Perovskite Solar Cells Based on Polymerized Chlorophyll Films as Environmentally Friendly Hole-Transporting Layers.

Hole-transporting layers (HTLs) play a crucial role in the performance of inverted, p-i-n perovskite solar cells (PSCs). Chlorophylls (Chls) are naturally abundant organic photoconductors on earth, with good charge carrier mobility and appropriate Fermi energy levels that make them promising candidates for use in photovoltaic devices. However, Chls films prepared using the solution method exhibit lower carrier mobility compared to other organic polymer films, which limits their application in PSCs. To address this issue, Chls molecules are chemically linked to reduce the charge transfer barrier, thus the transfer of charges between molecules is transformed to intramolecular charge transfer. This study synthesizes and characterizes two polymerized Chl films, PolyCuChl and PolyNiChl, as HTLs of CH3 NH3 PbI3 -based PSCs. PSCs based on the electrochemical polymerization of PolyChl HTLs demonstrate an enhanced power conversion efficiency (PCE) of up to 19.0%, which is the highest efficiency among devices based on Chl materials. Furthermore, these devices demonstrated exceptional long-term stability. These results highlight the potential of polymerized Chl films as a viable alternative to conventional HTLs in PSCs. The approach utilizes abundant, environmentally friendly, and versatile Chl derivatives, and can be extended to develop next-generation HTL materials for improved PSC performance.

Porphyra tenera Protects against PM2.5-Induced Cognitive Dysfunction with the Regulation of Gut Function.

To evaluate the biological effects of Porphyra tenera (P. tenera), we tried to confirm the possibility that the intake of P. tenera could modulate cognitive and intestinal functions in PM2.5-induced cognitive decline mice. P. tenera attenuated PM2.5-induced learning and memory impairment through antioxidant and anti-inflammatory effects by regulating the mitochondrial function and TLR-initiated NF-κB signaling. In addition, P. tenera effectively alleviated Aß production/tau phosphorylation by inhibiting the JNK phosphorylation. Also, the bioactive constituents of P. tenera determined the sulfated galactan, mycosporine-like amino acids (MAAs), and chlorophyll derivatives. Moreover, the bioactive compounds of P. tenera by gut fermentation protected against gut dysbiosis and intestinal tight junction damage with a decrease in inflammatory response and short-chain fatty acid production. Based on these results, our findings suggest that P. tenera with sulfated galactan and MAAs is a potential material for cognitive function improvement.

Shengxuening Extracted from Silkworm Excrement Mitigates Myelosuppression via SCF-Mediated JAK2/STAT3 Signaling.

Shengxuening (SXN) is a Chinese patent medicine with main ingredients (including chlorophyll derivatives and sodium iron chlorophyllin) extracted from silkworm excrement. SXN exhibited efficacy in clinical trials of renal anemia and iron deficiency anemia; however, the specific mechanisms remain unclear. This study found that SXN increased the number of peripheral blood cells and improved the bone marrow morphology in myelosuppressed mouse model, reversed the reduction in body weight and spleen indices, and increased the serum levels of erythropoietin and granulocyte-macrophage colony-stimulating factor. Quantitative real-time PCR array and Western blot analysis showed the enhanced expression of stem cell factor (SCF), JAK2, and STAT3 in the liver. These results suggested that SXN promoted the recovery of hemopoietic function in myelosuppressed models by increasing the secretion of hematopoietic factors and activating the JAK2/STAT3 pathway. Therefore, this medicine may be applied as therapeutic pharmaceutical drug to mitigate myelosuppression.

Chlorophyll Derivatives from Marine Cyanobacteria with Lipid-Reducing Activities.

Marine organisms, particularly cyanobacteria, are important resources for the production of bioactive secondary metabolites for the treatment of human diseases. In this study, a bioassay-guided approach was used to discover metabolites with lipid-reducing activity. Two chlorophyll derivatives were successfully isolated, the previously described 132-hydroxy-pheophytin a (1) and the new compound 132-hydroxy-pheofarnesin a (2). The structure elucidation of the new compound 2 was established based on one- and two-dimensional (1D and 2D) NMR spectroscopy and mass spectrometry. Compounds 1 and 2 showed significant neutral lipid-reducing activity in the zebrafish Nile red fat metabolism assay after 48 h of exposure with a half maximal effective concentration (EC50) of 8.9 ± 0.4 µM for 1 and 15.5 ± 1.3 µM for 2. Both compounds additionally reduced neutral lipid accumulation in 3T3-L1 multicellular spheroids of murine preadipocytes. Molecular profiling of mRNA expression of some target genes was evaluated for the higher potent compound 1, which indicated altered peroxisome proliferator activated receptor gamma (PPARγ) mRNA expression. Lipolysis was not affected. Different food materials (Spirulina, Chlorella, spinach, and cabbage) were evaluated for the presence of 1, and the cyanobacterium Spirulina, with GRAS (generally regarded as safe) status for human consumption, contained high amounts of 1. In summary, known and novel chlorophyll derivatives were discovered from marine cyanobacteria with relevant lipid-reducing activities, which in the future may be developed into nutraceuticals.

Antiradical Activity of Porphyrins with a Diisobornylphenol Fragment at the Macrocycle Periphery.

This article focuses on the antiradical activity of a number of 2,6-diisobornylphenol-porphyrin conjugates with various spacers between the porphyrin and phenolic fragments in the model reaction of ethylbenzene oxidation initiated by azoisobutyric acid dinitrile. The study has shown that the electronic effects of the groups directly related to the 2,6-diisobornylphenol fragment exert the predominant influence both on the reactivity of the phenolic hydroxyl group in interaction with free radicals and on the antiradical activity of the molecule as a whole. The antiradical activity of the molecule is generally less affected by the nature of the substituents in the porphyrin macrocycle, mainly due to a change in the stoichiometric inhibition coefficient in the presence of relatively easily oxidizable groups. It was found that the length of the spacer between the porphyrin and phenolic fragments does not affect the antiradical activity of the conjugate.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

Inhibitors for cholesterol ester accumulation in macrophages from Chinese cabbage.

The cholesterol ester accumulates in macrophages in the early stage of atherosclerotic lesions, leading to the formation of foam cells. We examined the inhibitory effects of the crude extracts of 22 edible plants on foam cell formation and isolated nine chlorophyll derivatives as potent inhibitors from Chinese cabbage. The results of the present study suggest that the chlorophyll derivatives contained in edible plants may be useful for the prevention and treatment of atherosclerosis.

Interactions of chlorophyll-derived photosensitizers with human serum albumin are determined by the central metal ion.

Two structurally similar derivatives of chlorophyll a, chlorophyllide a (Chlide) and zinc-pheophorbide a (Zn-Pheide), differing only in central metal ion (Mg2+ or Zn2+, respectively) substituting the tetrapyrrole ring, were investigated with regard to their binding to human serum albumin (HSA). Chlide and Zn-Pheide are very promising photosensitizers with potential application in photodynamic therapy, therefore it is desirable to investigate their interactions with serum proteins. The studies included absorption and steady-state fluorescence spectroscopy, as well as molecular docking. It was found that both investigated compounds form complexes with HSA. Experimental data revealed two classes of binding sites for each compound. The affinities (Ka) for the first class were in the range of 105 and 106 M-1 for Chlide and Zn-Pheide, respectively, while the second class was characterized by the affinities of the order of 104 M-1 for both derivatives. Molecular docking simulations together with displacement studies revealed that the primary binding site of the studied compounds is the heme site, localized in the subdomain IB, however the best characterized binding sites of HSA, namely the Sudlow's sites I and II are also involved. The interactions between the derivatives of chlorophyll and HSA were found to be predominantly hydrophobic and to a lesser extent hydrogen bonding. Our results demonstrate that the centrally bound metal ion determines both the affinity and mode of binding to HSA, which may be a feature differentiating these compounds in terms of their pharmacokinetics.Communicated by Ramaswamy H. Sarma.

Biomimetic Materials Based on Poly-3-hydroxybutyrate and Chlorophyll Derivatives.

Electrospinning of biomimetic materials is of particular interest due to the possibility of producing flexible layers with highly developed surfaces from a wide range of polymers. Additionally, electrospinning is characterized by a high simplicity of implementation and the ability to modify the produced fibrous materials, which resemble structures found in living organisms. This study explores new electrospun materials based on polyhydroxyalkanoates, specifically poly-3-hydroxybutyrate, modified with chlorophyll derivatives. The research investigates the impact of chlorophyll derivatives on the morphology, supramolecular structure, and key properties of nonwoven materials. The obtained results are of interest for the development of new flexible materials with low concentrations of chlorophyll derivatives.

Insights into the mechanism of action of the chlorophyll derivative 13-2-hydroxypheophytine a on reducing neutral lipid reserves in zebrafish larvae and mice adipocytes.

Obesity is a worldwide epidemic and natural products may hold promise in its treatment. The chlorophyll derivative 13-2-hydroxypheophytine (hpa) was isolated in a screen with zebrafish larvae to identify lipid reducing molecules from cyanobacteria. However, the mechanisms underlying the lipid-reducing effects of hpa in zebrafish larvae remain poorly understood. Thus, investigating the mechanism of action of hpa and validation in other model organisms such as mice represents important initial steps. In this study, we identified 14 protein targets of hpa in zebrafish larvae by thermal proteome profiling, and selected two targets (malate dehydrogenase and pyruvate kinase) involved in cellular metabolism for further validation by enzymatic measurements. Our findings revealed a dose-dependent inhibition of pyruvate kinase by hpa exposure using protein extracts of zebrafish larvae in vitro, and in exposure experiments from 3 to 5 days post fertilization in vivo. Analysis of untargeted metabolomics of zebrafish larvae detected 940 mass peaks (66 increased, 129 decreased) and revealed that hpa induced the formation of various phospholipid species (phosphoinositol, phosphoethanolamine, phosphatidic acid). Inter-species validation showed that brown adipocytes exposed to hpa significantly reduced the size of lipid droplets, increased maximal mitochondrial respiratory capacity, and the expression of PPARy during adipocyte differentiation. In line with our data, previous work described that reduced pyruvate kinase activity lowered hepatic lipid content via reduced pyruvate and citrate, and improved mitochondrial function via phospholipids. Thus, our data provide new insights into the molecular mechanism underlying the lipid reducing activities of hpa in zebrafish larvae, and species overlapping functions in reduction of lipids.

Efficient Green Light Acclimation of the Green Algae Picochlorum sp. Triggering Geranylgeranylated Chlorophylls.

In analogy to higher plants, eukaryotic microalgae are thought to be incapable of utilizing green light for growth, due to the "green gap" in the absorbance profiles of their photosynthetic pigments. This study demonstrates, that the marine chlorophyte Picochlorum sp. is able to grow efficiently under green light emitting diode (LED) illumination. Picochlorum sp. growth and pigment profiles under blue, red, green and white LED illumination (light intensity: 50-200 µmol m-2 s-1) in bottom-lightened shake flask cultures were evaluated. Green light-treated cultures showed a prolonged initial growth lag phase of one to 2 days, which was subsequently compensated to obtain comparable biomass yields to red and white light controls (approx. 0.8 gDW L-1). Interestingly, growth and final biomass yields of the green light-treated sample were higher than under blue light with equivalent illumination energies. Further, pigment analysis indicated, that during green light illumination, Picochlorum sp. formed unknown pigments (X1-X4). Pigment concentrations increased with illumination intensity and were most abundant during the exponential growth phase. Mass spectrometry and nuclear magnetic resonance data indicated, that pigments X1-X2 and X3-X4 are derivatives of chlorophyll b and a, which harbor C=C bonds in the phytol side chain similar to geranylgeranylated chlorophylls. Thus, for the first time, the natural accumulation of large pools (approx. 12 mg gDW -1) of chlorophyll intermediates with incomplete hydrogenation of their phytyl chains is demonstrated for algae under monochromatic green light (Peak λ 510 nm, full width at half maximum 91 nm). The ability to utilize green light offers competitive advantages for enhancing biomass production, particularly under conditions of dense cultures, long light pathways and high light intensity. Green light acclimation for an eukaryotic microalgae in conjunction with the formation of new aberrant geranylgeranylated chlorophylls and high efficiency of growth rates are novel for eukaryotic microalgae. Illumination with green light could enhance productivity in industrial processes and trigger the formation of new metabolites-thus, underlying mechanisms require further investigation.

Climate change influences chlorophylls and bacteriochlorophylls metabolism in hypersaline microbial mat.

This study aimed to determine the effect of the climatic change on the phototrophic communities of hypersaline microbial mats. Ocean acidification and warming were simulated alone and together on microbial mats placed into mesocosms. As expected, the temperature in the warming treatments increased by 4 °C from the initial temperature. Surprisingly, no significance difference was observed between the water pH of the different treatments despite of a decrease of 0.4 unit pH in the water reserves of acidification treatments. The salinity increased on the warming treatments and the dissolved oxygen concentration increased and was higher on the acidification treatments. A total of 37 pigments were identified belonging to chlorophylls, carotenes and xanthophylls families. The higher abundance of unknown chlorophyll molecules called chlorophyll derivatives was observed in the acidification alone treatment with a decrease in chlorophyll a abundance. This change in pigmentary composition was accompanied by a higher production of bound extracellular carbohydrates but didn't affect the photosynthetic efficiency of the microbial mats. A careful analysis of the absorption properties of these molecules indicated that these chlorophyll derivatives were likely bacteriochlorophyll c contained in the chlorosomes of green anoxygenic phototroph bacteria. Two hypotheses can be drawn from these results: 1/ the phototrophic communities of the microbial mats were modified under acidification treatment leading to a higher relative abundance of green anoxygenic bacteria, or 2/ the highest availability of CO2 in the environment has led to a shift in the metabolism of green anoxygenic bacteria being more competitive than other phototrophs.

Potential Application of Photosensitizers With High-Z Elements for Synergic Cancer Therapy.

The presence of heavy elements in photosensitizers (PS) strongly influences their electronic and photophysical properties, and hence, conjugation of PS with a suitable element is regarded as a potential strategy to improve their photodynamic properties. Moreover, PS conjugated to metal ion or metal complex and heavy atoms such as halogen have attracted considerable attention as promising agents for multimodal or synergistic cancer therapy. These tetrapyrrole compounds depending on the type and nature of the inorganic elements have been explored for photodynamic therapy (PDT), chemotherapy, X-ray photon activation therapy (PAT), and radiotherapy. Particularly, the combination of metal-based PS and X-ray irradiation has been investigated as a promising novel approach for treating deep-seated tumors, which in the case of PDT is a major limitation due to low light penetration in tissue. This review will summarize the present status of evidence on the effect of insertion of metal or halogen on the photophysical properties of PS and the effectiveness of various metal and halogenated PS investigated for PDT, chemotherapy, and PAT as mono and/or combination therapy.

Characterization of complex photosynthetic pigment profiles in European deciduous tree leaves by sequential extraction and reversed-phase high-performance liquid chromatography.

Leaf pigments, including chlorophylls and carotenoids, are important biochemical indicators of plant photosynthesis and photoprotection. In this study, we developed, optimized, and validated a sequential extraction and liquid chromatography-diode array detection method allowing for the simultaneous quantification of the main photosynthetic pigments, including chlorophyll a, chlorophyll b, ß-carotene, lutein, neoxanthin, and the xanthophyll cycle (VAZ), as well as the characterization of plant pigment derivatives. Chromatographic separation was accomplished with the newest generation of core-shell columns revealing numerous pigment derivatives. The sequential extraction allowed for a better recovery of the main pigments (+25 % chlorophyll a, +30 % chlorophyll b, +42 % ß-carotene, and 61% xanthophylls), and the characterization of ca. 5.3 times more pigment derivatives (i.e., up to 62 chlorophyll and carotenoid derivatives including isomers) than with a single-step extraction. A broad working range of concentrations (300-2,000 ng.mL-1) was achieved for most pigments and their derivatives and the limit of detection was as low as a few nanograms per milliliter. The method also showed adequate trueness (RSD < 1%) and intermediate precision (RSD < 5%). The method was developed and validated with spinach leaves and their extracts. The method was successfully performed on leaf pigment extracts of European deciduous tree species. Within a case study using Fagus sylvatica L. leaves, pigment derivatives revealed a high within-individual tree variability throughout the growing season that could not be detected using the main photosynthetic pigments alone, eventually showing that the method allowed for the monitoring of pigment dynamics at unprecedented detail.

Zinc-Substituted Pheophorbide A Is a Safe and Efficient Antivascular Photodynamic Agent.

The present study focuses on the photodynamic activity of zinc-substituted pheophorbide a against human endothelial cells. Previously, zinc pheophorbide a has been shown to be a very potent photosensitizer but also a strong albumin binder. Binding to albumin significantly reduces its availability to cancer cells, which may necessitate the use of relatively high doses. Here we show that zinc pheophorbide a is very effective against vascular endothelial cells, even in its albumin-complexed form. Albumin complexation increases the lysosomal accumulation of the drug, thus enhancing its efficiency. Zinc pheophorbide a at nanomolar concentrations induces endothelial cell death via apoptosis, which in many cases is considered a desirable cell death mode because of its anti-inflammatory effect. Additionally, we demonstrate that in comparison to tumor cells, endothelial cells are much more susceptible to photodynamic treatment with the use of the investigated compound. Our findings demonstrate that zinc pheophorbide a is a very promising photosensitizer for use in vascular-targeted photodynamic therapy against solid tumors, acting as a vascular shutdown inducer. It can also possibly find application in the treatment of a range of vascular disorders. Numerous properties of zinc pheophorbide a are comparable or even more favorable than those of the well-known photosensitizer of a similar structure, palladium bacteriopheophorbide (TOOKAD®).

Light-Activated Biomedical Applications of Chlorophyll Derivatives.

Tetrapyrroles are the basis of essential physiological functions in most living organisms. These compounds represent the basic scaffold of porphyrins, chlorophylls, and bacteriochlorophylls, among others. Chlorophyll derivatives, obtained by the natural or artificial degradation of chlorophylls, present unique properties, holding great potential in the scientific and medical fields. Indeed, they can act as cancer-preventing agents, antimutagens, apoptosis inducers, efficient antioxidants, as well as antimicrobial and immunomodulatory molecules. Moreover, thanks to their peculiar optical properties, they can be exploited as photosensitizers for photodynamic therapy and as vision enhancers. Most of these molecules, however, are highly hydrophobic and poorly soluble in biological fluids, and may display undesired toxicity due to accumulation in healthy tissues. The advent of nanomedicine has prompted the development of nanoparticles acting as carriers for chlorophyll derivatives, facilitating their targeted administration with demonstrated applicability in diagnosis and therapy. In this review, the chemical and physical properties of chlorophyll derivatives that justify their usage in the biomedical field, with particular regard to light-activated dynamics are described. Their role as antioxidants and photoactive agents are discussed, introducing the most recent nanomedical applications and focusing on inorganic and organic nanocarriers exploited in vitro and in vivo.

Synthesis and evaluation of novel chlorophyll a derivatives as potent photosensitizers for photodynamic therapy.

Chlorophyll a exhibits excellent photosensitive activity in photosynthesis. The unstability limited its application as photoensitizer drug in photodynamic therapy. Here a series of novel chlorophyll a degradation products pyropheophorbide-a derivatives were synthesized and evaluated for lung cancer in PDT. These compounds have strong absorption in 660-670 nm with high molar extinction coefficient, and fluorescence emission in 660-675 nm upon excitation with 410-415 nm light. They all have much higher ROS yields than pyropheophorbide-a, and compound 10 was even higher than [3-(1-hexyloxyethyl)]-pyrophoeophorbide a (HPPH). Distinctive phototoxicity was observed in vitro and the inhibition effect was in light dose-dependent and drug dose-dependent style. They can effectively inhibit the growth of lung tumor in vivo. Among them, compound 8 and 11 have outstanding photodynamic anti-tumor effects without obvious skin photo-toxicity, so they can act as new drug candidates for photodynamic therapy.

Development of an analytical method for chlorophyll pigments separation by reversed-phase supercritical fluid chromatography.

Chlorophyll pigments give the green colour to plants, which is a quality attribute of food and vegetables. However, the chemical structure of native chlorophyll can change during varied processes (drying, freezing, extraction) applied to plants, which produce degradation compounds that could have a brown and unwanted colour. Systematic experiments have been conducted in supercritical fluid chromatography with a C18 stationary phase to understand and model the chromatographic behaviour of the compounds with respect to the nature of the modifier (MeOH, ACN, and MeOH/ACN 50/50) and its percentage, from 10% to 100%. Specific retention changes were observed, which provide numerous analytical conditions to achieve compound separation. The chromatographic profile of the extract containing native chlorophyll a, b and numerous phytylated chlorophyll derivatives (pheophytin a, a', b, b'; hydroxypheophytin a, a', b, b'; pyropheophytin and lactone derivatives) is strongly impacted by the nature of the modifier and, because of the complexity of the extract, the optimal conditions obtained are unusual for supercritical fluid chromatography. An original method development using an optimization criterion was discussed for the analyses of samples, leading to a fast analytical method with a very low backpressure and a flow rate gradient, but a simplest and rapid method is also suggested for samples displaying fewer derivatives.

A Naturally Derived Carrier for Photodynamic Treatment of Squamous Cell Carcinoma: In Vitro and In Vivo Models.

Photodynamic therapy (PDT) is a non-invasive treatment strategy that includes the combination of three components-a photosensitizer, a light source, and tissue oxygen. PDT can be used for the treatment of skin diseases such as squamous cell carcinoma. The photosensitizer used in this study is the naturally derived chlorophyll derivative chlorin e6 (Ce6), which was encapsulated in ultradeformable ethosomes. Singlet oxygen production by Ce6 upon laser light irradiation was not significantly affected by encapsulation into ethosomes. PDT of squamous cell carcinoma cells treated with Ce6 ethosomes triggered increased mitochondrial superoxide levels and increased caspase 3/7 activity, resulting in concentration- and light-dose-dependent cytotoxicity. Ce6 ethosomes showed good penetration into 3D squamous cell carcinoma spheroids, which upon laser light irradiation exhibited reduced size, proliferation, and viability. The PDT effect of Ce6 ethosomes was specific and showed higher cytotoxicity against squamous cell carcinoma spheroids compared to normal skin fibroblast spheroids. In addition, PDT treatment of squamous cell carcinoma xenografts grown on chorioallantoic membranes of chick eggs (CAM) exhibited reduced expression of Ki-67 proliferation marker and increased terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, indicating reduced proliferation and activation of apoptosis, respectively. The results demonstrate that Ce6-loaded ethosomes represent a convenient formulation for photodynamic treatment of squamous cell carcinoma.

Ethosomes and lipid-coated chitosan nanocarriers for skin delivery of a chlorophyll derivative: A potential treatment of squamous cell carcinoma by photodynamic therapy.

Photodynamic therapy (PDT) is a localized treatment strategy used for skin cancers such as squamous cell carcinoma (SCC), the second most common form of skin cancer. PDT combines a photosensitizer, laser source and tissue oxygen. In this study, the selected photosensitizer, ferrous chlorophyllin (Fe-CHL) was loaded in ethosomes and lipid coated chitosan (PC/CHI) nanocarriers to enhance skin delivery of Fe-CHL for potential PDT of squamous carcinoma. The nanocarrier formulations were characterized and studied for their skin retention and penetration depth of Fe-CHL across mouse skin ex vivo using high performance liquid chromatography and confocal microscopy. Confocal microscope images of mouse skin showed deeper penetration of ethosomes down to the dermis when compared to PC/CHI that was confined to the epidermis, although they showed no significant difference in skin retention. Immunohistochemistry (IHC) staining with ki67 and TUNEL show maintained skin structure and no cytotoxic effects of the nanocarrier gel formulations before laser exposure to mouse skin. The nanocarriers were also studied for their PDT effect against human SCC monolayer and three-dimensional (3-D) spheroids. When compared to ethosomes, PC/CHI showed higher cytotoxicity in MTT assay and live confocal microscopy showed cell disintegration after laser exposure. For 3-D spheroids, PC/CHI also showed higher cytotoxicity using acid phosphatase assay and a decrease in spheroid size was observed using light microscopy. In conclusion, both types of nanocarriers can be used for their potential treatment of SCC using PDT depending on the tumour localization in the skin.