxidation. Regarding endocytosis, AgNP–PS conjugates are taken up by endosomes, which then progress to lysosomes. These lysosomes have a lower pH that favours the release of Ag+ ions, which initiates reactions between H2O2 found in ROS and AgNPs, leading to the release of Ag+ ions in vivo [47, 120].
The AgNP–PSs conjugates target the tumour cells via passive and active targeting or both. Passive targeting relies on the small size and surface properties of nanocarriers, allowing them to penetrate and accumulate in tumour tissues through the EPR effect, which results from the leaky vasculature of tumours [121]. Compared to cancerous tissues, healthy tissues have more stable and less permeable blood vessels, which reduces the absorption of NPs [50]. Active targeting entails the conjugation of certain ligands to the NP surface, allowing for targeted delivery to cancer cells by binding to overexpressed receptors or markers on their surface [121]. A synergic benefit of loading the PS in the AgNP is that it does not need to be released from the nanocarrier, as both the molecular O2 and generated 1O2 can diffuse in and out of the nanocarrier [61, 122].
AgNPs–PS conjugates are more potent for PDT, as these nanoconjugates are more stable and can address the hydrophobicity and poor bioavailability of many free molecular PSs, such as curcumin [18, 61]. The LCSCs express specific markers (e.g., CD44, CD133, ALDHA1, PROM1 and OCT4), which make them more resistant to standard therapies due to an increase in tumour stemness. Therefore, the dual disruption of the stemness pathways (such as Notch, SHH, WNT and RTKs) and apoptosis induction is vital to cancer eradication [123]. This concept is confirmed when phytochemical‐capped AgNPs, Bidens pilosa‐synthesised Cum–PEG–BpAgNPs, improved the solubility and PDT activity of curcumin, targeting LCSCs under 470 nm laser light [18]. In addition, targeting of LCSCs relies on passive accumulation and surface functionalisation. Based on this, the clinical LCSC‐targeting agents in clinical development include RO4929097 (Notch inhibitor), disulfiram/Cu (ALDH inhibitor) and ABBV‐399 (anti‐MET antibody‐drug conjugate). Table 4 provides a summary of some of the targeting mechanisms of LCSCs and general lung cancer cells [18, 123, 124].
Moreover, AgNPs enhance the overall efficacy of PDT through the efficient transfer of energy from excited plasmonic AgNPs to molecular O2 and/or organic PSs via the Förster resonance energy transfer (FRET) mechanism [61, 125]. In addition, light upconversion occurs when the upconversion NPs (UCNPs) absorb NIR radiation and emit a shorter wavelength light, leading to the excitation of organic PSs [26].

6
Challenges, Limitations and Future Perspectives
Although green‐synthesised nanoconjugates have gained popularity in recent years, there remain challenges in translating the green‐synthesised AgNPs from the lab to the clinic. These include scalability, reproducibility, safety and regulatory compliance [126]. Therefore, future research must work on standardising extraction and synthesis protocols, while exploring different AgNPs shapes and new materials for PDT‐based therapies, as seen with the fourth‐generation PSs. Enhanced purification techniques need to be developed, along with thorough in vivo toxicological and pharmacokinetic studies for safer profiles (i.e., possible chronic impact of AgNPs in body tissues and systems) [102]. Finally, there is a need to develop industrial‐scale green synthesis technologies and engage with regulatory agencies to establish clear guidelines [126].
Other drawbacks include PDT being unable to deal with disseminated metastases and having difficulty in affecting tumours surrounded by necrotic tissue or dense and deep tumours [62].
Future directions should continue to explore combinatorial therapies to overcome resistance, such as coupling AgNPs–PS with chemotherapy drugs (e.g., cisplatin, carboplatin, docetaxel, etc.) and gene therapies. There is a need to assess pharmacokinetics and biodistribution, particularly for targeting drug‐resistant cancers via phytochemical‐mediated pathways [127]. A recent development in cancer research is the use of targeted tumour vasculature, particularly nucleic acid‐integrated nanohybrids [128].
There is a need for a better understanding of the pathological mechanisms that regulate LCSCs' maintenance, and how these mechanisms cause these cells to resist conventional therapies, and drive recurrence and metastasis. This will develop a more efficient and targeted therapy to eradicate this key tumour cell subpopulation [129].

7
Conclusions
PDT has emerged as a promising non‐invasive strategy for lung cancer treatment; however, its clinical application faces challenges such as poor PS delivery, low tumour selectivity and insufficient ROS generation. The integration of nanotechnology, particularly AgNPs, offers a transformative solution to these limitations. Green‐synthesised AgNPs, leveraging plant‐derived phytochemicals as reducing and stabilising agents, exhibit remarkable biocompatibility, cost‐effectiveness and reduced cytotoxicity compared to conventional synthesis methods. These NPs enhance PS stability, bioavailability and tumour targeting through passive mechanisms like the improved permeability and EPR effect or active targeting via ligand‐receptor interactions. In addition, AgNPs amplify PDT efficacy by leveraging plasmonic effects, NP and light upconversion, significantly boosting ROS generation and enabling deeper tissue penetration. Preclinical studies highlight the potential of plant‐mediated AgNPs, such as those derived from D. anomala and M. oleifera, which demonstrate selective cytotoxicity against lung cancer cells while sparing healthy tissues.
However, challenges persist, such as the long‐term biosafety concerns, including the potential long‐term toxicity of AgNPs, optimisation of synthesis parameters (e.g., pH, temperature, reaction time) and the need to address tumour heterogeneity and resistance mechanisms. Scalability of the green synthesis may be affected by the composition of plant extracts, which may vary due to season, geography and extraction methods. This could affect the standardisation of large‐scale production and clinical translation. Future research must prioritise combinatorial approaches, integrating AgNPs–PS conjugates with chemotherapy, immunotherapy, or gene therapy to overcome multi‐drug resistance and eradicate LCSCs. In addition, increased pharmacokinetic studies and clinical trials are needed to validate the safety and efficacy of these nanoconjugates in humans. Through bridging the gap between green nanotechnology and precision medicine, AgNPs–PS conjugates hold great potential to significantly advance PDT, which will offer improved survival rates and quality of life for lung cancer patients worldwide.

Author Contributions

Njabulo Henry Sibanda: conceptualisation, writing original draft, visualisation. Anine Crous: conceptualisation, reviewing and editing, supervision, funding acquisition. Blassan P. George: conceptualisation, reviewing and editing, supervision, funding acquisition. All authors read and approved the final manuscript.

Consent
The authors have nothing to report.

Conflicts of Interest
The authors declare no conflicts of interest.