Peptide-Guided Photodynamic Therapy via Integrin αvβ6 in Pancreatic Cancer

TL;DR

This study explores a new photodynamic therapy approach for pancreatic cancer using a peptide that targets a specific receptor on cancer cells.

Contribution

The novelty lies in using a peptide-conjugated photosensitizer targeting integrin αvβ6 to improve PDT specificity for pancreatic cancer.

Findings

The peptide-guided photosensitizer showed uptake and activity in PDAC spheroids.

In vivo results in mouse models showed moderate efficacy despite tumor microenvironment challenges.

Optimization of dosing and models is needed for better clinical translation.

Abstract

Photodynamic therapy (PDT) is a technique based on the use of photosensitizers activated by light to destroy cancer cells in the presence of oxygen. This enables localized cancer treatment and, in some settings, fluorescence-guided visualization. However, the efficacy and clinical translation of PDT have been limited by the low specificity of traditional photosensitizers. The aim of the study is to create a ligand-guided PDT approach for pancreatic ductal adenocarcinoma (PDAC) using a peptide-conjugated photosensitizer binding to integrin αvβ6, which is a receptor linked to tumor growth and prevalent in PDAC cells. Current treatment options for this tumor are limited, with surgical resection and chemotherapy only effective when the tumor is detected early. Given the limited treatment options for PDAC, PDT via αvβ6 offers a new pathway for precision treatment. The cyclic peptide cyclo[FRGDLAFp(NMe)K], recognized for its high affinity to αvβ6, was chosen to guide a phthalocyanine-class photosensitizer toward αvβ6-expressing PDAC models. The PDT approach was further refined by developing 3D spheroid models and in vivo BxPc3 xenograft models in NOD/SCID mice, where its therapeutic efficacy was assessed. In the absence of a non-targeted control photosensitizer, a contribution from non-specific accumulation and EPR effects in the in vivo setting cannot be fully ruled out. This study highlights the potential of a peptide-guided photosensitizer, demonstrating uptake and photodynamic activity in spheroids, with moderate in vivo results addressing tumor microenvironment challenges. Optimization of PDT dosing, laser precision, and preclinical models, such as patient-derived xenografts, are crucial to enhance clinical translation.

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1. Introduction

Pancreatic adenocarcinoma is a highly aggressive cancer, typically diagnosed in individuals over the age of 70. It is one of the leading causes of cancer-related deaths globally, with a stable incidence over the past few decades, particularly in developed countries and among men [1]. The pancreaticoduodenectomy remains the surgical approach with the highest potential for success, although it is typically not an option for patients diagnosed at later stages [2]. Palliative treatment for advanced pancreatic cancer has traditionally involved chemotherapy, with gemcitabine and recently FOLFIRINOX, showing varying survival benefits [3]. However, these treatments highlight the ongoing need for more effective strategies to address this challenging disease. Efforts are focused on improving targeted therapy for pancreatic cancer. Photodynamic therapy (PDT) involves the selective accumulation of a photosensitizing agent in target tissues, which upon activation by light of a specific wavelength, generates cytotoxic reactive oxygen species, predominantly singlet oxygen leading to oxidative damage and subsequent apoptosis or necrosis of the targeted cells. Innovations in advanced photosensitizers and biomedical engineering aim to enhance the delivery and activation of these agents specifically within tumor tissues, improving treatment precision and efficacy [4]. PDT for pancreatic cancer has been tested with various photosensitizing agents (PSs), such as hematoporphyrin derivative (HpD), dihematoporphyrin ether (DHE), and Photofrin. While these agents showed moderate tumor selectivity, they caused significant side effects. Reducing PS doses and shielding the duodenum mitigated some of these issues. Preclinical studies highlight PDT potential, emphasizing the need for selecting optimal PSs and light delivery methods. Targeting specific tumor-associated molecules could improve selectivity, enhancing its therapeutic efficacy and reducing off target effects [5]. Integrin αvβ6 has emerged as a promising target for enhancing the precision in pancreatic ductal adenocarcinoma (PDAC). αvβ6 is a cell-surface receptor that plays a critical role in regulating cellular processes such as adhesion, migration, and invasion, all of which are vital for cancer progression [6]. The expression of the αvβ6 integrin is normally absent or minimally expressed in healthy tissues, while αvβ6 is upregulated during tissue remodeling, particularly in carcinogenesis. This aberrant expression makes αvβ6 an attractive candidate for targeted therapies aimed at improving the specificity of PDT in PDAC [7]. The integrin αvβ6 was exploited by using the highly specific cyclic peptide cyclo[FRGDLAFp(NMe)K] to guide a phthalocyanine-class photosensitizer directly to PDAC tumors. By developing 3D spheroid models and in vivo BxPc3 xenograft models in NOD/SCID mice, we aimed to refine this PDT strategy and evaluate its therapeutic potential [8]. Phthalocyanines, including silicon phthalocyanine (SiPc), constitute a prominent class of photosensitizers characterized by structural resemblance to porphyrins such as protoporphyrin IX (PpIX). Their extended macrocyclic ring system allows absorption at longer wavelengths, facilitating enhanced tissue penetration and improved efficacy in deep-seated photodynamic applications [9]. Silicon phthalocyanines have shown strong photosensitizing efficacy in preclinical and clinical studies. Their absorption in the far-red spectrum (700 nm) enables deeper tissue penetration (up to 1–1.5 cm), with minimal interference from hemoglobin and water, supporting the use of PDT to target residual tumor cells beyond the resection margin [10]. PDAC shows highly infiltrative growth and poorly defined margins. As a result, complete surgical resection is often not possible, and local recurrence is common [11]. PDT offers a strategy to eliminate residual tumor cells at the resection margin [12]. In this study, we developed a silicon phthalocyanine photosensitizer conjugated to dual αvβ6-targeting peptides. Efficacy was assessed both in PDAC spheroids, which replicate critical aspects of the tumor microenvironment, and in an animal model to validate in vivo efficacy.

2. Results

2.1. Synthesis of cyclo[FRGDLAFp(NMe)K]

The synthesis of the cyclic peptide cyclo[FRGDLAFp(NMe)K], previously described in the literature [13], was performed via Fmoc-based solid-phase peptide synthesis (SPPS) on 2-chlorotrityl chloride resin, followed by on-resin cyclization using PyBop/DIPEA. The schematic synthesis protocol of cyclo[FRGDLAFp(NMe)K] is reported in Supplementary Materials (Scheme 1). The cyclopeptide was obtained with a yield of 40% and a purity of 99% (Figure S1).

Supplementary Figure S4 provides additional validation of the preferential affinity of the peptide for αvβ6, as demonstrated by competitive binding assays showing a markedly lower IC_50_ for αvβ6 (22 nM) compared with αvβ3 (14.6 µM).

2.2. Conjugation of the SiPc with 2 cyclo[FRGDLAFp(NMe)K] and Stability of the Final Product

The cyclopeptide was conjugated to SiPc, through the ε- amino groups of the Lys side-chains, reacting SiPc-NHS, prepared following the previously reported method [14], with two equivalents of cyclo[FRGDLAFp (NMe)K] [15] (details in Supplementary Materials). The reaction mixture was purified by reverse-phase HPLC, affording the desired conjugate as a blue powder. The overall yield was 32%, and the final product showed a purity of 96%, as determined by HPLC. The identity of the compound, SiPc–2c[FRGDLAFp(NMe)K], was confirmed by mass spectrometry (Figures S2 and S3).

The optical absorption of the photosensitizer was evaluated in both 5% DMSO/PBS and 5% DMSO/serum solutions. No significant spectral changes were observed in the serum medium over 24 h. This consistent behavior demonstrates that the photosensitizer maintains its optical characteristics in environments representative of physiological conditions (Figure 1).

2.3. Expression of Integrin αvβ6 in PDAC Cells and Spheroids

High expression of the transmembrane protein αvβ6 in the BxPc3 cell line was confirmed by flow cytometry and western blot (Figure 2a,b).

In contrast, integrin αvβ6 was remarkably absent in both MiaPaca2 and Panc1 cell lines, which were then used as negative controls.

The observed differences in integrin expression highlight the importance of αvβ6 as a biomarker in pancreatic carcinoma. This pattern specifically supports the selection of BxPc3 and Capan1 as appropriate in vitro models for investigating integrin-directed therapeutic strategies and emphasizes the role of αvβ6-related signaling in tumor progression. Based on these findings, BxPc3 was chosen for subsequent photodynamic therapy studies.

To further characterize the cellular system, three-dimensional spheroid cultures were generated from BxPc3 and MiaPaca2 cell lines, which exhibit high and low levels of integrin αvβ6 expression, respectively. (Figure 3a).

Representative confocal images of BxPc3 spheroids are shown in Figure 3b,c, while images of MiaPaca2 spheroids are not shown. Semi-quantitative analysis revealed an increase in fluorescence intensity upon drug treatment. Specifically, comparison between treated and untreated spheroids imaged under identical conditions showed an approximately twofold increase in signal (treated-to-untreated ratio of 1.86), indicating enhanced probe accumulation following treatment.

In addition, fluorescence quantification using a standard signal-to-background ratio (SBR) definition, in which the background was measured in spheroid-free regions, yielded an SBR of approximately 5.7 for treated spheroids, indicating clear signal emergence relative to background (Figure S6). Taken together, these complementary metrics reflect both the magnitude of signal increase induced by treatment and the extent to which the probe signal is distinguishable from background. In 3D spheroid models, fluorescence-based measurements should be interpreted as conservative, semi-quantitative indicators of probe accumulation.

2.4. Photodynamic Therapy on PDAC Spheroids

Spheroids composed of BxPc3 cells subjected to full photodynamic treatment (photosensitizer plus laser irradiation) exhibited evident loss of structural integrity and partial cell death, as observed by bright-field microscopy (Figure 4a–d). In contrast, spheroids exposed to control conditions (drug only or laser only) retained a compact and well-defined morphology comparable to untreated controls. Consistent with these morphological observations, photodynamic treatment resulted in a cytotoxicity level of approximately 30%, which was significantly higher than all control conditions (Figure 4e).

Semi-quantitative morphological scoring further highlighted treatment-dependent differences in spheroid integrity. Control spheroids were predominantly classified as intact and compact (score 0), whereas photodynamically treated spheroids displayed increasing degrees of disintegration, ranging from partial loss of compactness to pronounced structural deformation and fragmentation, corresponding to higher morphological scores.

Since the data obtained from the three-dimensional model was promising, the study progressed in vivo, where a xenograft model using the BxPc3 cell line in NOD/SCID mice was established.

2.5. Photodynamic Therapy on Xenograft Model

In Figure 5a, a slowdown in tumor volume growth is observed in the data shown in red, which corresponds to animals that received high-dose PDT (116 J/cm^2^), compared to all other groups, including control animals and those treated with low-dose PDT.

When analyzing individual animal data with the exponential growth model as shown in Figure 5 (y = y_0_e^kx^), the high-dose PDT treatment group showed a marked reduction in tumor growth rate compared to other groups. This finding (Figure 5b,c) is reflected in the parameters K (the exponential growth rate of tumor volume) and Tau (the characteristic growth time of the tumor).

The growth rate K, measured in days^−1^ indicates how quickly the tumor volume expands. Higher K values mean faster growth, as more tumor volume is added each day.

The characteristic growth time Tau, in days, represents the time it takes for the tumor volume to increase by a factor of e (about 2.718). A smaller Tau suggests faster growth, while a larger Tau indicates slower tumor progression.

In the high-dose PDT group, the K value is lower, and the Tau value is higher, indicating a slower tumor growth rate compared to other groups. This pattern suggests that high-dose PDT effectively slows tumor progression by extending the time needed for the tumor to grow by a factor of e. The reduction in growth rate highlights the significant therapeutic impact of high-dose PDT in tumor expansion.

Importantly, no qualitative evidence of off-target effects was observed, and representative images of control animals included in Supplementary Figure S5 show no detectable signal accumulation or macroscopic alterations in non-target tissues under the experimental conditions used.

Regarding animal welfare assessment, it was noted that, among all animals included in each experimental group, one animal in the laser-irradiated group and three animals in the group receiving drug administration followed by complete irradiation (PDT) exhibited a transient body weight loss of 6–10% in the days immediately following treatment; in all cases, body weight was rapidly regained, with no evidence of persistent adverse effects or prolonged distress.

2.6. Ex Vivo Analysis

As already reported in literature [15], ex vivo analysis of tumor tissues stained with hematoxylin and eosin staining revealed similar results across the different treatment groups, showing a tumor morphology which is characteristic of pancreatic adenocarcinoma with abundant stromal component and traces of mucin (Figure 6a,b).

The tumor masses were well vascularized, with the endothelial area accounting for 1.9 ± 0.45% of the total tumor area. A reduction in the intensity and distribution of the proliferation marker Ki67 was observed qualitatively in tumor sections from mice treated with the compound and subjected to PDT, compared with untreated controls, where Ki67 staining was more diffuse throughout the tumor and more intense at the periphery.

Expression of integrins αvβ3, αvβ5, and αvβ6 was performed by Western Blot analysis on tumor tissues. The aim was to better characterize the model and understand the involvement of these integrins in the photosensitizer activity. Tumor tissues from animals in the different groups were compared with cells pellet to assess the expression levels on both cells and tissue, respectively β3, β5, and β6 (Figure 7).

It has to be stated that the presence of β3 and β5 integrins in the analyzed tumor tissues prevents a definitive determination of whether the photosensitizer, which was designed to engage β6, binds exclusively to this integrin subtype [16]. Moreover, the stromal components which constitute the extracellular matrix and where integrins are high expressed are still present after tumor digestion from animal models. In summary, detection of integrins within stromal compartments is expected and physiologically normal; it does not compromise the assessment of treatment specificity. The presence of other integrin subtypes in the tumor microenvironment is a physiological feature and does not, by itself, limit the specificity of targeting [17].

3. Discussion

This study demonstrates the activity of the photosensitizer in tumor spheroids, confirming the value of three-dimensional models for assessing drug efficacy and resistance. The presence of multiple integrins and stromal components reflects the physiological heterogeneity of the tumor microenvironment while preserving the specificity of the ligand-guided photosensitizer [18].

The need to develop a selective and specific probe with a high soluble photosensitizer drove the design of the molecule used in this study. Additionally, the compound was designed to exhibit high selectivity for the integrin αvβ6, which is known to be a promising target for pancreatic adenocarcinoma treatment [19]. This design was achieved by synthesizing a cyclic nonapeptide containing the RGD sequence, which was then conjugated to a silicon phthalocyanine, improving its water solubility and enhancing its stability, optical properties, and serum compatibility [20]. The conjugation of both axial positions of silicon phthalocyanine with cyclic peptides, above and below the plane, helped overcome aggregation issues typical of hydrophobic photosensitizers, making it a more viable candidate for therapeutic applications [21]. Silicon(IV) phthalocyanines constitute a well-documented class of photosensitizers, and the preservation of the phthalocyanine core ensures retention of their established photophysical properties, including strong absorption in the therapeutic window and efficient singlet oxygen generation.

Regarding the expression of the integrins, theBxPc3 cell line confirmed the expected levels of integrin β3 and β6 among the studied pancreatic cancer lines. The drug was internalized by the BxPc3 spheroids, which highly express αvβ6. The photosensitizer demonstrated a certain level of activity at the highest treatment dose [22]. Consistently, semi-quantitative fluorescence analysis in BxPc3 spheroids showed an increase in signal upon treatment and a clear separation from background, supporting effective probe accumulation in a 3D tumor model, where optical attenuation and limited penetration inherently constrain contrast.

Silicon(IV) phthalocyanines constitute a well-documented class of photosensitizers, and the preservation of the phthalocyanine core ensures retention of their established photophysical properties, including strong absorption in the therapeutic window and efficient singlet oxygen generation [23,24].

The use of BxPc3 pancreatic cancer spheroids in this study provides a more physiologically relevant model of pancreatic adenocarcinoma, closely mimicking in vivo features such as dense ECM, complex spatial organization, and gradients of oxygen and nutrients [25]. Three-dimensional spheroid models effectively recapitulate the desmoplastic stroma, ECM composition (including collagen I, fibronectin, hyaluronic acid, and laminin) and cellular heterogeneity observed in patient tumors, aspects that are largely absent in conventional two-dimensional cultures [26].

In vitro results on BxPc3 spheroids highlight the potential of αvβ6-directed photodynamic therapy (PDT) in pancreatic cancer. The photosensitizer induced significant cytotoxicity (30%), supporting preferential therapeutic activity in 3D models. The maintenance of viability in spheroids larger than 300 μm, without necrotic cores prior to treatment, indicates adequate oxygenation for PDT activation, suggesting that this experimental platform realistically evaluates photosensitizer penetration, oxygen distribution, and cellular response, providing predictive insights for in vivo applications [27]. Moreover, the integrin-directed photosensitizer demonstrated selective cytotoxicity, with minimal activity on αvβ6-negative cells, highlighting its potential to reduce off-target toxicity and support clinical applications in tumors with high αvβ6 expression [28]. These findings further validate the use of spheroid models for preclinical screening and for guiding optimization strategies aimed at improving photosensitizer penetration, distribution, and treatment selectivity.

Qualitative assessments of photosensitizer uptake and cytotoxicity in BxPc3 pancreatic cancer spheroids, even when precise subcellular localization is limited by imaging constraints, highlight the barriers to effective drug penetration in fibrotic tumors. The dense extracellular matrix and abundant stromal content characteristic of these tumors restrict diffusion and lead to heterogeneous photosensitizer distribution, resulting in variable photodynamic responses. This heterogeneity aligns with the moderate in vivo efficacy observed, despite pronounced in vitro cytotoxicity, and reflects the known impact of stromal desmoplasia on drug delivery and therapeutic outcomes in PDAC models [29]. This highlights the challenges in drug delivery, particularly in fibrotic tumors like pancreatic adenocarcinoma, which may limit the penetration of therapeutic agents [30].

The increased expression of integrin αvβ5 and possible involvement of β3, driven by stromal components, further complicates the interpretation of targeting specificity and therapeutic effect. While probes designed to interact with αvβ6 demonstrate integrin-associated effects in vitro and in vivo, the presence of other integrin subtypes in the tumor microenvironment raises the possibility of off-target interactions or compensatory mechanisms that may influence treatment response [31]. Therefore, further competition experiments in vitro and in vivo are necessary to better understand the full range of integrin interactions contributing to the observed effects.

The in vivo photodynamic therapy experiments demonstrated that administration of a high PDT dose slowed tumor growth, confirming effective delivery of the photosensitizer to the tumor, its successful activation, and its therapeutic action within the well-oxygenated tumor microenvironment. The finding that tumor masses became softer 3–5 days after PDT, confirming a photodynamic reaction but without histological evidence of necrosis, should be interpreted as evidence of effective stromal and extracellular matrix remodeling rather than direct tumor cell ablation. In pancreatic adenocarcinoma, the dense fibrotic stroma is a major barrier to drug delivery and therapeutic efficacy [32]. PDT-induced softening of tumor tissue reflects photochemical disruption of stromal components, such as collagen and fibroblasts, which can enhance subsequent drug penetration and may sensitize tumors to further therapy, even in the absence of overt necrosis [33].

PDAC tumors often pose a challenge to drug penetration due to their dense, stromal nature [34]. However, the high binding affinity of the PS for αvβ6, combined with the RGD sequence in the peptide, which allows binding to other integrins, likely contributed to its greater uptake at the tumor site. Accordingly, in the absence of a direct non-targeted comparator, the in vivo photodynamic response is interpreted as consistent with an αvβ6-directed delivery, rather than as definitive evidence of receptor-mediated targeting.

Definitive confirmation of receptor-mediated targeting in vivo will require additional experiments including a non-targeted or scrambled control compound; while this was not feasible within the present study design, animal numbers were deliberately minimized in accordance with ethical considerations, and such controls are recognized as an important priority for future investigations. In fact in the absence of a non-targeted control photosensitizer, the contribution of non-specific accumulation and EPR effects cannot be fully excluded in the in vivo setting.

In this context, the use of a subcutaneous xenograft model in the present study allowed a controlled and reproducible evaluation of molecular targeting and light–drug interactions, while minimizing confounding variables related to complex tumor architecture, surgical procedures, and heterogeneous optical delivery that are inherent to more advanced PDAC models.

The lower cytotoxic effect observed in vivo relative to in vitro is an expected and widely reported phenomenon in PDT, reflecting physiological barriers such as limited photosensitizer bioavailability, tumor hypoxia, and reduced effective light dose in living tissues. However, the molecular targeting and delivery efficiency in translational settings are crucial, where photosensitizer accumulation, oxygenation, and light availability collectively can determine therapeutic response.

In addition to these considerations, the future need emerges to create different setups for photodynamic therapy dosing, starting from the high dose used in this study and increasing it, or by evaluating repeated treatments over time [35]. The advantage of repeated treatments would be to gradually target the tumor mass, making it progressively less intact and thus more responsive to the therapy [36].

High-dose PDT increases reactive oxygen species generation, but its effectiveness is limited by hypoxia and dense stroma [37]. Strategies to overcome hypoxia include co-delivery of oxygen-generating agents (e.g., perfluorocarbons, microbubbles) and nanoplatforms that locally release oxygen or modulate tumor metabolism to reduce oxygen consumption, thereby improving ROS yield and cytotoxicity [38]. The development of this αvβ6-directed phthalocyanine conjugate represents a promising first step toward selective photodynamic therapy, as it demonstrates effective stromal remodeling, enhanced drug penetration, and targeted cytotoxicity in 3D pancreatic cancer models [39].

4. Materials and Methods

4.1. Chemical Synthesis

The cyclic peptide cyclo[FRGDLAFp(NMe)K] was synthesized via Fmoc-based SPPS with on-resin cyclization and final deprotection. Peptide synthesis was performed on 2- 2-chlorotrityl chloride resin (Sigma-Aldrich, Milan, Italy substitution grade: 1.0–1.5 mmol/g). Fmoc-Asp-OAll (1.2 equiv) was loaded onto the resin in dry DCM using DIPEA (Sigma-Aldrich, Milan, Italy) (3 equiv). Then peptide was elongated by double coupling Fmoc-amino acids (2 eq) using HOBt/PyBop (2 eq) (Sigma-Aldrich, Milan, Italy) as coupling agents. Fmoc deprotection was performed by 20% piperidine in DMF with 0.1 M HOBt. After peptide elongation, the OAll protecting group was selectively removed using Pd(PPh_3_)4/phenylsilane (Sigma-Aldrich, Milan, Italy) under inert conditions. Head-to-tail cyclization was then performed on resin using PyBOP/DIPEA (Sigma-Aldrich, Milan, Italy).

The cyclopeptide was cleaved from the resin using a mixture of trifluoroacetic acid (TFA), water, and triisopropylsilane (TIS) for 3 h. The crude product was analyzed by UPLC (Waters ACQUITY UPLC, Milford, CT, USA) on a BEH C18 column (Waters, 2.1 × 50 mm^2^, 1.7 μm particle size) with a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (TFA), from 5% to 95% acetonitrile, at a flow rate of 0.5 mL/min and detection at 220 nm. The pure cyclopeptide was analyzed by UPLC-MS (Waters ACQUITY UPLC) using a BEH C18 column (Waters, Milford, CT, USA 2.1 × 50 mm^2^, 1.7 μm particle size) and with a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (TFA), from 5% to 95% acetonitrile, at a flow rate of 0.5 mL/min and detection by ESI-MS in positive ion mode. Purity (99%) was estimated by integration of the area of the chromatogram peaks revealed at 220 nm. Peptide identity was verified by mass spectrometry. MS (ESI^+^): m/z calculated for C_51_H_75_N_12_O_9_ [M + H]^+^ = 1046.58, found 1046.93; [M + 2H]^2+^ =523.79, found 524.14.

SiPc was conjugated to two molecules of cyclo[FRGDLAFp(NMe)K] via NHS chemistry. A solution of synthetized SiPc-NHS [14] 10 mg, 0.0075 mmol) and triethylamine (TEA, 9.5 µL, 0.068 mmol) in 10 mL of DMF was added to a solution of cyclo[FRGDLAFp(NMe)K] (10 mg, 0.00956 mmol) in 10 mL of DMF. The reaction mixture was stirred at room temperature for 2 h. After completion, the crude product was precipitated and washed three times with ethyl ether. The resulting solid was purified by preparative HPLC using a Waters XTerra™ Prep RPdC8 19/100 column, with a gradient elution of acetonitrile in 0.1 M ammonium acetate buffer (pH 7), increasing from 30% to 80% acetonitrile over 15 min at a flow rate of 20 mL/min. A blue powder was obtained in 32% yield with a purity of 96%. MS (ESI^+^): m/z calculated for MS (ESI+): m/z calculated for C_162_H_212_N_38_O_28_Si, [M + 2H]^2+^ =1583.80, found 1583.76, [M + 3H]^3+^ = 1056.20, found 1056.34.

4.2. Optical Characterization of the Photosensitizer SiPc-2c[FRGDLAFp (NMe)K]

The absorbance of the photosensitizer was measured using a quartz cuvette (Hellma, Mullheim, Germany, 1 cm path length) and a UV/VIS spectrophotometer (PerkinElmer, Waltam, MA, USA Lambda 35). For the analysis, a 4 µM solution of the compound was prepared in two different solvents: 5% DMSO in PBS and 5% DMSO in serum (fetal bovine serum, FBS, Gibco, ThermoFisher, Waltham, MA, USA). The solution in serum was analyzed at multiple time points (2, 4, 6, and 24 h) to evaluate the stability of the compound over time.

4.3. PDAC Cell Cultures

The human pancreatic adenocarcinoma cell lines BxPc3 and Capan1 were selected as αvβ6 integrin positive cells. In contrast, MiaPaca2 and PANC1, known from the literature to express low levels of αvβ6, were used as negative controls. Capan1, MiaPaca2, PANC1 and BxPc3 cells were purchased from ATCC (Manassas, VA, USA)

All cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The absence of mycoplasma contamination was confirmed using the EZ-PCR Mycoplasma Test Kit (Sartorius, Gottingen, Germany). Cells were maintained in T175 flasks and passaged at a maximum dilution of 1:4 to ensure optimal growth for both in vitro and in vivo studies.

4.4. αvβ6 and αvβ3 Expression by Flow Cytometry

Expression of αvβ6 and αvβ3 was analyzed in different cell lines in order to investigate the selective affinity of the peptide for integrins. For each condition, 5 × 10^5^ cells per tube were incubated at 4 °C for 1 h in DMEM supplemented with 0.1% BSA and 1 mM Mg^2+^. The primary antibody was applied at a 1:50 dilution, followed by a 45 min incubation with a secondary antibody at a 1:100 dilution.

For αvβ6 detection, the primary antibody used was Anti-Integrin αvβ6 Mouse Monoclonal (MAB2077Z, Millipore, Darmstadt, Germany clone 6A2987), and the secondary antibody was Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Catalog #A-21131, Invitrogen, Carlsbad, CA, USA).

To assess αvβ3 expression in BxPc3, PANC1, MiaPaca2, and Capan1 cells, the same protocol was followed using PBS with 0.1% BSA as the incubation buffer. The primary antibody for αvβ3 was a directly conjugated Human Integrin αvβ3 Alexa Fluor^®^ 488 Antibody (Monoclonal Mouse IgG1, Clone 23C6, Catalog # FAB3050G, ReDSystem, a Bio-Techne, Minneapolis, MN, USA brand), followed by a secondary antibody labeled with Alexa Fluor 647.

Flow cytometry was performed using a BD Celesta FACS instrument (BD Biosciences, San Jose, CA, USA) to quantify integrin expression across the cell lines.

4.5. αvβ6 and αvβ3 Expression by Western Blot

Cell lysates were prepared by first collecting cell pellets for western blot analysis. The culture medium was removed, and cells were rinsed with cold PBS, then detached using a non-dissociating solution, inactivated with fresh medium, and centrifuged at 11,000 rpm for 5 min at 4 °C. Approximately 2.5 × 10^6^ cells were isolated, re-centrifuged at 1100 rpm, and stored at −80 °C until protein extraction. U87MG and HT29 cell lines were used as controls, serving as positive and negative references for integrin expression. U87MG cells express β3 integrin but lack β6, whereas HT29 cells express β6 integrin but not β3 [40]. These controls were included to validate the specificity of integrin detection and to compare integrin profiles across experimental cell lines.

For protein extraction, frozen cell pellets were resuspended in a minimal volume of lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 1 mM MgCl_2_, 1% Triton X-100, 10% glycerol) supplemented with 1 mM PMSF and a protease inhibitor cocktail. A PBS-based solution containing SDS and Triton X-100 was used to enhance cell lysis. Samples were vortexed for 30 min, sonicated, and centrifuged at 13,200 RPM for 20 min at 4 °C. The supernatants were collected and stored at −80 °C for further analysis. Protein concentration was determined using the BCA (Bicinchoninic Acid) assay.

Western blot analysis was performed on BxPc3, PANC1, and MiaPaca2 cell lysates, with U87MG and HT29 serving as controls for αvβ3 and αvβ6 integrins, respectively. Primary antibodies included Anti-Integrin αvβ3 (Integrin β3 (D7X3P) XP^®^ Rabbit mAb, Cell Signaling) at a 1:500 dilution and Anti-Integrin αvβ6 (clone EM05201, ZooMAb^®^ Rabbit Monoclonal ZRB1104) at a 1:1000 dilution, incubated overnight at 4 °C. The secondary antibody, a rabbit anti-IgG HRP-conjugated antibody, was used at a 1:15,000 dilution in 5% BSA in TBST and incubated for 1 h at room temperature. Protein transfer was carried out on PVDF membranes pre-activated in methanol to ensure effective protein binding and signal development. Bands were visualized using the ChemiDoc imaging system and analyzed with Image Lab software 6.1.

4.6. Formation and Imaging of BxPc3 and MiaPaca2 Spheroids

A total of 1 × 10^3^ BxPc3 cells and 1 × 10^3^ MIAPaCa-2 cells were seeded per well in a 96-well spheroid microplate (Corning, NY, USA REF 4515; black plate with a clear, round, ultra-low attachment bottom) using complete RPMI medium. Spheroid formation was monitored over a 7-day period, and spheroid diameter was measured using a spinning disk Tie 2 microscope (Nikon, Tokyo, Japan) in bright-field mode.

Both BxPc3 and MIAPaCa-2 cells formed compact spheroids within 24 h post-seeding. Spheroids were then incubated with the photosensitizing compound for 2 h, followed by a PBS wash. Subsequently, they were stained with DAPI (to label nuclei) and WGA (Wheat Germ Agglutinin, to label the plasma membrane) and imaged using a confocal microscope at the University of Turin laboratories.

Spheroid diameter was measured over time. For imaging, spheroids were stained with DAPI and WGA to label nuclei and cell membranes, respectively, and visualized by confocal microscopy. Due to the limited light penetration depth of confocal microscopy, acquired images represent only a semi-superficial layer of the spheroids. Semi-quantitative image analysis was performed using ImageJ 1.5. For each image, regions of interest (ROIs) were manually drawn within the spheroid to measure signal intensity and in adjacent spheroid-free areas to determine background intensity. Mean intensity values were extracted using the “Analyze” and “Measure” functions in ImageJ, and the signal-to-background ratio (SBR) was calculated as the ratio of mean signal intensity to mean background intensity.

4.7. PDT Laser Equipment and Laser Fluence Parameters

The FC-650/450 mW laser operated at a wavelength of 650 nm ± 5 nm in continuous wave mode, delivering over 450 mW of power through a 400 µm fiber core with an SMA905 connector and 1 m fiber length. Stable power output was maintained within a 5% RMS (Root Mean Square, power) variation over 4 h. Laser warm-up time was under 5 min. Laser operated within a temperature range of 10–40 °C and supported modulation options from 1 Hz to 30 kHz. Power (measured in watts, W) is essential for effective tissue penetration in laser therapy, alongside wavelength. Higher power allows for deeper photon penetration and shortens treatment duration by delivering more photons efficiently, according to the formula E = P × t, where energy (E) is a function of power (P) and time (t). An adjustable arm was used to hold the fiber in place, enabling precise control over power output, sample distance, and irradiation time for optimal treatment outcomes.

Regarding PDT irradiation parameters for in vitro spheroids were incubated for 2 h with the photosensitizer in the dark and then irradiated with the FC-650 laser (Changchun New Industries optoelectronics, Changchun, China) (650 nm, nominal output 450 mW, 400 µm fiber). The fiber tip was positioned 6 cm above the plate, the power density at the spheroid surface was 100 mW/cm^2^. Spheroids received a total energy density (fluence) of 40 J/cm^2^, corresponding to 400 s of irradiation.

Regarding PDT irradiation parameters for in vivo tumors each mouse received 10 nmol Silicon Phthalocyanine-2c[FRGDLAFp(NMe)K] intravenously. PDT was performed 4 h later using a laser beam expanded to a 1 cm^2^ spot covering the tumor. The power density at the tumor surface was 150 mW/cm^2^. Animals were assigned to two groups receiving different energy densities (fluence): 116 J/cm^2^ (irradiation time 773 s) and 56 J/cm^2^ (irradiation time 373 s).

4.8. Photodynamic Therapy on BxPc3 Spheroids

After a 2 h dark incubation with the photosensitizer, the spheroids were irradiated with a 650 nm laser (model FC-650, 450 mW output power, fiber core diameter 400 µm) at a dose of 40 J/cm^2^. The plate was then returned to the incubator for 24 h. Following this incubation, the photodynamic effect was assessed using a Spinning Disk microscope (Nikon, Tokyo, Japan) in bright-field mode, and a fluorescence-based cytotoxicity assay was performed to evaluate treatment efficacy (CyQUANT™ LDH Cytotoxicity Assay—Fluorescence Kit. (Catalog Nos. C20302 and C20303). Pub. No. MAN0018656 Rev. A.0.

The CyQUANT LDH Cytotoxicity Assay measures cytotoxicity based on lactate dehydrogenase (LDH) release. LDH is a cytosolic enzyme released into the culture medium when the cell membrane is damaged. In this assay, LDH catalyzes the conversion of lactate to pyruvate, resulting in the production of NADH. Subsequently, NADH reduces resazurin to resorufin, a highly fluorescent compound, which can be quantified by measuring fluorescence (excitation at 560 nm, emission at 590 nm).

The level of fluorescence directly correlates with the amount of LDH released, providing an indication of cytotoxicity.

The following formula was used to determine cytotoxicity percentage:

$$[eqn]$$

where Spontaneous LDH activity is the baseline LDH release in untreated cells, and the Maximum LDH activity corresponds to total LDH release following complete cell lysis. The term Compound-treated LDH activity represents the LDH released from cells treated with the test compound. This formula yields the percentage of cells with membrane damage attributed to the treatment, providing a measure of treatment-induced cytotoxicity.

Spheroid structural integrity was evaluated using a semi-quantitative morphological scoring system based on brightfield and fluorescence microscopy images. Spheroids were classified according to a four-level scale (0–3), where 0 corresponds to intact, compact spheroids with smooth and well-defined borders; 1 to mild disintegration characterized by border irregularities and partial loss of compactness; 2 to moderate disintegration with evident structural deformation and partial fragmentation; and 3 to severe disintegration marked by loss of spheroidal architecture and extensive fragmentation [41].

Morphological scoring was used as a qualitative–semiquantitative complement to LDH-based cytotoxicity measurements to provide structural context to biochemical evidence of cell damage. Consistent with this approach, previous studies have employed image-based morphometric analyses to characterize spheroid structural alterations following photodynamic therapy, supporting the use of morphological metrics as a valid complement to biochemical assays.

4.9. Animal Study

Procedures were performed according to the national and international laws on animal research (L.D. 26/2014; Directive 2010/63 EU) and in accordance with ARRIVE guidelines. Animal studies were approved by Italian Ministerial authorizations under the project 27/2024-PR. Female NOD/SCID mice were obtained from Envigo, Italy. At arrival, during the acclimation period (which lasted 5 days) and experimental procedures animals were housed in polysulfone Tecniplast cages type III (up to 4 animals/cage).

During the entire period of the study the animals were maintained in conditioned and at limited access environments (mean temp: 22 °C; mean relative humidity: 55%; controlled light to give a daily 12h photoperiod). Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.

Other items were included to enrich the cage environment, such as clipper homes, suspended tunnels, chocolate mini treats and sunflower seeds.

Animal model setup: study involved 32 female NOD/SCID mice (5 weeks old at arrival), obtained from Envigo, Italy. This immunodeficient mouse model was selected for its suitability in xenograft studies of human pancreatic cancer.

• Experimental Design

In this study BxPc3 cells were washed with serum-free medium, resuspended in 0.1 mL of serum-free medium, and injected into the right flank of 6-week-old mice under sevoflurane anesthesia.

NOD/SCID mice were subcutaneously inoculated with BxPc3 cells and divided into four groups (n = 8 per group): a high-dose PDT group, a low-dose PDT group, a drug control, and a laser control. Control experiments were designed to isolate the photodynamic contribution of the photosensitizer by including irradiation-only and photosensitizer-only conditions, based on extensive prior evidence that non-targeted silicon phthalocyanines exhibit limited in vivo efficacy due to nonspecific distribution and rapid clearance. Each tumor-bearing animal received 1.5 × 10^6^ BxPc3 cells, and tumor volumes were measured three times a week to track growth. Treatments began when tumor volumes reached 60–90 mm^3^, prior to the onset of exponential growth.

• Drug and laser treatment

Each animal received an intravenous dose of 10 nmol of Silicon Phthalocyanine-2c[FRGDLAFp(NMe)K] in a solution of PBS containing 10% DMSO. PDT treatment was conducted 4 h after drug administration. Group 1 received laser irradiation at the dose of 116 J/cm^2^, while Group 2 received irradiation at the dose of 56 J/cm^2^. Groups 3 and 4 served as controls, receiving only the drug or laser treatment, respectively.

For the laser treatment, according to the experimental schedule, mice were anesthetized with sevoflurane, and the tumor area was shaved to allow laser exposure. The tumor site was exposed to laser light. Post-irradiation, tumor progression was monitored for up to 60 days or until tumors reached 800 mm^3^. Tumor volumes were measured with caliper, and humane endpoints were applied as necessary. After treatment, animals from group 1 and 2 received Buprenorphine s.c. (0.05 mg/kg) twice per day (every 8–10 h), for three days.

Moreover, animals from all the groups were offered modified water with Buprenorphine 0.5 mg/kg, from the irradiation day, for 7 days.

• Monitoring of tumor growth

The tumor growth was monitored three times per week, starting from day 7 post-inoculation, using caliper measurements. Tumor volume was calculated using the formula: (L × W^2^)/2, where L is the longest dimension and W is the shortest.

Mice were euthanized upon reaching predefined humane endpoints: ≥20% body weight loss, tumor volume ≥ 1.5 cm^3^, tumor ulceration, visible signs of severe or sustained distress, tail necrosis, or at 60 days post-tumor induction.

Tumor growth kinetics were analyzed by nonlinear regression using GraphPad Prism (GraphPad Software 10). Tumor volume measurements from each individual animal were plotted as a function of time and fitted separately using a single-phase exponential growth model defined by the equation y = y_0_e^kx^, where y denotes the tumor volume at time x, y_0_ represents the estimated tumor volume at the initial time point, and k is the exponential growth rate constant. Model fitting was performed using nonlinear least-squares regression (ordinary least squares).

4.10. Histological Analysis and Protein Expression

Tumor tissues were excised and processed into frozen OCT. Sections of 10 µm were prepared from anterior, middle, and posterior regions of the tumors and stained with standard Hematoxylin and Eosin (H&E) staining. For the evaluation of tumor vasculature and proliferation index, the immunofluorescence (IF) staining of the endothelial marker CD105 and Ki67, respectively, were carried out.

Briefly, air dried-tumor sections were fixed acetone for 10 min at RT. Sections were then washed in PBS and permeabilized in 0.3% Triton X100 for 15 min at RT. After incubation with 3% BSA in PBS blocking buffer for 1 h at RT, sections were directly stained overnight at 4 °C with either the monoclonal rat anti-CD105 antibody (Abcam) or rabbit anti-Ki67 antibody (Abcam) diluted in 1% BSA in PBS. Sections were washed and further stained with anti-rat or -rabbit AlexaFluo555-conjugated secondary antibodies (Life Technologies) for 1 h at RT. After three washes, sections were counterstained with DAPI (Life Technologies) and mounted with aqueous mounting medium (Dako). Images were acquired on ScanScope FL (Aperio, Leica Microsystems) and image analysis was performed with the use of ImageJ software. CD105 positive area was calculated as a percentage of the tumor area and expressed as mean ± SD of the percentage calculated on 3 stained sections.