Preliminary Study of New Gallium-68 Radiolabeled Peptide Targeting NRP-1 to Detect Brain Metastases by Positron Emission Tomography.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = -1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

A new algorithm for a better characterization and timing of the anti-VEGF vascular effect named "normalization".

Antiangiogenics are widely used in cancer treatment in combination with chemotherapy and radiotherapy for their vascular effects. Antiangiogenics are supposed to induce morphological and functional changes in the chaotic tumor vasculature that would help enhance the therapeutic efficacy of chemotherapy and radiotherapy through the amelioration of the drug delivery or the oxygenation in the tumor, respectively. However, finding the best treatment sequence is not an easy task to achieve and no consensus has yet been established because of the lack of knowledge regarding when and for how long the vascular network is ameliorated. The aim of this work was to develop a dedicated image processing algorithm able to analyze the vascular structures on optical microscopy images of the vascular network and to follow its fine modifications in vivo, over time. We applied this algorithm to follow the evolution of the vascular parameters (vascularized tissue surface, branches, sprouts and length), in response or not to anti-VEGF therapy (10 mg/kg/day) and determine precisely whether there is really a vascular "normalization" with anti-VEGF therapy in comparison with the parameters extracted from healthy vascular networks. We found that for this determination, the choice of region of interest to analyze is critical as it is important to compare only microcirculation areas and avoid areas with arteriole-venule-capillary hierarchy. The algorithm analysis allowed us to define a vascular "normalization" in treated tumors, between 8 and 12 days of bevacizumab treatment that was confirmed by standard immunohistochemical analysis, microvascular permeability assessment and immunohistological blood perfusion assessment.

Non-targeted effects of radiation therapy for glioblastoma.

Radiotherapy is recommended for the treatment of brain tumors such as glioblastoma (GBM) and brain metastases. Various curative and palliative scenarios suggest improved local-regional control. Although the underlying mechanisms are not yet clear, additional therapeutic effects have been described, including proximity and abscopal reactions at the treatment site. Clinical and preclinical data suggest that the immune system plays an essential role in regulating the non-targeted effects of radiotherapy for GBM. This article reviews current biological mechanisms for regulating the non-targeted effects caused by external and internal radiotherapy, and how they might be applied in a clinical context. Optimization of therapeutic regimens requires assessment of the complexity of the host immune system on the activity of immunosuppressive or immunostimulatory cells, such as glioma-associated macrophages and microglia. This article also discusses recent preclinical models adapted to post-radiotherapy responses. This narrative review explores and discusses the current status of immune responses both locally via the "bystander effect" and remotely via the "abscopal effect". Preclinical and clinical observations demonstrate that unirradiated cells, near or far from the irradiation site, can control the tumor response. Nevertheless, previous studies do not address the problem in its global context, and present gaps regarding the link between the role of the immune system in the control of non-targeted effects for different types of radiotherapy and different fractionation schemes applied to GBM. This narrative synthesis of the scientific literature should help to update and critique available preclinical and medical knowledge. Indirectly, it will help formulate new research projects based on the synthesis and interpretation of results from a non-systematic selection of published studies.

Tumor-Infiltrating Lymphocyte Scoring in Neoadjuvant-Treated Breast Cancer.

Neoadjuvant chemotherapy (NAC) is now the standard of care for patients with locally advanced breast cancer (BC). TIL scoring is prognostic and adds predictive value to the residual cancer burden evaluation after NAC. However, NAC induces changes in the tumor, and the reliability of TIL scoring in post-NAC samples has not yet been studied. H&E- and dual CD3/CD20 chromogenic IHC-stained tissues were scored for stromal and intra-tumoral TIL by two experienced pathologists on pre- and post-treatment BC tissues. Digital TIL scoring was performed using the HALO® image analysis software (version 2.2). In patients with residual disease, we show a good inter-pathologist correlation for stromal TIL on H&E-stained tissues (CCC value 0.73). A good correlation for scoring with both staining methods (CCC 0.81) and the digital TIL scoring (CCC 0.77) was also observed. Overall concordance for TIL scoring in patients with a complete response was however poor. This study reveals there is good reliability for TIL scoring in patients with detectable residual tumors after NAC treatment, which is comparable to the scoring of untreated breast cancer patients. Based on the good consistency observed with digital TIL scoring, the development of a validated algorithm in the future might be advantageous.

Targeting Glioblastoma-Associated Macrophages for Photodynamic Therapy Using AGuIX®-Design Nanoparticles.

Glioblastoma (GBM) is the most difficult brain cancer to treat, and photodynamic therapy (PDT) is emerging as a complementary approach to improve tumor eradication. Neuropilin-1 (NRP-1) protein expression plays a critical role in GBM progression and immune response. Moreover, various clinical databases highlight a relationship between NRP-1 and M2 macrophage infiltration. In order to induce a photodynamic effect, multifunctional AGuIX®-design nanoparticles were used in combination with a magnetic resonance imaging (MRI) contrast agent, as well as a porphyrin as the photosensitizer molecule and KDKPPR peptide ligand for targeting the NRP-1 receptor. The main objective of this study was to characterize the impact of macrophage NRP-1 protein expression on the uptake of functionalized AGuIX®-design nanoparticles in vitro and to describe the influence of GBM cell secretome post-PDT on the polarization of macrophages into M1 or M2 phenotypes. By using THP-1 human monocytes, successful polarization into the macrophage phenotypes was argued via specific morphological traits, discriminant nucleocytoplasmic ratio values, and different adhesion abilities based on real-time cell impedance measurements. In addition, macrophage polarization was confirmed via the transcript-level expression of TNFα, CXCL10, CD-80, CD-163, CD-206, and CCL22 markers. In relation to NRP-1 protein over-expression, we demonstrated a three-fold increase in functionalized nanoparticle uptake for the M2 macrophages compared to the M1 phenotype. The secretome of the post-PDT GBM cells led to nearly a three-fold increase in the over-expression of TNFα transcripts, confirming the polarization to the M1 phenotype. The in vivo relationship between post-PDT efficiency and the inflammatory effects points to the extensive involvement of macrophages in the tumor zone.

Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment.

Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures.

Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity.

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-ß-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

Multiscale Selectivity and in vivo Biodistribution of NRP-1-Targeted Theranostic AGuIX Nanoparticles for PDT of Glioblastoma.

BACKGROUND: Local recurrences of glioblastoma (GBM) after heavy standard treatments remain frequent and lead to a poor prognostic. Major challenges are the infiltrative part of the tumor tissue which is the ultimate cause of recurrence. The therapeutic arsenal faces the difficulty of eradicating this infiltrating part of the tumor tissue while increasing the targeting of tumor and endogenous stromal cells such as angiogenic endothelial cells. In this aim, neuropilin-1 (NRP-1), a transmembrane receptor mainly overexpressed by endothelial cells of the tumor vascular system and associated with malignancy, proliferation and migration of GBM, highlighted to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (VTP). METHODS: The multiscale selectivity was investigated for KDKPPR peptide moiety targeting NRP-1 and a porphyrin molecule as photosensitizer (PS), both grafted onto original AGuIX design nanoparticle. AGuIX nanoparticle, currently in Phase II clinical trials for the treatment of brain metastases with radiotherapy, allows to achieve a real-time magnetic resonance imaging (MRI) and an accumulation in the tumor area by EPR (enhanced permeability and retention) effect. Using surface-plasmon resonance (SPR), we evaluated the affinities of KDKPPR and scramble free peptides, and also peptides-conjugated AGuIX nanoparticles to recombinant rat and human NRP-1 proteins. For in vivo selectivity, we used a cranial window model and parametric maps obtained from T2*-weighted perfusion MRI analysis. RESULTS: The photophysical characteristics of the PS and KDKPPR molecular affinity for recombinant human NRP-1 proteins were maintained after the functionalization of AGuIX nanoparticle with a dissociation constant of 4.7 µM determined by SPR assays. Cranial window model and parametric maps, both revealed a prolonged retention in the vascular system of human xenotransplanted GBM. Thanks to the fluorescence of porphyrin by non-invasive imaging and the concentration of gadolinium evaluated after extraction of organs, we checked the absence of nanoparticle in the brains of tumor-free animals and highlighted elimination by renal excretion and hepatic metabolism. CONCLUSION: Post-VTP follow-ups demonstrated promising tumor responses with a prolonged delay in tumor growth accompanied by a decrease in tumor metabolism.

Approaches to physical stimulation of metallic nanoparticles for glioblastoma treatment.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor. Despite new knowledges on the genetic characteristics, conventional therapy for GBM, tumor resection followed by radiotherapy and chemotherapy using temozolomide is limited in efficacy due to high rate of recurrence. GBM is indeed one of the most complex and difficult cancer to treat mainly due to its highly invasive properties and the standard treatments are thus rarely curative. Major challenges in the treatment of GBM are the limitation of irreversible brain damage, the infiltrative part of the tumor which is the ultimate cause of recurrence, the difficulty of identifying tumor margins and disseminated tumor cells, and the transport across the blood-brain barrier in order to obtain a sufficient therapeutic effect for pharmalogical agents. Considering these limitations, this review explores the in vivo potential of metal-based nanoparticles for hyperthermia, radiotherapy and photodynamic therapy. This article describes and clearly outlines the recent in vivo advances using innovative therapeutic metallic nanoparticles such as iron oxide, silver, gadolinium and gold nanoparticles.

Fighting Hypoxia to Improve PDT.

Photodynamic therapy (PDT) has drawn great interest in recent years mainly due to its low side effects and few drug resistances. Nevertheless, one of the issues of PDT is the need for oxygen to induce a photodynamic effect. Tumours often have low oxygen concentrations, related to the abnormal structure of the microvessels leading to an ineffective blood distribution. Moreover, PDT consumes O2. In order to improve the oxygenation of tumour or decrease hypoxia, different strategies are developed and are described in this review: 1) The use of O2 vehicle; 2) the modification of the tumour microenvironment (TME); 3) combining other therapies with PDT; 4) hypoxia-independent PDT; 5) hypoxia-dependent PDT and 6) fractional PDT.

Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma.

Despite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall.

A peptide competing with VEGF165 binding on neuropilin-1 mediates targeting of a chlorin-type photosensitizer and potentiates its photodynamic activity in human endothelial cells.

Destruction of the neovasculature is essential for efficient tumor eradication by photodynamic therapy (PDT). Since the over-expression of receptors for vascular endothelial growth factor (VEGF) is correlated with tumor angiogenesis and subsequent growth, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenyl-chlorin, TPC), via a spacer (6-aminohexanoic acid, Ahx), to a VEGF receptor-specific heptapeptide (ATWLPPR). ATWLPPR and TPC-Ahx-ATWLPPR bound exclusively to neuropilin-1 (NRP-1) recombinant chimeric protein (IC50=19 and 171 microM, respectively) but were devoid of affinity for VEGF receptor type 2 (VEGFR-2, KDR), to which ATWLPPR was initially thought to bind. TPC-Ahx-ATWLPPR was incorporated up to 25-fold more in human umbilical vein endothelial cells (HUVEC) than TPC over a 24-h period, and the addition of 8 mM ATWLPPR induced a significant decrease of this uptake (P<0.05), corroborating a receptor-mediated incorporation. Slightly less cytotoxic in the dark, TPC-Ahx-ATWLPPR exhibited enhanced in vitro photodynamic activity (10.4-fold), compared to TPC. Pharmacokinetic analysis in nude mice xenografted with U87 human malignant glioma cells revealed relevant tumor levels as soon as 1 h after intravenous injection of TPC-Ahx-ATWLPPR, and a rapid elimination from the blood compartment. Moreover, TPC-Ahx-ATWLPPR was not degraded in vivo up to 2 h after intravenous injection. Taken together, our results demonstrate that TPC-Ahx-ATWLPPR is a much more potent photosensitizer in vitro than TPC, in NRP-1-expressing cells. Thus, it may efficiently potentiate the vascular effect of PDT in vivo.

p27(Kip1) is a microtubule-associated protein that promotes microtubule polymerization during neuron migration.

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive, and the present work describes how p27(Kip1) controls cell-cycle-unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27(Kip1) show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, p27(Kip1) is a microtubule-associated protein that promotes polymerization of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27(Kip1) controls actomyosin contractions that drive both forward translocation of the nucleus and growth cone splitting. Thus, p27(Kip1) cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons.

Photodynamic therapy targeting neuropilin-1: Interest of pseudopeptides with improved stability properties.

The general strategy developed aims to favor the vascular effect of photodynamic therapy by targeting tumor vasculature. Since angiogenic endothelial cells represent an interesting target to potentiate this vascular effect, we previously described the conjugation of a photosensitizer to a peptide targeting neuropilins (NRPs) over-expressed specially in tumor angiogenic vessels and we recently characterized the mechanism of photosensitization-induced thrombogenic events. Nevertheless, in glioma-bearing nude mice, we demonstrated that the peptide moiety was degraded to various rates according to time after intravenous administration. In this study, new peptidases-resistant pseudopeptides were tested, demonstrating a molecular affinity for NRP-1 and NRP-2 recombinant chimeric proteins and devoid of affinity for VEGF receptor type 1 (Flt-1). To argue the involvement of NRP-1, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor. We evidenced a statistically significant decrease of the different peptides-conjugated photosensitizers uptake after RNA interference-mediated silencing of NRP-1. Peptides-conjugated photosensitizers allowed a selective accumulation into cells. In mice, no degradation was observed in plasma in vivo 4h after intravenous injection by MALDI-TOF mass spectrometry. This study draws attention to this potential problem with peptides, especially in the case of targeting strategies, and provides useful information for the future design of more stable molecules.

Peptide-conjugated chlorin-type photosensitizer binds neuropilin-1 in vitro and in vivo.

The strategy developed aims to favor the vascular effect of photodynamic therapy (PDT) by targeting tumor vasculature. This approach is considered by coupling a photosensitizer (PS) to an heptapeptide targeting neuropilin-1 (NRP-1). We previously demonstrated that this new conjugated PS, which binds to recombinant NRP-1 protein, was a much more potent PS compared to the non-conjugated PS in human umbilical vein endothelial cells (HUVEC) expressing NRP-1, due to the coupling of the peptide moiety. To argue the involvement of NRP-1 in the conjugated PS cellular uptake, MDA-MB-231 breast cancer cells were used, strongly over-expressing NRP-1 receptor, and we evidenced a significant decrease of the conjugated PS uptake after RNA interference-mediated silencing of NRP-1. In mice xenografted ectopically with U87 human malignant glioma cells, we demonstrated that only the conjugated PS allowed a selective accumulation in endothelial cells lining tumor vessels. Vascular endothelial growth factor (VEGF) plasma and tumor levels could not prevent the recognition of the conjugate by NRP-1. The vascular effect induced by the conjugated PS, was characterized by a reduction in tumor blood flow around 50% during PDT. In vivo, the photodynamic efficiency with the conjugated PS induced a statistically significant tumor growth delay compared to the non-coupled PS. The peptide-conjugated chlorin-type PS uptake involves NRP-1 and this targeting strategy favors the vascular effect of PDT in vivo.

Response surface methodology: an extensive potential to optimize in vivo photodynamic therapy conditions.

PURPOSE: Photodynamic therapy (PDT) is based on the interaction of a photosensitizing (PS) agent, light, and oxygen. Few new PS agents are being developed to the in vivo stage, partly because of the difficulty in finding the right treatment conditions. Response surface methodology, an empirical modeling approach based on data resulting from a set of designed experiments, was suggested as a rational solution with which to select in vivo PDT conditions by using a new peptide-conjugated PS targeting agent, neuropilin-1. METHODS AND MATERIALS: A Doehlert experimental design was selected to model effects and interactions of the PS dose, fluence, and fluence rate on the growth of U87 human malignant glioma cell xenografts in nude mice, using a fixed drug-light interval. All experimental results were computed by Nemrod-W software and Matlab. RESULTS: Intrinsic diameter growth rate, a tumor growth parameter independent of the initial volume of the tumor, was selected as the response variable and was compared to tumor growth delay and relative tumor volumes. With only 13 experimental conditions tested, an optimal PDT condition was selected (PS agent dose, 2.80 mg/kg; fluence, 120 J/cm(2); fluence rate, 85 mW/cm(2)). Treatment of glioma-bearing mice with the peptide-conjugated PS agent, followed by the optimized PDT condition showed a statistically significant improvement in delaying tumor growth compared with animals who received the PDT with the nonconjugated PS agent. CONCLUSIONS: Response surface methodology appears to be a useful experimental approach for rapid testing of different treatment conditions and determination of optimal values of PDT factors for any PS agent.

Tissue distribution and pharmacokinetics of an ATWLPPR-conjugated chlorin-type photosensitizer targeting neuropilin-1 in glioma-bearing nude mice.

Destruction of the neovasculature is essential for efficient tumor eradication by photodynamic therapy (PDT). The PDT anti-vascular effect can be promoted by developing addressed photosensitizers localized preferentially to the tumor vascular compartment. A new photosensitizer conjugated to an heptapeptide [H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH (ATWLPPR)] targeting neuropilin-1, a Vascular Endothelial Growth Factor (VEGF) co-receptor, has been synthesized. It was administered intravenously for an easier access to endothelial cells lining the vasculature in human malignant glioma-bearing nude mice. Plasma pharmacokinetic parameters were derived from plasma concentration-time data using a non-compartmental analysis and validated a relatively rapid elimination from the blood compartment with an elimination rate constant of 0.062 h(-1) and a biological half-life of 11.0 h. The photosensitizer was mainly concentrated in organs such as liver, spleen and kidneys, which are rich in reticuloendothelial cells. In these organs, the elimination profiles of the photosensitizer were comparable, with half-lives as short as 12.2, 15.1 and 19.7 h, respectively. The peptidic moiety of the conjugated photosensitizer was degraded to various rates depending on the organ considered, most of the degradation process occurred in organs of the reticuloendothelial system. A metabolic product resulting from the enzymatic cleavage of the peptide bond between Ala and Thr was detected in plasma at all the examined time points from 2 h post-injection. The conjugated photosensitizer accumulated rapidly and at high levels in the tumor, with 2.3% of injected dose per gram of tumor tissue at 1 h after injection. Taking into account the aspecific uptake of the degradation product, the tumor levels of total photoactivable compounds might exhibit an interesting photodynamic activity. On the contrary, levels of total photoactivable compounds remained low in the skin. This study provides essential information for the choice of the time interval not to exceed to activate the photosensitizer.

Metabolic profile of a peptide-conjugated chlorin-type photosensitizer targeting neuropilin-1: an in vivo and in vitro study.

Because angiogenic endothelial cells of the tumor vasculature represent an interesting target to potentiate the antivascular effect of photodynamic therapy, we recently described the conjugation of a photosensitizer [5-(4-carboxyphenyl)-10,15,20-triphenylchlorin (TPC)], via a spacer [6-aminohexanoic acid (Ahx)], to a vascular endothelial growth factor receptor-specific heptapeptide [H-Ala-Thr-Trp-Leu-Pro-Pro-Arg-OH (ATWLPPR)] and showed that TPC-Ahx-ATWLPPR binds to neuropilin-1. Because peptides often display low stability in biological fluids, we examined the in vivo and in vitro stability of this conjugate by high-performance liquid chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry. TPC-Ahx-ATWLPPR was stable in vitro in human and mouse plasma for at least 24 h at 37 degrees C but, following i.v. injection in glioma-bearing nude mice, was degraded in vivo to various rates, depending on the organ considered. TPC-Ahx-A was identified as the main metabolic product, and biodistribution studies suggested that its appearance in plasma mainly resulted from the degradation of the peptidic moiety into organs of the reticuloendothelial system. According to in vitro cell culture experiments, TPC-Ahx-ATWLPPR was also significantly degraded after incorporation in human umbilical vein endothelial cells (HUVEC), mainly into TPC-Ahx-A and to a lesser extent into TPC-Ahx-AT and TPC-Ahx-ATWLPP. TPC-Ahx-ATWLPPR mostly localized into lysosomes, and when HUVEC were treated with the lysosomal enzymes' inhibitor ammonium chloride, this resulted in a significant decrease of the peptide degradation. This study provides essential information for the choice of the time of activation of the photosensitizer (drug-light interval) not to be exceeded and for the future design of more stable molecules.

Recent improvements in the use of synthetic peptides for a selective photodynamic therapy.

Photodynamic therapy (PDT) is a relatively new cytotoxic treatment, predominantly used in anti-cancer approaches, that depends on the retention of photosensitizers in tumor and their activation after light exposure. Photosensitizers are photoactive compounds such as porphyrins and chlorins that upon photoactivation, effect strongly localized oxidative damage within the target cells. The ability to confine activation of the photosensitizer by restricting illumination to the tumor allows for a certain degree of selectivity. Nevertheless, the targeted delivery of photosensitizers to defined cells is a major problem in PDT of cancer, and one area of importance is photosensitizer targeting. Alterations or increased levels in receptor expression of specific cellular type occur in the diseased tissues. Therefore, photosensitizers can be covalently attached to molecules such as peptides, leading to a receptor-mediated targeting strategy. These active-targeting approaches may be particularly useful for anti-vascular PDT. Moreover, it has been shown that the photocytotoxicity of photodynamic drugs could be enhanced by delivering high amounts of a photosensitizer into subcellular organelles such as the nucleus where nucleic acids represent target molecules sensitive to photodamage. The recent progresses in the use of active-targeting strategy with synthetic peptides and the interest of using an active-targeting strategy in PDT, which could allow efficient cellular internalization of photosensitizers, are described in this review.