Double-PEGylated Cyclopeptidic Photosensitizer Prodrug Improves Drug Uptake from In Vitro to Hen's Egg Chorioallantoic Membrane Model.

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs.

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free "click" chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Squalene-PEG: Pyropheophorbide-a nanoconstructs for tumor theranostics.

Novel nanoscale drug delivery biomaterials are of great importance for the diagnosis and treatment of different cancers. We have developed a new pegylated squalene (SQ-PEG) derivative with self-assembly properties. Supramolecular assembly with a lipophilic photosensitizer pyropheophorbide-a (Ppa) by nanoprecipitation gave nanoconstructs SQ-PEG:Ppa with an average size of 200 nm in diameter and a drug loading of 18% (w/w). The composite material demonstrates nanoscale optical properties by tight packing of Ppa within Sq-PEG:Ppa resulting in 99.99% fluorescence self-quenching. The biocompatibility of the nanomaterial and cell phototoxicity under light irradiation were investigated on PC3 prostate tumor cells in vitro. SQ-PEG:Ppa showed excellent phototoxic effect at low light dose of 5.0 J/cm2 as a consequence of efficient cell internalization of Ppa by the nanodelivery system. The diagnostic potential of SQ-PEG:Ppa nanoconstructs to deliver Ppa to tumors in vivo was demonstrated in chick embryo model implanted with U87MG glioblastoma micro tumors.

Squalene-PEG-Exendin as High-Affinity Constructs for Pancreatic Beta-Cells.

Novel drug delivery systems targeting native, transplanted, or cancerous beta-cells are of utmost importance. Herein, we present new exendin-4 derivatives with modified unnatural amino acids at strategic positions within the polypeptide sequence. The modified peptides allowed modular orthogonal chemical modifications to attach imaging agents and amphiphilic squalene-PEG groups. The resulting conjugates, SQ-PEG-ExC1-Cy5 and SQ-PEG-ExC40-Cy5 fluorescence probes, display low nanomolar affinity to GLP-1R in fluorescence-based binding assays with EC50 at 1.1 ± 0.2 and 0.8 ± 0.2 nM, respectively. Naturally expressing GLP-1R MIN6 cells and recombinantly transfected CHL-GLP-1R positive cells were specifically targeted by all of the new beta-cell probes in vitro. Specific islet targeting was observed after i.v. injection of SQ-PEG-ExC1-Cy5 with SQ-PEG in normoglycemic mice ex vivo. Semiquantitative biodistribution analysis by epifluorescence indicated prolonged blood half-life (3.8 h) for the amphiphilic Ex conjugate. Liver and pancreas were identified as main biodistribution organs for SQ-PEG-ExC1-Cy5.

Enhanced prostate cancer targeting by modified protease sensitive photosensitizer prodrugs.

Prodrugs combining macromolecular delivery systems with site-selective drug release represent a powerful strategy to increase selectivity of anticancer agents. We have adapted this strategy to develop new polymeric photosensitizer prodrugs (PPP) sensitive to urokinase-like plasminogen activator (uPA). In these compounds (to be referred to as uPA-PPPs) multiple copies of pheophorbide a are attached to a polymeric carrier via peptide linkers that can be cleaved by uPA, a protease overexpressed in prostate cancer (PCa). uPA-PPPs are non-phototoxic in their native state but become fluorescent and produce singlet oxygen after uPA-mediated activation. In the present work, we studied the influence of side-chain modifications, molecular weight, and overall charge on the photoactivity and pharmacokinetics of uPA-PPPs. An in vitro promising candidate with convertible phototoxicity was then further investigated in vivo. Systemic administration resulted in a selective accumulation and activation of the prodrug in luciferase transfected PC-3 xenografts, resulting in a 4-fold increase in fluorescence emission over time. Irradiation of fluorescent tumors induced immediate tumor cell eradication as shown by whole animal bioluminescence imaging. PDT with uPA-PPP could therefore provide a more selective treatment of localized PCa and reduce side effects associated with current radical treatments.

On the cutting edge: protease-sensitive prodrugs for the delivery of photoactive compounds.

Most invasive diseases such as cancer or rheumatoid arthritis are characterized by the upregulation of diverse proteases. Since the early 1970s this phenomenon has been exploited for the selective delivery of a variety of drugs. However, only recently have we and others tried to translate this concept into photomedicine. After a short overview of proteases and the proteolytic imbalance in cancer, we will discuss strategies, their potential and limitations to exploit upregulation of proteases for the selective delivery of in vivo fluorescence reporters and photosensitizers. These strategies can be roughly divided into horizontal, i.e. peptide-based, and vertical, i.e. macromolecular approaches. In the former, a short peptide-based substrate is directly tagged to the photoactive compound or used as a linker between the photoactive compound and a substance that alters its photoactivity. In the latter, the protease sensitive sequence serves as linker between a polymeric carrier and the photoactive payload. Such a macromolecular approach may further benefit from passive targeting through the enhanced penetration and retention effect.

c(RGDfK)- and ZnTriMPyP-Bound Polymeric Nanocarriers for Tumor-Targeted Photodynamic Therapy.

Active targeting strategies are currently being extensively investigated in order to enhance the selectivity of photodynamic therapy. The aim of the present research was to evaluate whether the external decoration of nanopolymeric carriers with targeting peptides could add more value to a photosensitizer formulation and increase antitumor therapeutic efficacy and selectivity. To this end, we assessed PLGA-PLA-PEG nanoparticles (NPs) covalently attached to a hydrophilic photosensitizer 5-[4-azidophenyl]-10,15,20-tri-(N-methyl-4-pyridinium)porphyrinato zinc (II) trichloride (ZnTriMPyP) and also to c(RGDfK) peptides, in order to target αv ß3 integrin-expressing cells. In vitro phototoxicity investigations showed that the ZnTriMPyP-PLGA-PLA-PEG-c(RGDfK) nanosystem is effective at submicromolar concentrations, is devoid of dark toxicity, successfully targets αv ß3 integrin-expressing cells and is 10-fold more potent than related nanosystems where the PS is occluded instead of covalently bound.

Intracellular reactive oxygen species in monocytes generated by photosensitive chromophores activated with blue light.

OBJECTIVES: Disinfection of the tooth pulp-canal system is imperative to successful endodontic therapy. Yet, studies suggest that 30-50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death mediated by generated reactive oxygen species (ROS) has been reported, but red-light also may thermally damage resident tissues. In the current study, we tested the hypothesis that several blue light chromophores (380-500 nm) generate intracellular reactive oxygen species but are not cytotoxic to mammalian cells. METHODS: THP1 monocytes were exposed to 10 microM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (27J/cm(2), 60s). After activation, intracellular ROS were measured using a dihydrofluorescein diacetate technique, and cytotoxicity was determined by measuring mitochondrial activity with the MTT method. RESULTS: All photosensitizers produced intracellular ROS levels that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Photosensitizers except riboflavin exhibited cytotoxicity above 10 microM, and all except pheophorbide-a-PLL were more cytotoxic after blue light irradiation. SIGNIFICANCE: The current study demonstrated the possible utility of blue light chromophores as producers of ROS that would be useful for endodontic disinfection.

Synergies of VEGF inhibition and photodynamic therapy in the treatment of age-related macular degeneration.

PURPOSE: Photodynamic therapy (PDT) and the administration of compounds acting against vascular endothelial growth factor (anti-VEGF) are approved for the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). Experimental evidence that the combined use of both treatment options may improve therapeutic outcome is presented. METHODS: Fertilized chick eggs were incubated until day 12 of embryo development (EDD12) and were treated by PDT using two different photosensitizing agents (liposomal formulation of BPD-MA; m-THPP encapsulated in polymeric nanoparticles) and were visualized using an epifluorescence microscope. Vascular occlusion of the treated zones of the chorioallantoic membrane (CAM) was assessed by fluorescence angiography 24 and 48 hours after treatment. Alternatively, PDT-treated areas were exposed to a soluble VEGF receptor antagonist (sFlt-1) 6 hours after treatment and were analyzed. RESULTS: Vascular occlusion in the PDT-treated areas was observed with both photosensitizers 24 hours after treatment. Reperfusion of preexisting blood vessels and first signs of revascularization were visible 48 hours after PDT. Topical administration of sFlt-1 to the treated areas augmented occlusion and limited subsequent angiogenesis in a dose-dependent manner. CONCLUSIONS: The combined use of PDT and of agents targeting angiogenic cytokines may synergistically improve therapeutic outcome after combined treatment in patients with CNV secondary to AMD.

Polymeric photosensitizer prodrugs for photodynamic therapy.

A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(L)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme.

Hypericin-loaded nanoparticles for the photodynamic treatment of ovarian cancer.

A photodynamic approach has been suggested to improve diagnosis and therapy of ovarian cancer. As Hypericin (Hy), a natural photosensitizer (PS) extracted from Hypericum perforatum, has been shown to be efficient in vitro and in vivo for the detection or treatment of other cancers, Hy could also be a potent tool for the treatment and detection of ovarian cancer. Due to its hydrophobicity, systemic administration of Hy is problematic. Thus, polymeric nanoparticles (NPs) of polylactic acid (PLA) or polylactic-co-glycolic acid (PLGA) were used as a drug delivery system. Hy-loaded NPs were produced with the following characteristics: (i) size in the 200-300 nm range, (ii) negative zeta potential, (iii) low residual PVAL and (iv) drug loading from 0.03 to 0.15% (w/w). Their in vitro photoactivity was investigated on the NuTu-19 ovarian cancer cell model derived from Fischer 344 rats and compared to free drug. Hy-loaded PLA NPs exhibited a higher photoactivity than free drug. Increasing light dose or incubation time with cells induced an enhanced activity of Hy-loaded PLA NPs. Increased NP drug loading had a negative effect on their photoactivity on NuTu-19 cells: at the same Hy concentration, the higher was the drug loading, the lower was the phototoxic effect. The influence of NP drug loading on the Hy release from NPs was also investigated.

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro.

BACKGROUND: In dentistry, antibacterial photodynamic therapy (a-PDT) has shown promising results for inactivating bacterial biofilms causing carious, endodontic and periodontal diseases. In the current study, we assessed the ability of eosin Y exposed to 3 irradiation protocols at inactivating Enterococcus faecalis biofilms, in vitro. METHODS: E. faecalis biofilms formed on hydroxyapatite disks were incubated with eosin Y (10-80µM), then activated with blue light using different irradiation protocols. Biofilms exposed to continuous exposure were incubated for 40min before being light-activated for 960 s. For the intermittent exposure, biofilms were exposed 4 times to the light/photosensitizer combination (960 s total) without renewing the photosensitizer. For repeated a-PDT, the same light dose was delivered in a series of 4 irradiation periods separated by dark periods; fresh photosensitizer was added between each light irradiation. After treatment, bacteria were immediately labeled with LIVE/DEAD BacLight Bacterial Viability kit and viability was assessed by flow cytometry (FCM). Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). RESULTS: The viability of E. faecalis biofilms exposed to 10µM eosin Y, was significantly reduced compared to controls (light only-eosin Y only). After a second exposure to blue light-activated eosin Y, viability significantly decreased from 58% to 12% whereas 6.5% of the bacterial biofilm remained live after a third exposure (p<0.05). Only 3.5% of the bacterial population survived after the fourth exposure. CONCLUSIONS: The results of this study indicate that blue light-activated eosin Y can photoinactivate E. faecalis biofilms grown on hydroxyapatite disks. Also, repeated exposures to blue light-activated eosin Y were shown to significantly improve efficacy. Further studies seem warranted to optimize the antibacterial activity of blue light-activated eosin Y on major oral pathogens.

Improved photodynamic activity of porphyrin loaded into nanoparticles: an in vivo evaluation using chick embryos.

Hydrophobic porphyrins are potentially interesting molecules for the photodynamic therapy (PDT) of solid cancers or ocular vascularization diseases. Their pharmaceutical development is, however, hampered by their lipophilicity, which renders formulation difficult especially when intravenous administration is needed. Encapsulation of a lipophilic derivative of porphyrin, the meso-tetra(p-hydroxyphenyl)porphyrin (p-THPP), into polymeric biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles proved to enhance its photodynamic activity against mammary tumour cells when compared to free drug. In order to further investigate these carriers, the efficacy of the encapsulated drug was assessed on the chick embryo chorioallantoic membrane (CAM) model. First, we identified a suitable solvent for the drug in terms of p-THPP solubility and tolerability by chick embryos. This solution was used as a reference. Then, the fluorescence pharmacokinetics and the photodynamic effects of the porphyrin on CAM vessels were evaluated after intravenous administration of either a p-THPP solution (free drug) or the drug loaded into nanoparticles. The results showed that: (i) the drug remained longer in the vascular compartment when incorporated into nanoparticles and (ii) vascular effects of p-THPP after light irradiation were enhanced with nanoparticle carriers. These results are discussed taking into account the extravasation of intravascular circulating photosensitizers and its influence on PDT performance.

Flow cytometric assessment of Streptococcus mutans viability after exposure to blue light-activated curcumin.

BACKGROUND: Streptococcus mutans biofilms are considered as primary causative agents of dental caries. Photodynamic antimicrobial chemotherapy (PACT) has been recently proposed as a strategy for inactivating dental biofilms. This study aimed to investigate the effect of blue light-activated curcumin on S. mutans viability and to explore its potential as a new anti-caries therapeutic agent. The effect of different concentrations and incubation times of photo-activated curcumin on the survival of S. mutans in planktonic and biofilm models of growth was assessed by flow cytometry. METHODS: Streptococcus mutans in planktonic suspensions or biofilms formed on hydroxyapatite disks were incubated for 5 or 10min with curcumin prior to blue light activation. Bacteria were labeled with SYTO 9 and propidium iodide before viability was assessed by flow cytometry. Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). RESULTS: For planktonic cultures, 0.2µM of light-activated curcumin significantly reduced S. mutans viability (p<0.05). For biofilm cultures, light-activated curcumin at concentration of 40-60µM only suppressed viability by 50% (p<0.05). Independently of the mode of growth, incubation time has no significant effect on PACT efficiency. CONCLUSION: This study indicates that blue light-activated curcumin can efficiently inactivate planktonic cultures of S. mutans whereas biofilms were more resistant to treatment. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to replicate and grow after cell sorting. Further studies seem warranted to optimize the efficacy of light-activated curcumin against S. mutans biofilms.

Antimicrobial activity and cytotoxicity of 3 photosensitizers activated with blue light.

INTRODUCTION: Pulp repair is less likely to occur when dentin or pulpal tissue remains infected after caries excavation. Yet there are currently few options to kill residual bacteria without damaging resident cells. The current study has evaluated the effect of 3 blue light-activated chemicals on the viability of lactobacilli, odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD21), and human embryonic stem cells (hESC H1). METHODS: Bacteria were incubated for 15 minutes with curcumin, eosin Y, or rose bengal and then irradiated with blue light (240 seconds). Bacteria were labeled with LIVE/DEAD BacLight Bacterial Viability kit, and viability was assessed by fluorescence-activated cell sorting. Cytotoxicity assays were performed on MDPC-23 cells, OD21, and hESC H1 cells grown in 24-well plates and exposed to the same photosensitizer-light combination. After 24 hours, cellular response was measured by using the methyl-thiazol-diphenyl-tetrazolium assay. Results were statistically analyzed by using one-way analysis of variance and Tukey multiple comparison intervals (α = 0.05). RESULTS: Bacterial viability was significantly reduced after exposure to different combinations of light and photosensitizers; mitochondrial activity of cultured cells remained unaffected when exposed to the same conditions, suggesting a good therapeutic index in vitro. CONCLUSIONS: Blue light-mediated disinfection is promising for the development of new treatment strategies designed to promote pulp repair after carious exposure.

Blue light-mediated inactivation of Enterococcus faecalis in vitro.

In dentistry, residual infection remains a major cause of failure after endodontic treatment; many of these infections involve Enterococcus faecalis. In the current study, we explored the possibility that blue light activated photosensitizers could be used, in principle, to inactivate this microbe as an adjunct disinfection strategy for endodontic therapy. Three blue light absorbing photosensitizers, eosin-Y, rose bengal, and curcumin, were tested on E. faecalis grown in planktonic suspensions or biofilms. Photosensitizers were incubated for 30 min with bacteria then exposed to blue light (450-500 nm) for 240 s. Sodium hypochlorite (3%) was used as a control. After 48 h, the viability of E. faecalis was estimated by measuring colony-forming units post-exposure vs. untreated controls (CFU/mL). Blue light irradiation alone did not alter E. faecalis viability. For planktonic cultures, blue light activated eosin-Y (5 µM), rose bengal (1 µM), or curcumin (5 µM) significantly (p<0.05) reduced E. faecalis viability compared to exposure to the unirradiated photochemicals. For biofilm cultures, concentrations of light-activated eosin-Y, rose bengal, and curcumin of 100, 10, and 10 µM respectively, completely suppressed E. faecalis viability (p<0.05). Although the current results are limited to an in vitro model, they support further exploration of blue light activated antimicrobials as an adjunct therapy in endodontic treatment.

Production of reactive oxygen species from photosensitizers activated with visible light sources available in dental offices.

OBJECTIVES: The aim of this study was to assess the ability of commonly available red- or blue-light dental sources to generate reactive oxygen species (ROS) from photosensitive chemicals that might be useful for photodynamic antimicrobial chemotherapy (PACT). BACKGROUND: Although the use of red diode lasers is well documented, there is limited information on how useful blue-light sources might be for PACT in dental contexts. MATERIALS AND METHODS: A diode laser (Periowave; see Table 1 for material and equipment sources) emitting red light (660-675 nm) was used to activate toluidine blue; riboflavin and pheophorbide-a polylysine (pheophorbide-a-PLL) were photoactivated using an Optilux 501 curing unit emitting blue light (380-500 nm). Ozone gas (generated by OzoTop, Tip Top Tips, Rolle, Switzerland), sodium hypochlorite, and hydrogen peroxide were used for comparison. ROS production was estimated using an iodine-triiodide colorimetric assay, and ROS levels were plotted versus concentration of chemicals to determine each chemical's efficiency in ROS production. One-way ANOVA with Tukey post hoc analysis (alpha = 0.05) was used to compare the efficiencies of ROS production for the various chemicals. RESULTS: Sodium hypochlorite, hydrogen peroxide, and ozone gas produced ROS spontaneously, whereas pheophorbide-a-PLL, riboflavin, and toluidine blue required light exposure. The efficiency of ROS production was higher for pheophorbide-a-PLL and toluidine blue than for ozone gas or riboflavin (p < 0.05). Hydrogen peroxide was the least efficient ROS producer. CONCLUSIONS: The results of the current study support the use of blue- or red-light-absorbing photosensitizers as candidates to produce ROS for clinical applications. Blue-light photosensitizers were as efficient as red-light photosensitizers in producing ROS and more efficient than the oxidant chemicals currently used for dental disinfection.

Toward the understanding of the photodynamic activity of m-THPP encapsulated in PLGA nanoparticles: correlation between nanoparticle properties and in vivo activity.

Biodegradable polymeric nanoparticles (NP) are promising delivery systems for photosensitizers (PS). A hydrophobic PS, the meso-tetra(p-hydroxyphenyl)porphyrin (m-THPP) was encapsulated into NP made of poly(D,L-lactide-co-glycolide) using an emulsification-diffusion method, and poly(vinyl alcohol) (PVAL) as stabilizing agent during the emulsification step. Three batches of NP with mean diameters of 117, 285, and 593 nm, respectively, were prepared. NP of 117 nm exhibited the highest rate of reactive oxygen species production and the fastest m-THPP release determined by the transfer of m-THPP to serum proteins in vitro. Similarly, the 117 nm batch exhibited the highest in vivo photodynamic activity, as established in the chick embryo chorioallantoic membrane model. Our data suggest that m-THPP release before illumination is an important step for in vivo photodynamic activity especially for large NP. The NP size and the amount of residual PVAL at the NP surface were shown to control both the PS release and activity.

Benefits of nanoencapsulation for the hypercin-mediated photodetection of ovarian micrometastases.

The high recurrence and lethality of ovarian cancer at advanced stages is problematic, especially due to the development of numerous micrometastases scattered throughout the abdominal cavity. Fluorescence photodetection (PD) used in combination with surgical resection of malignant tissues has been suggested to improve recovery. Based on promising in vivo results for the detection of bladder cancer, hypericin (Hy), a natural photosensitizer (PS), stands as a good candidate for the photodetection of ovarian cancer. However, due to its hydrophobicity, systemic administration of Hy is problematic. Polymeric nanoparticles (NPs) help to overcome these delivery and stability problems and enable intravenous administration of Hy. In this study, Hy-loaded NPs of polylactic acid were produced with the following properties: (i) mean size of 268 nm, (ii) negative zeta potential, (iii) low residual surfactant and (iv) drug loading of 3.7 % (w/w). The potential of hypericin-loaded nanoparticles for the fluorescence photodetection of ovarian metastases in Fischer 344 rats bearing ovarian tumours was compared to free drug. The selectivity of Hy administered with both formulations was assessed first by fluorescence endoscopy, and then quantified after tissue extraction. The results showed an improved selective accumulation of Hy in ovarian micrometastases when NPs were used.