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Italians appear to be snubbing beaches this summer, amid claims they are rebelling against the high prices charged by the owners of private beach concessions.

Going to the beach and renting cabins, loungers and parasols – usually at the same location – has long been an ingrained habit of Italian summer holiday culture.

But this year’s season began with a notable fall in beachgoer numbers after private resorts along Italy’s two long stretches of coastline recorded a decrease of between 15% and 25% in June and July compared with the same period in 2024.

The problem is not so much the weekend, when beach resorts are often congested, especially those close to cities such as Rome, but during the week. Those who do go are also spending less on food and drink.

Fabrizio Licordari, the president of Assobalneari Italia, an association representing beach clubs, blamed the decline on the high cost of living and its consequences on spending power.

“Even with two salaries, many families struggle to reach the end of the month,” he told Ansa news agency. “In such circumstances, it’s natural that the first expenses to be cut are those for leisure, entertainment and holidays.”

The drop in attendance, however, also coincides with increases in the cost of private beach resorts and the growing rebellion against their dominance of Italian shorelines, which has left very little space for free beaches.

The cost of renting a sunlounger is a recurring topic of discussion, and rightly so – on average, it costs 17% more than it did four years ago, according to figures this week from the consumer group Altroconsumo. On beaches in the Lazio region, for example, it is difficult to rent two loungers and an umbrella for less than €30 (£26) a day. That rises to about €90 in the popular resort of Gallipoli in Puglia.

The actor Alessandro Gassmann stoked the debate after sharing a photo of a beach with deserted loungers on his Instagram page and writing alongside it: “I read that the season is not going well. Maybe it’s because the prices are exaggerated and the country’s economic situation is forcing Italians to choose free beaches? Lower the prices and maybe things will get better.”

Maurizio Rustignoli, the president of Fiba, the Italian beach resorts federation, argued that reports of high price rises were “misleading” and that, where they occurred, it was by only a small percentage. He added that people in return benefited from services including security and lifeguard supervision.

But the consumers association Codacons said going to beach resorts had become “a drain” on people’s finances and accused the concession owners of “shedding crocodile tears”.

The beaches might be losing custom, but areas in the mountains, especially the Dolomites, have had a significant rise in visitor numbers, with some areas fearing overtourism. According to a report this week in the newspaper Il Messaggero, more Italians are venturing to the mountains for their holidays, partly as a way to escape increasingly hot summers caused in part by the climate crisis.

The Ivy faces legal action from a waiter who claims he was refused details about how the upmarket restaurant chain calculated his share of tips and service charges, despite a new law aimed at ensuring they are shared more fairly and transparently.

The waiter, who asked not to be identified, quit the Ivy in June, claiming that he had been given an “totally unfair” share of tips and service charges, and is claiming constructive dismissal against the Ivy’s owner at an employment tribunal.

The Ivy said it “absolutely refute[s]” the waiter’s allegations and will challenge them at the tribunal.

The case could shine a light on how diners’ gifts are shared out at the Ivy, which is owned by the tycoon Richard Caring. It could also clarify what level of detail businesses should provide to their workers on the allocation of service charges, such as how their share compares with others’ and how their allocation was decided.

The waiter, who worked part-time, claims he was allocated £46.34 in gratuities and service charges for 43 hours’ work in March, a sum that was later increased to £97.45. He claims that that share, which came from a total pot of £31,562 handed over by diners that month in his branch of the Ivy, was far lower than it should have been.

The waiter estimates that he was one of about 43 employees at the branch – including waiting staff, the kitchen team and management – meaning that his hours would have equated to about 2% of the total worked by staff that month. Yet he received less than a 300th of the service charge and tips collected. The Ivy disputed his calculations, arguing they were inaccurate and misleading. It said it used an independent consultancy to calculate how tips and service charges were shared. Lawyers working for the chain said he was a “disgruntled and discredited” former employee.

Under the law implemented last October, employers in Britain must share out 100% of service charges collected in a venue to workers there. It must be done in a “fair and transparent manner” and employees have the right to know “how tips are allocated and distributed”.

Diners at the Ivy, which was spun out of the London celebrity haunt and operates nearly 40 restaurants in the UK and Ireland, pay about £21 for fish and chips and £4.50 for a cup of coffee. Caring is attempting a £1bn sale of the business, which is owned by his Troia (UK) Restaurants parent company.

The Ivy said it had complied with the Employment (Allocation of Tips) Act 2023 and operated “a fair and transparent” scheme for distributing tips and service charge, which was “overseen by employee representatives and an independent, third-party business used by many restaurants in the hospitality industry”.

A spokesperson for Troia (UK) Restaurants said: “We absolutely refute all the claims that are being made and will provide all the evidence necessary to disprove these allegations to the employment tribunal.

“We introduced a fair and transparent scheme after consultations with staff that has been well received and is overseen by employee representatives and an independent, third-party business used by many restaurants in the hospitality industry.”

The Ivy said it shared out service charges via a system called a “tronc”, with different team members allocated “tronc points” that determine their share of the service charge each month. However, employees are not given an explanation of exactly how their tronc points are determined or how they compare with other team members’. Diners at the branch of the Ivy where the waiter worked pay an optional 12.5% service charge, with some adding tips on top. The firm says card and cash tips go directly to the individual who was given them.

The Ivy claimed that it was unable to give details on how tronc points were allocated because doing so would breach its employees’ rights.

The law states that gratuities must be shared out with regard to a code of practice and says there should be a written policy available to workers explaining “how tips are allocated and distributed and what steps the employer takes to ensure tips are handled fairly and transparently”.

Michael Newman, an employment law expert at Leigh Day, said: “This legislation was introduced to try and make the system fairer and the company has either managed to avoid it or the legislation hasn’t achieved its purpose. This case could clarify if employers need to provide more detail on how they distribute the service charge.”

The Ivy waiter’s payslips do not make clear which payments relate to personal tips and which to service charges and the company does not disclose how its waiters’ share of the service charge compares with kitchen staff, managers or other workers. It also does not reveal to workers how many individuals at a restaurant have shared the service charge. The Ivy has said that the worker “was provided with full details of his tronc allocation and how it was calculated, which included information which went beyond what he was entitled to receive pursuant to the relevant legislation”.

The waiter had worked on and off for the Ivy since November 2023 and said he sought clarification of his service charge and tips from last autumn and into this spring. He was given a warning about behaviour in April, which he claims included forgetting guests drinks and not emptying ice buckets. The business disputes those claims. He said that at about the same time he filed a formal demand for information on his service charge allocation, before resigning in June.

The tribunal hearing is due in April 2026.

Introduction

Prostate cancer (PCa) is one of the most common malignancies in men and a major cause of cancer-related deaths globally.¹ Its incidence is several-fold higher in developed countries than in low- and middle-income countries (LMICs), primarily due to differences in access to prostate-specific antigen (PSA) screening and advanced imaging technologies.^2–4 While modalities such as Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) facilitate early diagnosis and intervention in high-resource settings, delayed diagnosis remains common in LMICs owing to limited healthcare infrastructure.^5,6 These disparities highlight the need for innovative, affordable, and precise imaging tools to enhance early detection, surgical guidance, and treatment monitoring of PCa.

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, is significantly overexpressed in most PCa and has become a validated molecular target for both imaging and therapy.⁷ In 2020, the FDA approved ⁶⁸Ga-PSMA-11 as the first PET imaging agent for PCa.⁸ Subsequently, in 2022, ¹⁷⁷Lu-PSMA-617 (Pluvicto) was approved for the treatment of metastatic castration-resistant PCa (mCRPC).⁹ However, these agents rely on ionizing radiation, which poses safety concerns and provides limited spatial resolution, thereby restricting their applicability for real-time surgical navigation or optical pathological assessment.

Although PSMA-617 and PSMA-11 both share the glutamate-urea-lysine pharmacophore, their physicochemical characteristics limit further adaptation into nanoprobe platforms. PSMA-617 is highly hydrophobic and lacks suitable functional groups for conjugation, while PSMA-11 incorporates large chelating moieties that can hinder surface modification and compromise probe assembly.¹⁰ In contrast, ACUPA (2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid)—a glutamate-urea-based ligand engineered with a reactive thiol group—retains high affinity for PSMA and allows site-specific conjugation to nanoparticles via thiol–maleimide chemistry.^11,12 This structural simplicity and functional accessibility make ACUPA particularly suitable for the construction of targeted nanoprobes with enhanced stability and biocompatibility.

Molecular imaging enables non-invasive, dynamic visualization of physiological and pathological processes at the molecular level both in vivo and in freshly excised ex vivo specimens.^13,14 Fluorescence imaging, particularly in the near-infrared (NIR) spectrum, has emerged as a powerful complement to conventional modalities such as ultrasound, MRI, and PET.^15,16 Compared to visible light (400–700 nm) and NIR-I (700–900 nm), NIR-II fluorescence imaging (1000–1700 nm) offers markedly reduced tissue scattering, minimal autofluorescence, and superior penetration depth, achieving subcellular resolution at centimeter-scale tissue depths.^17–19

Recent studies have demonstrated that conjugation of specific molecular targets with NIR fluorophores enables precise imaging of cancerous lesions, including PCa, in both live animal models and excised tissues.^17,19–26 Despite this promise, PSMA-targeted probes operating in the NIR-II window remain scarce. Most clinically explored agents, such as OTL-78 and Cy-KUE-OA, emit in the NIR-I range, limiting their efficacy in deep-tissue applications.^27,28 In contrast, organic semiconducting polymers (OSPs) like OSP₁₂ exhibit high quantum yield, tunable emission properties, and photostability, making them ideal candidates for constructing next-generation NIR-II imaging probes.²⁹

In this study, we developed a novel PSMA-targeted NIR-II fluorescent nanoprobe, PSMA-OSP₁₂ nanoparticles (NPs), by covalently conjugating ACUPA to an OSP₁₂-based polymer micelle via thiol–maleimide click chemistry using DSPE-PEG-Mal. This design enables simultaneous integration of high tumor specificity, strong NIR-II emission, and excellent biocompatibility. We systematically characterized its physicochemical properties, in vitro and in vivo targeting capabilities, biodistribution, and biosafety. Furthermore, we demonstrated its applicability for intraoperative fluorescence navigation and ex vivo pathological visualization, addressing key limitations in current PCa imaging strategies.

Materials and Methods

Patient Samples

Three PCa patients, diagnosed postoperatively via pathological examination, provided paraffin-embedded tissue sections of tumor and adjacent normal tissue. Hematoxylin and eosin (HE) staining and PSMA immunohistochemical (IHC) staining (PSMA/GCPII antibody, 13163-1-AP, Proteintech, USA) were performed to assess PSMA protein expression levels. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Ethics Committee of Shenzhen Hospital, Chinese Academy of Medical Sciences (JS2024-7-1).

Databases and Bioinformatics Analyses

TIMER 2.0 (http://timer.cistrome.org/): A comprehensive online database based on TCGA and other datasets, used to analyze the expression differences of PSMA (FOLH1) across 32 types of tumors, including prostate cancer.³⁰

Prostate Cancer Atlas (https://prostatecanceratlas.org/): This database integrates RNA sequencing data from PCa samples, enabling pseudotime trajectory analysis to investigate disease progression from normal tissue to localized and metastatic tumors.³¹ In this study, we used it for pseudotime trajectory analysis and to examine the expression differences of PSMA across different molecular types of PCa.

GEPIA2 (http://gepia.cancer-pku.cn/index.html): GEPIA2 is an interactive database combining TCGA and GTEx datasets, comprising 9736 tumor and 8587 normal samples. In this study, it was used to analyze the prognostic impact of PSMA expression on PCa overall survival (OS) and disease-free survival (DFS).³²

Synthesis of PSMA-OSP₁₂ NPs NIR-II Probe

The NIR-II fluorescent polymer OSP₁₂ was synthesized as previously described.²⁹ For nanoprobe preparation, 10 mg of DSPE-PEG(2000)-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], DSPE-PEG-Mal, C139H271N4O57P, Avanti Polar Lipids, USA) and 1 mg of OSP12 were co-dissolved in tetrahydrofuran (THF, analytical grade, Sigma-Aldrich) and rapidly added into 10 mL of deionized water under continuous sonication (Branson 2510, USA) for 20 minutes. The resulting dispersion was dialyzed against deionized water (MWCO 8–14 kDa, Spectrum Labs, USA) for 24 h, filtered through a 0.22 μm PES membrane (Millipore, USA), and concentrated to 5 mL using centrifugal ultrafiltration (Corning^® Spin-X, MWCO 3 kDa, 3000 rpm, 5 min). To introduce the targeting ligand, 1.5 mg of ACUPA-SH (S)-2-[3-((S)-5-amino-1-carboxypentyl)ureido]pentanedioic acid with a terminal thiol group, C₁₇H₂₉N₃O₆S, MW 407.44, New Research Biosciences, China) and 500 μL of 10× PBS (pH 7.4) were added to the dispersion and stirred overnight at 4 °C in the dark. The thiol–maleimide reaction enabled efficient coupling, and the final PSMA-OSP₁₂ NPs were purified by centrifugation and resuspended in PBS.

The excitation and emission spectra of PSMA-OSP₁₂ NPs were recorded using a fluorescence spectrometer (FLS1000, Edinburgh Instruments, UK). For in vitro NIR-II fluorescence imaging, three formulations—ACUPA-SH, OSP₁₂ NPs, and PSMA-OSP₁₂ NPs—were diluted to 100 μg/mL in deionized water and loaded into 1.5 mL Eppendorf tubes. To assess concentration-dependent fluorescence properties, PSMA-OSP₁₂ NPs were further diluted to final concentrations of 10, 20, 40, 80, and 160 μg/mL and imaged under LP filters at 1000, 1100, 1200, and 1300 nm, respectively.

All fluorescence imaging, captured using an InGaAs camera (NIRvana 640, Princeton Instruments, USA), was performed under standardized conditions using an 808 nm continuous-wave diode laser with a power density of 60 mW/cm² and an exposure time of 100 ms. Long-pass (LP) filters at 1000–1300 nm were employed depending on experimental needs. All raw fluorescence images were acquired without post-acquisition enhancement, contrast adjustment, or pixel-wise background subtraction to ensure data authenticity and reproducibility. For quantitative analysis, regions of interest (ROIs) were manually delineated over tumors and representative background organs using Fiji-ImageJ (NIH, USA). The mean fluorescence intensity (MFI) within each ROI was measured and used to calculate imaging contrast metrics.

PSMA Binding Specificity Assay

To evaluate the binding specificity of PSMA-OSP₁₂ NPs, a cell-based NIR-II fluorescence imaging assay was performed using PSMA-positive and PSMA-negative prostate cancer cell lines. PSMA-positive 22Rv1 cells and PSMA-negative PC-3 cells were seeded into black, clear-bottom 96-well plates (Corning^®) at a density of 1 × 10⁴ cells per well and cultured overnight at 37 °C in a humidified 5% CO₂ incubator.

Cells were divided into four treatment groups: Control 1: PC-3 cells incubated with PSMA-OSP₁₂ NPs (10–160 μg/mL); Control 2: 22Rv1 cells incubated with non-targeted OSP₁₂ NPs (10–160 μg/mL); Blocking group: 22Rv1 cells pretreated with ACUPA-SH (10 μM, 1 h),²⁸ followed by incubation with PSMA-OSP₁₂ NPs (10–160 μg/mL); Experimental group: 22Rv1 cells directly incubated with PSMA-OSP₁₂ NPs (10–160 μg/mL). All groups were incubated at 37 °C for 24 hours. After incubation, cells were washed three times with PBS to remove unbound materials. Each condition was performed in triplicate (n = 3), and statistical analysis was conducted using two-way ANOVA.

In vivo Fluorescence Imaging and Biodistribution in Xenograft Models

Male BALB/c nude mice (4–6 weeks old, purchased from Viton Lihua, Foshan, China) were used to establish subcutaneous prostate cancer xenografts. A total of 1 × 10⁷ 22Rv1 cells suspended in PBS containing 25% Matrigel (Corning, Cat# 354237) were injected into the right flank of each mouse. Tumor volumes were calculated using the formula V = ab² / 2, where a and b denote the long and short diameters of the tumor. When tumors reached approximately 300 mm³, mice were randomized into three groups (n = 3 per group): Control group: injected with non-targeted OSP₁₂ NPs; Blocking group: pre-treated with ACUPA-SH (10 mM) 1 hour prior to PSMA-OSP₁₂ NPs administration;^33,34 Experimental group: injected with PSMA-OSP₁₂ NPs alone. Each mouse received 200 μL of 1 mg/mL nanoprobe solution via tail vein injection. In vivo NIR-II fluorescence imaging was performed using an InGaAs camera with 808 nm laser excitation, 1100 nm long-pass filter, and 100 ms exposure time at 10 min, 4 h, 12 h, 24 h, and 48 h post-injection. Additionally, at 30 h, animals were imaged under different spectral filters to assess tissue penetration and emission profiles. Tumor-to-Background Ratio (TBR) and Tumor-to-Liver Ratio (TLR) were calculated as: TBR = MFI_tumor / MFI_background; TLR = MFI_tumor / MFI_liver.

Ex vivo Tumor Imaging of PSMA-OSP₁₂ NPs

To evaluate the tumor-targeting capability of PSMA-OSP₁₂ NPs under controlled ex vivo conditions, an NIR-II fluorescence imaging assay was performed on tumor tissue slices. A total of six tumor-bearing mice were randomly divided into two groups (n = 3 per group). After excision, tumors were immediately sliced into approximately 2 mm thick sections using a sterile blade under cold PBS. Slices were transferred into plates and blocked with 5% fetal bovine serum (FBS) for 10 minutes at room temperature to reduce nonspecific binding. The samples were then incubated with either PSMA-OSP₁₂ NPs or non-targeted OSP₁₂ NPs at a final dye concentration of 200 μg/mL in PBS for 2 hours at room temperature in the dark. Following incubation, tumor slices were washed thoroughly three times with PBS to remove unbound nanoparticles. Quantitative fluorescence values were compared between groups using Student’s t-test.

Biosafety Evaluation of PSMA-OSP₁₂ NPs

To assess the biosafety of PSMA-OSP₁₂ NPs, a series of in vivo experiments were conducted. Male BALB/c nude mice were randomly divided into two groups (n=5 per group): one receiving PSMA-OSP₁₂ NPs (1 mg/mL, 200 μL) and the other receiving an equivalent volume of PBS as a control. The following parameters were evaluated:

Body Weight: Mice were monitored for changes in body weight from day 0 to day 14 post-injection, with measurements taken every two days to evaluate systemic toxicity; Hematological and Serum Biochemical Analysis: Blood samples were collected via the tail vein at days 0, 7, and 14 post-injection to measure red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA). These parameters were used to assess potential effects on hematological, hepatic, and renal functions; Histopathological Analysis: At day 14, mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) were harvested for HE staining to evaluate pathological changes. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee (TOPGM-IACUC-2023-0111).

Statistical Analysis

Data analysis was performed using OriginPro 2018C (OriginLab) and Fiji-ImageJ (NIH, USA). All quantitative data were analyzed using GraphPad Prism 9.0 and are expressed as mean ± standard deviation (SD). Statistical comparisons between two groups were performed using unpaired two-tailed Student’s t-test, while one-way or two-way ANOVA was used for multiple group comparisons, followed by Dunnett’s or Tukey’s post hoc tests as appropriate. Survival analysis was conducted using the Log rank test, and univariate and multivariate Cox proportional hazards models were applied to assess independent prognostic factors. P values less than 0.05 were considered statistically significant (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Results

Expression and Prognostic Significance of PSMA (FOLH1) in PCa

We utilized the TIMER database to analyze the expression of PSMA (Encoded by FOLH1) in different cancerous and normal tissues. The results revealed that PSMA expression is significantly upregulated in certain cancers. Notably, PSMA expression in Prostate Adenocarcinoma (PRAD) far exceeds its levels in other tumor tissues, highlighting its potential significance (Figure 1a). Using the Prostate Cancer Atlas database, pseudotime analysis was performed to map the progression of PCa from normal tissue (n=167) to primary tumors (n=499), then to androgen receptor-positive prostate cancer (ARPC, n=418), neuroendocrine PCa (NEPC, n=34), and ultimately to double-negative PCa (DNPC, n=22) (Figure 1b). Quantitative analysis revealed a significant upregulation of PSMA expression in the early stages (Primary tumors and ARPC) of PCa, followed by a gradual decline as the disease progresses to NEPC and DNPC (Figure 1c), which is similar to the progression trajectory of PCa. This intriguing pattern suggests that PSMA could serve as a crucial marker for early diagnosis and therapeutic intervention in PCa (Figure 1c). To further validate the expression levels of PSMA, cancerous and adjacent normal tissues from three clinical patients (n=3 per group) were collected. Although only three patient samples were analyzed by IHC, all exhibited strong PSMA expression in tumor tissues compared to adjacent normal tissues, consistent with the public database results. Due to the limited sample size, no statistical analysis was performed on the IHC data, and these results serve as qualitative validation rather than independent statistical confirmation. Comprehensive assessments through HE and IHC confirmed significantly higher PSMA expression in tumor tissues compared to normal tissues (Figure 1d and e). Lastly, Kaplan-Meier survival analysis with patients stratified by PSMA expression (based on the best-cut-off value) revealed a trend toward lower OS and DFS in the high PSMA expression group. Specifically, DFS was significantly lower in the high-expression group compared to the low-expression group (P = 0.00034, Figure 1f and g). Univariate Cox regression analysis demonstrated that high PSMA expression was significantly associated with worse DFS (BCR-free survival), with a hazard ratio (HR) of 2.41 (95% CI: 1.46–3.95, P = 0.001, Figure 1h and i). However, after adjusting for age, Gleason score, pathological T stage, and N stage in multivariate Cox regression analysis, the association between PSMA expression and DFS was no longer statistically significant (P = 0.070, Figure 1h and i). These results suggest that while PSMA expression may have prognostic value, it is not an independent influencing factor for DFS when other clinical variables are considered.

Expression of PSMA (FOLH1) in Different Clinical Subgroups

To investigate the relationship between PSMA expression and clinical characteristics of PCa, we analyzed datasets from the UALCAN database across various clinical subgroups. PSMA expression was significantly higher in T1-T3 stages compared to normal tissues, but no significant differences were observed among the T stages (Supplementary Figure 1a).In contrast, expression levels in PCa patients across different ethnic groups were consistently and significantly higher than in normal individuals (Supplementary Figure 1b). Notably, patients with metastases exhibited significantly elevated PSMA expression compared to those normal (Supplementary Figure 1c). In relation to Gleason scores, PSMA levels demonstrated an increasing trend with higher scores, but at a Gleason score as high as 10, PSMA expression shows no statistically significant difference compared to that in normal controls (Supplementary Figure 1d), which reflects similarities with the trend of low PSMA expression in NEPC and DNPC (Figure 1b and 1c). Analysis of molecular subtypes revealed that PSMA was generally overexpressed across most subtypes (ERG-fusion, ETV1-fusion, ETV4-fusion, FOXA1-mutation, and SPOP-mutation subtypes), with the exception of the FLI-fusion and IDH1-mutation subtypes, where expression levels were similar to those of normal tissue (Supplementary Figure 1e). Additionally, a relationship between TP53 mutations and PSMA expression was identified, indicating potential relevance in PCa progression (Supplementary Figure 1f). These findings suggest that PSMA expression correlates with clinical features such as age, metastatic status, Gleason score, and molecular subtypes, highlighting its potential as a biomarker for diagnosis and prognosis in PCa.

Synthesis and Construction of the PSMA-Targeted Probe and Basic Optical Characterization

Based on the findings above, PSMA has been identified as a viable molecular target for specific diagnostic and therapeutic applications. We developed a PSMA-targeted probe using the previously reported NIR material OSP₁₂ NPs.²⁹ The probe was constructed by self-assembling OSP₁₂ with DSPE-PEG-Mal to form nanoparticles, which were subsequently conjugated to the PSMA-targeting ligand ACUPA via thiol–maleimide coupling, resulting in the formation of the specific PSMA-targeted probe, PSMA-OSP₁₂ NPs (Figure 2a). The optical properties of PSMA-OSP₁₂ NPs, analyzed through spectral analysis, revealed that the probe exhibits excitation at 792.0 nm and emission at 1049.0 nm, extending its tail to approximately 1500 nm. This demonstrates the imaging capability of PSMA-OSP₁₂ NPs in the NIR-II region (Figure 2b). We used 100 μg/mL of ACUPA-SH, OSP₁₂ NPs, and PSMA-OSP₁₂ NPs under 1000 LP and 100 ms exposure conditions. ACUPA-SH exhibited negligible fluorescence signal, while the other two groups showed comparable fluorescence intensities (Figure 2c and d). Dynamic light scattering (DLS) analysis revealed that the hydrodynamic diameter of the PSMA-OSP₁₂ NPs was 66.61 ± 21.10 nm, with a polydispersity index (PDI) of 0.100, indicating a moderately uniform size distribution. In contrast, the non-targeted OSP₁₂ NPs showed a smaller average diameter of 59.53 ± 18.30 nm and a lower PDI of 0.094, confirming that the conjugation of the PSMA-targeting ligand ACUPA modestly increased particle size and dispersity (Supplementary Figure 2a and b).

Figure 2 Synthesis, optical characterization, and imaging performance of PSMA-OSP12 NPs. (a) Schematic illustration of PSMA-OSP12 NPs synthesis. OSP12 self-assembles with DSPE-PEG-Mal to form micelles with maleimide terminals, which are conjugated to ACUPA-SH via thiol–maleimide chemistry. (b) Excitation (blue) and emission (red) spectra of PSMA-OSP12, showing NIR-II fluorescence characteristics. (c) Comparative fluorescence imaging of ACUPA-SH, OSP12 NPs, and PSMA-OSP12 NPs under identical conditions (100 μg/mL; 1000 nm LP filter). (d) Quantification of fluorescence intensities from (c) (one-way ANOVA). (e) In vitro fluorescence imaging of PSMA-OSP12 NPs at serial concentrations (10–160 μg/mL) under different long-pass (LP) filters (1000–1300 nm). (f) In vivo fluorescence imaging of tumor-bearing mice injected with PSMA-OSP12 NPs under the same LP filters and exposure conditions. (g) Linear relationship between fluorescence intensity and probe concentration (R² = 0.997). (h) Quantified fluorescence intensity of tumors under different LP filters (one-way ANOVA vs 1100 nm LP). (i) Tumor-to-background ratio (TBR) under varying LP filters showing maximum imaging contrast at 1100 nm (one-way ANOVA). All imaging was performed using a laser power of 60 mW/cm² and an exposure time of 100 ms. ****P < 0.0001, 1 cm. Abbreviation: NS, not significant.

Based on the feed molar ratio (ACUPA-SH: DSPE-PEG-Ma ≈ 1.25:1), the average PEG-lipid surface density (~1000–1500 chains per nanoparticle), and a reported thiol–maleimide conjugation efficiency of approximately 60%,³⁵ we estimate that each PSMA-OSP₁₂ NPs carries approximately 600–900 ACUPA molecules on its surface.

To evaluate photostability, both formulations were incubated in PBS at 4 °C and monitored for fluorescence intensity at Days 0, 3, and 7. As shown in Supplementary Figure 3a and b, the fluorescence signals of OSP₁₂ NPs and PSMA-OSP₁₂ NPs remained stable over the 7-day observation period. At Day 3, OSP12 NPs retained 89.24 ± 3.92% of their initial signal, while PSMA-OSP₁₂ NPs maintained 85.06 ± 2.20%. By Day 7, fluorescence retention was 74.50 ± 2.42% for OSP₁₂ NPs and 71.42 ± 2.35% for PSMA-OSP₁₂ NPs, respectively. These results confirm that the surface modification with ACUPA does not impair the inherent photostability of the OSP₁₂ NPs fluorophore and support its suitability for prolonged in vivo imaging.

Additionally, PSMA-OSP₁₂ NPs achieved high-definition imaging up to 1300 nm in the NIR-II region in vitro and in vivo (Figure 2e and f) and the fluorescence intensity of PSMA-OSP₁₂ NPs increased with its concentration (Figure 2g). In vivo imaging, the PSMA-OSP₁₂ NPs (1 mg/mL) achieved clear imaging under an 1100 nm filter, with a relatively strong fluorescence intensity (P <0.0001, Figure 2h) and a higher TBR of 7.61 ± 0.19, which was significantly higher than that of other groups (P <0.0001, Figure 2i).

In vitro Validation of PSMA-Specific Binding of PSMA-OSP₁₂ NPs

To select an appropriate cell line for xenograft tumor modeling, PSMA expression levels were first evaluated in five human prostate cell lines using flow cytometry (Supplementary Figure 4a). LNCaP and VCaP exhibited nearly 100% PSMA positivity, while 22Rv1 showed moderate-to-high expression levels (60–70%). In contrast, PSMA expression was low in PC-3 and RWPE-1 cells (2–4%). Although LNCaP cells exhibit high PSMA expression, their poor tumorigenic capacity in BALB/c nude mice has been widely reported in the literature³⁶ and was further validated by our preliminary in vivo experiments. Additionally, VCaP and RWPE-1 are rarely used for subcutaneous xenograft modeling due to their limited in vivo growth potential. Considering both PSMA expression and tumorigenicity, 22Rv1 cells were selected for subsequent xenograft model establishment (Supplementary Figure 4b). PC-3 cells, which lack PSMA expression, were used as the negative control cell line.³⁷

Based on the differential PSMA expression profiles observed in prostate cancer cell lines, we next evaluated the PSMA-targeting capability of PSMA-OSP₁₂ NPs using PSMA-positive 22Rv1 cells and PSMA-negative PC-3 cells. In vitro NIR-II fluorescence imaging was performed under four treatment conditions (Figure 3a).

Figure 3 In vitro binding specificity and blocking validation of PSMA-OSP12 NPs. (a) Fluorescence imaging of PSMA-positive 22Rv1 and PSMA-negative PC-3 cells under four treatment conditions: control 1, PSMA-negative PC-3 cells incubated with PSMA-OSP12 NPs; control 2, 22Rv1 cells incubated with free OSP12 NPs (non-targeted); blocking group, 22Rv1 cells pretreated with ACUPA-SH (10 μM, 1 h) prior to PSMA-OSP12 NPs exposure; and experimental group, 22Rv1 cells directly incubated with PSMA-OSP12 NPs (10–160 μg/mL, 24 h). (b) Quantification of fluorescence intensities revealed a significant, concentration-dependent increase in the experimental group. In contrast, control 1 (PC-3 + PSMA-OSP12 NPs) and control 2 (22Rv1 + free OSP12 NPs) showed no significant difference and maintained low signal levels. Blocking with ACUPA-SH markedly reduced fluorescence intensity, confirming the PSMA-specific binding of PSMA-OSP12 NPs (two-way ANOVA). (n = 3 per group). ****P < 0.0001.

In Control 1 (PC-3 + PSMA-OSP₁₂ NPs), PSMA-negative PC-3 cells exhibited minimal fluorescence, indicating negligible nonspecific binding. In Control 2 (22Rv1 + free OSP₁₂ NPs), 22Rv1 cells showed weak fluorescence when incubated with non-targeted OSP₁₂ NPs, confirming the absence of passive uptake. In the blocking group (22Rv1 pretreated with ACUPA-SH before PSMA-OSP₁₂ incubation), fluorescence signals were significantly suppressed across all concentrations, suggesting effective competitive inhibition of PSMA binding by free ligand. By contrast, the experimental group (22Rv1 + PSMA-OSP₁₂ NPs) exhibited a concentration-dependent increase in fluorescence intensity. Quantitative analysis (Figure 3b) demonstrated a strong linear correlation between probe concentration and signal intensity (Y = 263.1*X + 5770, R² = 0.9306), validating dose-dependent, receptor-mediated cellular uptake. These results confirm the high specificity of PSMA-OSP₁₂ toward PSMA-expressing tumor cells, highlighting its potential for quantitative molecular imaging applications.

Specific Tumor Targeting and Biodistribution Profile of PSMA-OSP₁₂ NPs

To comprehensively evaluate the in vivo tumor-targeting capability and biodistribution of PSMA-OSP₁₂ NPs, we established a human-derived 22Rv1 xenograft mouse model and compared three treatment groups: control (OSP₁₂ NPs), blocking (ACUPA pre-injection + PSMA-OSP₁₂ NPs), and experimental (PSMA-OSP₁₂ NPs).

In the control group, non-targeted OSP₁₂ NPs exhibited rapid hepatic accumulation with minimal tumor signal, which gradually decreased over time due to metabolic clearance. The blocking group, pretreated with ACUPA (10 mM, 1 h prior to probe administration), also showed limited tumor fluorescence, highlighting the successful inhibition of PSMA receptor binding (Figure 4a). In contrast, mice in the experimental group receiving PSMA-OSP₁₂ NPs demonstrated evident tumor localization beginning at 4 h post-injection, with fluorescence intensities progressively intensifying and peaking between 24–48 h. Quantitative analysis revealed significantly higher TBR in the experimental group compared to both control and blocking groups, with values reaching 7.19 ± 0.40 and 7.40 ± 1.28 at 24 h and 48 h, respectively (Figure 4b). TLR analysis similarly demonstrated enhanced tumor specificity, with liver signal gradually declining post-injection (Figure 4c). These findings confirm the selective in vivo binding of PSMA-OSP₁₂ NPs to PSMA-expressing tumors and the efficacy of ACUPA-mediated blocking in suppressing this interaction.

Figure 4 In vivo tumor targeting and biodistribution of PSMA-OSP12 NPs. (a) Representative NIR-II fluorescence images of 22Rv1 xenograft-bearing mice at multiple time points following intravenous injection of non-targeted OSP12 NPs (control group), PSMA-OSP12 NPs with ACUPA pre-blocking (blocking group), or PSMA-OSP12 NPs (experimental group). Imaging was performed under identical parameters (808 nm excitation, 1100 nm long-pass filter, 100 ms exposure). (b and c) Quantitative analysis of tumor-to-background ratio (TBR) and tumor-to-liver ratio (TLR) demonstrates significantly higher tumor accumulation and specificity in the PSMA-OSP12 NPs group relative to both control and blocking groups (two-way ANOVA) and Post hoc power analysis. ****P < 0.0001. Abbreviation: NS, not significant.

To further characterize tissue-level distribution, ex vivo imaging at 48 h post-injection revealed that PSMA-OSP₁₂ NPs predominantly accumulated in tumors, liver, and kidneys, whereas non-targeted OSP₁₂ NPs and the blocking group showed minimal tumor retention and increased hepatic uptake (Supplementary Figure 5a). Quantitative organ fluorescence analysis confirmed significantly elevated tumor signal in the PSMA-OSP₁₂ NPs group (Supplementary Figure 5b, P < 0.0001). HE and PSMA IHC validated tumor identity and confirmed PSMA positivity across all groups (Supplementary Figure 5c), ruling out differential antigen expression as a source of imaging discrepancy.

Excretion profiling further indicated that PSMA-OSP₁₂ NPs are primarily cleared via the hepatobiliary pathway. Serial NIR-II imaging of feces and urine collected daily up to 5 days post-injection revealed robust fluorescence signals in feces but negligible signal in urine, confirming predominant fecal elimination with minimal renal involvement (Supplementary Figure 6).

These collective results highlight the high tumor specificity, favorable biodistribution, and safe excretion profile of PSMA-OSP₁₂ NPs, underscoring its potential utility for NIR-II fluorescence-guided cancer imaging.

Tumor-Specific Visualization of ex vivo Tumor Tissues Using the PSMA-OSP₁₂ NPs Probe as a Molecular-Level Pathological Imaging Tool

To explore the applicability of PSMA-OSP₁₂ NPs for ex vivo tumor margin visualization, we employed a 22Rv1 xenograft prostate cancer model and conducted NIR-II fluorescence imaging of excised tumor sections. Following tumor resection, samples were sectioned into ~2 mm-thick slices, blocked with 5% FBS, incubated with 200 μg/mL of either PSMA-OSP₁₂ NPs or OSP₁₂ NPs (control), and washed thoroughly with PBS prior to imaging (Figure 5a).

Figure 5 Ex vivo tumor-specific visualization using PSMA-OSP12 NPs. (a) Schematic diagram of the ex vivo NIR-II imaging workflow. Tumor-bearing mice (22Rv1 xenografts, n = 6) were randomly divided into two groups (n = 3 per group). Excised tumors were sectioned into ~2 mm slices, blocked with 5% FBS for 10 min, then incubated with either OSP12 NPs (control group) or PSMA-OSP12 NPs (experimental group) at 200 μg/mL for 2 h at room temperature, followed by three PBS washes. Imaging was performed using an NIR-II system (808 nm laser, 1100 nm long-pass filter, 100 ms exposure). (b and c) Representative fluorescence images and corresponding intensity profiles of tumor slices showed markedly stronger signal in the PSMA-OSP12 NPs group. (d) Quantification of fluorescence intensity confirmed significantly enhanced tumor labeling in the PSMA-OSP12 NPs group compared to control (Student’s t-test, ****P < 0.0001). Scale bar, 500 μm.

As shown in Figure 5b, tumor slices incubated with PSMA-OSP₁₂ NPs exhibited robust fluorescence localized within tumor boundaries, indicative of specific probe-target interaction. In contrast, tumor slices incubated with non-targeted OSP₁₂ NPs showed weak fluorescence, which was largely eliminated during washing, suggesting limited retention due to nonspecific interactions. Fluorescence intensity profile analysis (Figure 5c) revealed that signal distribution in the PSMA-OSP₁₂ NPs group was substantially higher and well-defined relative to the control group.

Quantitative analysis confirmed a significantly enhanced fluorescence signal in the PSMA-OSP₁₂ NPs group compared to the OSP₁₂ NPs group (P < 0.0001, Figure 5d). These results demonstrate the capability of PSMA-OSP₁₂ NPs to achieve highly specific tumor labeling in tissue slices, underscoring its potential utility as a molecular-level pathological imaging tool for intraoperative guidance or ex vivo diagnostic applications.

Biosafety Evaluation of PSMA-OSP₁₂ NPs

To evaluate the in vivo biosafety profile of PSMA-OSP₁₂ NPs, a comprehensive toxicity assessment was performed in healthy mice over a 14-day period. Throughout the study, no significant changes in body weight or general health status were observed in mice treated with PSMA-OSP₁₂ NPs compared to those receiving PBS (Figure 6a), suggesting minimal systemic toxicity. To further assess hematological safety, blood samples were collected on days 0, 7, and 14 post-injection. Analysis of hematological parameters—including RBC, WBC, PLT—revealed no significant differences between the PSMA-OSP₁₂ NPs and PBS control groups (Figure 6b–d). These findings indicate that administration of PSMA-OSP₁₂ NPs does not disrupt normal hematopoiesis or immune homeostasis. In addition, serum biochemical indices related to liver and kidney function were examined. Key markers—ALT, AST, ALB, BUN, and CREA—remained within physiological ranges in both groups, with no significant alterations detected at any time point (Figure 6e–i), indicating no measurable hepatic or renal toxicity. At the end of the study (day 14), major organs (heart, liver, spleen, lung, and kidney) were harvested for histopathological evaluation. HE staining revealed no pathological abnormalities or tissue damage in the PSMA-OSP₁₂ NPs group compared to PBS controls (Figure 6j). Collectively, these results demonstrate that PSMA-OSP₁₂ NPs possess an excellent biocompatibility and safety profile, with no evidence of systemic, hematologic, or organ-specific toxicity under the tested conditions.

Figure 6 Biosafety evaluation of PSMA-OSP12 NPs. (a) Body weight monitoring of mice treated with PSMA-OSP12 NPs or PBS over a 14-day period showed no significant differences. (b–d) Hematological parameters, including red blood cell (RBC) count, white blood cell (WBC) count, and platelet (PLT) count, remained within normal ranges in both groups. (e–i) Serum biochemical markers related to liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA), showed no significant abnormalities in the PSMA-OSP12 NP group compared to controls. (j) Representative hematoxylin and eosin (HE) staining of major organs (heart, liver, spleen, lung, kidney) revealed no visible pathological lesions in either group. No statistically significant differences were observed across all parameters (n=5 per group, two-way ANOVA). Scale bar, 200 μm.

Discussion

PSMA has long been recognized as a valuable biomarker for PCa and other cancers diagnosis and therapy.^2,10,38–42 In this study, we extended the current understanding by leveraging large-scale datasets and pseudotime trajectory analysis to reveal dynamic patterns of PSMA expression across PCa subtypes. Our data confirmed the elevated expression of PSMA in early-stage PCa, which gradually declines in aggressive subtypes such as NEPC and DNPC (Figure 1). This decline may be attributed to androgen receptor (AR) pathway suppression and the prevalence of TP53 mutations, which promote dedifferentiation and lineage plasticity.^31,43–45 These results highlight the biological significance of PSMA as a biomarker and provide a compelling rationale for its application in molecular imaging.

Despite the clinical availability of PSMA-targeted radioligands, including ⁶⁸Ga-PSMA-11 and ¹⁷⁷Lu-PSMA-617, their reliance on radiation limits their use in real-time surgical navigation and intraoperative decision-making.^46–49 Moreover, their molecular structures hinder integration into nanoprobe platforms due to hydrophobicity, lack of reactive groups, or bulky chelators.^10,50,51 In contrast, ACUPA-SH, used in this study, retains high binding affinity and incorporates a thiol group, enabling site-specific conjugation via maleimide-thiol chemistry. This bioconjugation approach offers several advantages: it proceeds under mild aqueous conditions, forms stable covalent thioether bonds, and minimizes nonspecific interactions, ensuring consistent batch-to-batch reproducibility.^11,12,52 The resulting PSMA-OSP₁₂ NPs thus achieve precise ligand orientation, enhanced stability, and reliable performance in vivo.

To overcome the limitations of NIR-I dyes such as S0456 (used in OTL78; excitation/emission: 774–776/794–796 nm) and Cy7 (used in Cy-KUE-OA; emission at 776 nm), which suffer from shallow tissue penetration and high background autofluorescence, we selected OSP₁₂ as the fluorophore for our nanoprobe.^27,28,53,54 PSMA-OSP₁₂ NPs exhibits excitation at 792.0 nm and emission at 1049.0 nm, placing it firmly in the NIR-II window.²⁹ Compared with clinically available dyes, OSP₁₂ offers superior photostability, deeper tissue penetration, reduced scattering, and negligible autofluorescence. These optical advantages enabled PSMA-OSP₁₂ NPs to achieve a TBR of 7.40 ± 1.28 at 48 hours post-injection, significantly outperforming OTL-78 (TBR ~2.0) and Cy-KUE-OA (TBR ~3.8).^28,53 Recent advances such as FC-PSMA have demonstrated the feasibility of PSMA-targeted NIR-II imaging in preclinical models, but suffer from drawbacks including complex synthesis routes, suboptimal in vivo stability, or limited modularity.^34,55 In contrast, our strategy offers a more practical and translational pathway while maintaining excellent imaging performance. This extended imaging window supports flexible scheduling of fluorescence-guided surgery (FGS) and enhances tumor delineation even under standard surgical lighting conditions. Notably, the deeper tissue penetration (>1 cm) and high SBR of NIR-II imaging overcome the resolution limitations of traditional NIR-I agents and support real-time intraoperative use.

Beyond in vivo applications, PSMA-OSP₁₂ NPs demonstrated substantial utility in ex vivo settings. Rapid incubation of freshly resected PCa specimens allowed for high-contrast visualization of tumor margins without the need for systemic administration. This approach avoids individual variability in pharmacokinetics and seamlessly integrates with conventional pathological workflows.⁵⁶ While ex vivo fluorescence imaging has been explored in other malignancies such as lung and colorectal cancers, our study represents the first application of a NIR-II PSMA-targeted probe in prostate cancer.^23,24,57 This capability offers a novel solution for intraoperative margin assessment and immediate postoperative diagnostics.

Ensuring biosafety is essential for clinical translation. In this study, we conducted comprehensive toxicity evaluations, including body weight monitoring, hematological and biochemical analyses, and histopathological examinations. The results revealed no significant adverse effects in major organs, and PSMA-OSP₁₂ NPs were predominantly cleared via hepatobiliary excretion, minimizing the risk of long-term retention. These findings align with previous NIR-II nanoprobe safety studies and support the translational potential of PSMA-OSP₁₂ NPs.¹⁸

While our findings underscore the promise of PSMA-OSP₁₂ NPs, several translational challenges must be addressed. Current imaging systems are not universally compatible with NIR-II wavelengths, necessitating the development of affordable and user-friendly detection platforms such as fiber-optic or laparoscopic modules.^58,59 Additionally, the cost of producing high-quality NIR-II fluorophores remains substantial, although improved clinical outcomes may justify these investments.¹⁷ Finally, given the heterogeneity of PSMA expression, patient selection strategies and imaging-guided treatment algorithms will be essential for maximizing benefit. Importantly, NIR-II imaging is not intended to replace PET or other modalities, but rather to serve as a complementary tool offering superior spatial resolution and intraoperative utility.

Conclusions

In summary, this study presents PSMA-OSP₁₂ NPs as a novel NIR-II nanoprobe with high specificity for PSMA-positive prostate cancer. By integrating the favorable binding characteristics of ACUPA-SH, the optical advantages of OSP₁₂, and a stable maleimide-thiol conjugation strategy, we successfully constructed a nanoprobe with outstanding tumor-targeting ability, high spatial resolution, and deep tissue penetration. The probe demonstrates excellent performance both in vivo and ex vivo, and exhibits favorable biosafety, indicating its translational potential. Although further studies are required to optimize clinical integration, PSMA-OSP₁₂ NPs offer a promising platform for real-time fluorescence-guided surgery and rapid pathological assessment, representing an important step forward in the development of precision diagnostics for prostate cancer.

Data Sharing Statement

Data from public databases can be accessed at the following websites: TIMER: http://timer.cistrome.org/; Prostate Cancer Atlas: https://prostatecanceratlas.org/; GEPIA: http://gepia.cancer-pku.cn/index.html. All study materials are included within the manuscript. The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics Declarations

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Ethics Committee of Shenzhen Hospital, Chinese Academy of Medical Sciences (JS2024-7-1). Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient to publish this paper.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College Institutional Research Project Funding (NO. SZ2020ZD003), and Sanming Project of Medicine in Shenzhen (NO. SZSM202111003).

Disclosure

The authors declare no conflicts of interest.

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