New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

AB186 Inhibits Migration of Triple-Negative Breast Cancer Cells and Interacts with α-Tubulin.

Breast cancer is one of the leading causes of cancer-related death among females worldwide. A major challenge is to develop innovative therapy in order to treat breast cancer subtypes resistant to current treatment. In the present study, we examined the effects of two Troglitazone derivatives Δ2-TGZ and AB186. Previous studies showed that both compounds induce apoptosis, nevertheless AB186 was a more potent agent. The kinetic of cellular events was investigated by real-time cell analysis system (RTCA) in MCF-7 (hormone dependent) and MDA-MB-231 (triple negative) breast cancer (TNBC) cells, followed by cell morphology analysis by immuno-localization. Both compounds induced a rapid modification of both impedance-based signals and cellular morphology. This process was associated with an inhibition of cell migration measured by wound healing and transwell assays in TNBC MDA-MB-231 and Hs578T cells. In order to identify cytoplasmic targets of AB186, we performed surface plasmon resonance (SPR) and pull-down analyses. Subsequently, 6 cytoskeleton components were identified as potential targets. We further validated α-tubulin as one of the direct targets of AB186. In conclusion, our results suggested that AB186 could be promising to develop novel therapeutic strategies to treat aggressive forms of breast cancer such as TNBC.

In vivo characterization of physiological and metabolic changes related to isocitrate dehydrogenase 1 mutation expcression by multiparametric MRI and MRS in a rat model with orthotopically grafted human-derived glioblastoma cell lines.

The physiological mechanism induced by the isocitrate dehydrogenase 1 (IDH1) mutation, associated with better treatment response in gliomas, remains unknown. The aim of this preclinical study was to characterize the IDH1 mutation through in vivo multiparametric MRI and MRS. Multiparametric MRI, including the measurement of blood flow, vascularity, oxygenation, permeability, and in vivo MRS, was performed on a 4.7 T animal MRI system in rat brains grafted with human-derived glioblastoma U87 cell lines expressing or not the IDH1 mutation by the CRISPR/Cas9 method, and secondarily characterized with additional ex vivo HR-MAS and histological analyses. In univariate analyses, compared with IDH1-, IDH1+ tumors exhibited higher vascular density (p < 0.01) and better perfusion (p = 0.02 for cerebral blood flow), but lower vessel permeability (p < 0.01 for time to peak (TTP), p = 0.04 for contrast enhancement) and decreased T1 map values (p = 0.02). Using linear discriminant analysis, vascular density and TTP values were found to be independent MRI parameters for characterizing the IDH1 mutation (p < 0.01). In vivo MRS and ex vivo HR-MAS analysis showed lower metabolites of tumor aggressiveness for IDH1+ tumors (p < 0.01). Overall, the IDH1 mutation exhibited a higher vascularity on MRI, a lower permeability, and a less aggressive metabolic profile. These MRI features may prove helpful to better pinpoint the physiological mechanisms induced by this mutation.

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.

Growth-Inhibitory Effect of Chitosan-Coated Liposomes Encapsulating Curcumin on MCF-7 Breast Cancer Cells.

Current anticancer drugs exhibit limited efficacy and initiate severe side effects. As such, identifying bioactive anticancer agents that can surpass these limitations is a necessity. One such agent, curcumin, is a polyphenolic compound derived from turmeric, and has been widely investigated for its potential anti-inflammatory and anticancer effects over the last 40 years. However, the poor bioavailability of curcumin, caused by its low absorption, limits its clinical use. In order to solve this issue, in this study, curcumin was encapsulated in chitosan-coated nanoliposomes derived from three natural lecithin sources. Liposomal formulations were all in the nanometric scale (around 120 nm) and negatively charged (around -40 mV). Among the three lecithins, salmon lecithin presented the highest growth-inhibitory effect on MCF-7 cells (two times lower growth than the control group for 12 µM of curcumin and four times lower for 20 µM of curcumin). The soya and rapeseed lecithins showed a similar growth-inhibitory effect on the tumor cells. Moreover, coating nanoliposomes with chitosan enabled a higher loading efficiency of curcumin (88% for coated liposomes compared to 65% for the non-coated liposomes) and a stronger growth-inhibitory effect on MCF-7 breast cancer cells.

Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis, molecular docking study and in vitro biological activities.

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A165 binding to NRP-1 (IC50=39µM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.

Multifunctional ultrasmall nanoplatforms for vascular-targeted interstitial photodynamic therapy of brain tumors guided by real-time MRI.

Photodynamic therapy (PDT) for brain tumors appears to be complementary to conventional treatments. A number of studies show the major role of the vascular effect in the tumor eradication by PDT. For interstitial PDT (iPDT) of brain tumors guided by real-time imaging, multifunctional nanoparticles consisting of a surface-localized tumor vasculature targeting neuropilin-1 (NRP-1) peptide and encapsulated photosensitizer and magnetic resonance imaging (MRI) contrast agents, have been designed. Nanoplatforms confer photosensitivity to cells and demonstrate a molecular affinity to NRP-1. Intravenous injection into rats bearing intracranial glioma exhibited a dynamic contrast-enhanced MRI for angiogenic endothelial cells lining the neovessels mainly located in the peripheral tumor. By using MRI completed by NRP-1 protein expression of the tumor and brain adjacent to tumor tissues, we checked the selectivity of the nanoparticles. This study represents the first in vivo proof of concept of closed-head iPDT guided by real-time MRI using targeted ultrasmall nanoplatforms. From the clinical editor: The authors constructed tumor vascular peptide targeting multifunctional silica-based nanoparticles, with encapsulated gadolinium oxide as MRI contrast agent and chlorin as a photosensitizer, as a proof of concept novel treatment for glioblastoma in an animal model.

New Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1 for Anti-Vascular Targeted Photodynamic Therapy.

Photodynamic therapy (PDT) is a cancer treatment modality that requires three components, namely light, dioxygen and a photosensitizing agent. After light excitation, the photosensitizer (PS) in its excited state transfers its energy to oxygen, which leads to photooxidation reactions. In order to improve the selectivity of the treatment, research has focused on the design of PS covalently attached to a tumor-targeting moiety. In this paper, we describe the synthesis and the physico-chemical and photophysical properties of six new peptide-conjugated photosensitizers designed for targeting the neuropilin-1 (NRP-1) receptor. We chose a TPC (5-(4-carboxyphenyl)-10,15, 20-triphenyl chlorine as photosensitizer, coupled via three different spacers (aminohexanoic acid, 1-amino-3,6-dioxaoctanoic acid, and 1-amino-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid) to two different peptides (DKPPR and TKPRR). The affinity towards the NRP-1 receptor of the conjugated chlorins was evaluated along with in vitro and in vivo stability levels. The tissue concentration of the TPC-conjugates in animal model shows good distribution, especially for the DKPPR conjugates. The novel peptide-PS conjugates proposed in this study were proven to have potential to be further developed as future NRP-1 targeting photodynamic therapy agent.

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.

Added Value of Scintillating Element in Cerenkov-Induced Photodynamic Therapy.

Cerenkov-induced photodynamic therapy (CR-PDT) with the use of Gallium-68 (68Ga) as an unsealed radioactive source has been proposed as an alternative strategy to X-ray-induced photodynamic therapy (X-PDT). This new strategy still aims to produce a photodynamic effect with the use of nanoparticles, namely, AGuIX. Recently, we replaced Gd from the AGuIX@ platform with Terbium (Tb) as a nanoscintillator and added 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenylporphyrin (P1) as a photosensitizer (referred to as AGuIX@Tb-P1). Although Cerenkov luminescence from 68Ga positrons is involved in nanoscintillator and photosensitizer activation, the cytotoxic effect obtained by PDT remains controversial. Herein, we tested whether free 68Ga could substitute X-rays of X-PDT to obtain a cytotoxic phototherapeutic effect. Results were compared with those obtained with AGuIX@Gd-P1 nanoparticles. We showed, by Monte Carlo simulations, the contribution of Tb scintillation in P1 activation by an energy transfer between Tb and P1 after Cerenkov radiation, compared to the Gd-based nanoparticles. We confirmed the involvement of the type II PDT reaction during 68Ga-mediated Cerenkov luminescence, id est, the transfer of photon to AGuIX@Tb-P1 which, in turn, generated P1-mediated singlet oxygen. The effect of 68Ga on cell survival was studied by clonogenic assays using human glioblastoma U-251 MG cells. Exposure of pre-treated cells with AGuIX@Tb-P1 to 68Ga resulted in the decrease in cell clone formation, unlike AGuIX@Gd-P1. We conclude that CR-PDT could be an alternative of X-PDT.

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.

A state of the art in analytical quality-by-design and perspectives in characterization of nano-enabled medicinal products.

Quality-by-Design (QbD) guidance is a risk-based and proactive approach to drug development proposed in the early 2000s and now widely used in the pharmaceutical field in compliance with the ICH Q8-Q11 guidelines. Analytical Quality by Design (AQbD), introduced in 2010, is the adaptation of the QbD paradigm for the development of analytical methods. AQbD aims at optimizing the accuracy and robustness of analysis results by identifying and controlling critical analytical variables and method parameters over the entire protocol, including biological sample preparation, measurement technology and statistical analysis. Nevertheless, much remains to be done for a clear understanding and an efficient implementation of this new paradigm in practice. The first objective of this review is to propose a global clarification of the Analytical Quality by Design approach by reviewing its terminology and steps and by clarifying its relationships with the well-established QbD paradigm and ICH guidelines. Two new templates of documents have been proposed: a form designed for the definition of the analytical target profile and a connection matrix between expected metrological properties and analytical attributes. Finally, the open challenges in the characterization of nano-enabled medicinal products are examined from the AQbD angle.

Multi-tracer and multiparametric PET imaging to detect the IDH mutation in glioma: a preclinical translational in vitro, in vivo, and ex vivo study.

BACKGROUND: This translational study explores multi-tracer PET imaging for the non-invasive detection of the IDH1 mutation which is a positive prognostic factor in glioma. METHODS: U87 human high-grade glioma (HGG) isogenic cell lines with or without the IDH1 mutation (CRISP/Cas9 method) were stereotactically grafted into rat brains, and examined, in vitro, in vivo and ex vivo. PET imaging sessions, with radiotracers specific for glycolytic metabolism ([18F]FDG), amino acid metabolism ([18F]FDopa), and inflammation ([18F]DPA-714), were performed sequentially during 3-4 days. The in vitro radiotracer uptake was expressed as percent per million cells. For each radiotracer examined in vivo, static analyses included the maximal and mean tumor-to-background ratio (TBRmax and TBRmean) and metabolic tumor volume (MTV). Dynamic analyses included the distribution volume ratio (DVR) and the relative residence time (RRT) extracted from a reference Logan model. Ex vivo analyses consisted of immunological analyses. RESULTS: In vitro, IDH1+ cells (i.e. cells expressing the IDH1 mutation) showed lower levels of [18F]DPA-714 uptake compared to IDH1- cells (p < 0.01). These results were confirmed in vivo with lower [18F]DPA-714 uptake in IDH+ tumors (3.90 versus 5.52 for TBRmax, p = 0.03). Different values of [18F]DPA-714 and [18F] FDopa RRT (respectively 11.07 versus 22.33 and 2.69 versus - 1.81 for IDH+ and IDH- tumors, p < 0.02) were also observed between the two types of tumors. RRT [18F]DPA-714 provided the best diagnostic performance to discriminate between the two cell lines (AUC of 100%, p < 0.01). Immuno-histological analyses revealed lower expression of Iba-1 and TSPO antibodies in IDH1+ tumors. CONCLUSIONS: [18F]DPA-714 and [18F] FDopa both correlate with the presence of the IDH1 mutation in HGG. These radiotracers are therefore good candidates for translational studies investigating their clinical applications in patients.

Quantum dot-folic acid conjugates as potential photosensitizers in photodynamic therapy of cancer.

This study examined the in vitro potential of bioconjugated quantum dots (QDs) as photosensitizers for photodynamic therapy (PDT). According to our previous approaches using photosensitizers, folic acid appears to be an optimal targeting ligand for selective delivery of attached therapeutic agents to cancer tissues. We synthesized hydrophilic near infrared emitting CdTe(S)-type QDs conjugated with folic acid using different spacers. Photodynamic efficiency of QDs conjugated or not with folic acid was evaluated on KB cells, acting as a positive control due to their overexpression of FR-α, and HT-29 cells lacking FR-α, as negative control. A design of experiments was suggested as a rational solution to evaluate the impacts of each experimental factor (QD type and concentration, light fluence and excitation wavelength, time of contact before irradiation and cell phenotype). We demonstrated that, for concentrations lower than 10 nM, QDs displayed practically no cytotoxic effect without light exposure for both cell lines. Whereas QDs at 2.1 nM displayed a weak photodynamic activity, a concentration of 8 nM significantly enhanced the photodynamic efficiency characterized by a light dose-dependent response. A statistically significant difference in photodynamic efficiency between KB and HT-29 cells was evidenced in the case of folic acid-conjugated QDs. Optimal conditions led to an enhanced photocytotoxicity response, allowing us to validate the ability of QDs to generate a photodynamic effect and of folic acid-conjugated QDs for targeted PDT.

Terbium-Based AGuIX-Design Nanoparticle to Mediate X-ray-Induced Photodynamic Therapy.

X-ray-induced photodynamic therapy is based on the energy transfer from a nanoscintillator to a photosensitizer molecule, whose activation leads to singlet oxygen and radical species generation, triggering cancer cells to cell death. Herein, we synthesized ultra-small nanoparticle chelated with Terbium (Tb) as a nanoscintillator and 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenyl porphyrin (P1) as a photosensitizer (AGuIX@Tb-P1). The synthesis was based on the AGuIX@ platform design. AGuIX@Tb-P1 was characterised for its photo-physical and physico-chemical properties. The effect of the nanoparticles was studied using human glioblastoma U-251 MG cells and was compared to treatment with AGuIX@ nanoparticles doped with Gadolinium (Gd) and P1 (AguIX@Gd-P1). We demonstrated that the AGuIX@Tb-P1 design was consistent with X-ray photon energy transfer from Terbium to P1. Both nanoparticles had similar dark cytotoxicity and they were absorbed in a similar rate within the cells. Pre-treated cells exposure to X-rays was related to reactive species production. Using clonogenic assays, establishment of survival curves allowed discrimination of the impact of radiation treatment from X-ray-induced photodynamic effect. We showed that cell growth arrest was increased (35%-increase) when cells were treated with AGuIX@Tb-P1 compared to the nanoparticle doped with Gd.

The detrimental invasiveness of glioma cells controlled by gadolinium chelate-coated gold nanoparticles.

Glioblastoma are characterized by an invasive phenotype, which is thought to be responsible for recurrences and the short overall survival of patients. In the last decade, the promising potential of ultrasmall gadolinium chelate-coated gold nanoparticles (namely Au@DTDTPA(Gd)) was evidenced for image-guided radiotherapy in brain tumors. Considering the threat posed by invasiveness properties of glioma cells, we were interested in further investigating the biological effects of Au@DTDTPA(Gd) by examining their impact on GBM cell migration and invasion. In our work, exposure of U251 glioma cells to Au@DTDTPA(Gd) led to high accumulation of gold nanoparticles, that were mainly diffusely distributed in the cytoplasm of the tumor cells. Experiments pointed out a significant decrease in glioma cell invasiveness when exposed to nanoparticles. As the proteolysis activities were not directly affected by the intracytoplasmic accumulation of Au@DTDTPA(Gd), the anti-invasive effect cannot be attributed to matrix remodeling impairment. Rather, Au@DTDTPA(Gd) nanoparticles affected the intrinsic biomechanical properties of U251 glioma cells, such as cell stiffness, adhesion and generated traction forces, and significantly reduced the formation of protrusions, thus exerting an inhibitory effect on their migration capacities. Consistently, analysis of talin-1 expression and membrane expression of beta 1 integrin evoke the stabilization of focal adhesion plaques in the presence of nanoparticles. Taken together, our results highlight the interest in Au@DTDTPA(Gd) nanoparticles for the therapeutic management of astrocytic tumors, not only as a radio-enhancing agent but also by reducing the invasive potential of glioma cells.

Neuropilin-1 targeting photosensitization-induced early stages of thrombosis via tissue factor release.

PURPOSE: This article characterizes the vascular effects following vascular-targeted photodynamic therapy with a photosensitizer which actively targets endothelial cells. METHODS: This strategy was considered by coupling a chlorin to a heptapeptide targeting neuropilin-1 in human malignant glioma-bearing nude mice. A laser Doppler microvascular perfusion monitor was used to monitor microvascular blood perfusion in tumor tissue. Endothelial cells' ultra structural integrity was observed by transmission electron microscopy. The consequences of photosensitization on tumor vessels, tissue factor expression, fibrinogen consumption, and thrombogenic effects were studied by immunohistochemical staining. RESULTS: Treatment of glioma-bearing mice with the conjugate showed a statistically significant tumor growth delay. Vascular effect was characterized by a decrease in tumor tissue blood flow at about 50% baseline during treatment not related to variations in temperature. This vascular shutdown was mediated by tumor blood vessels' congestion. A pro-thrombotic behavior of targeted endothelial cells in the absence of ultra structural changes led to the induction of tissue factor expression from the earliest times post-treatment. Expression of tissue factor-initiated thrombi formation was also related to an increase in fibrinogen consumption. CONCLUSION: Using a peptide-conjugated photosensitizer targeting neuropilin-1, induction of tissue factor expression immediately post-treatment, led to the establishment of thrombogenic effects within the vessel lumen.

Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR(165) and molecules interfering with VEGF(165) binding to NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound, derived from d-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids arginine, and tryptophane or threonine. At 100 microM, all compounds exhibited a weak affinity for NRP-1, the most efficient being the bis-guanidinylated compound 32 (IC(50)=92 microM) which could be considered as a new NRP-1 non-peptidic ligand.

Overcoming the diverse mechanisms of multidrug resistance in lung cancer cells by photodynamic therapy using pTHPP-loaded PLGA-lipid hybrid nanoparticles.

Multidrug resistance (MDR) and the spread of cancer cells (metastasis) are major causes leading to failure of cancer treatment. MDR can develop in two main ways, with differences in their mechanisms for drug resistance, first drug-selected MDR developing after chemotherapeutic treatment, and metastasis-associated MDR acquired by cellular adaptation to microenvironmental changes during metastasis. This study aims to use a nanoparticle-mediated photodynamic therapy (NPs/PDT) approach to overcome both types of MDR. A photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP) was loaded into poly(D,L-lactide-co-glycolide) (PLGA)-lipid hybrid nanoparticles. The photocytotoxic effect of the nanoparticles was evaluated using two different MDR models established from one cell line, A549 human lung adenocarcinoma, including (1) A549RT-eto, a MDR cell line derived from A549 cells by drug-selection, and (2) detachment-induced MDR acquired by A549 cells when cultured as floating cells under non-adherent conditions, which mimic metastasizing cancer cells in the blood/lymphatic circulation. In the drug-selected MDR model, A549RT-eto cells displayed 17.4- and 1.8-fold resistance to Etoposide and Paclitaxel, respectively, compared to the A549 parental cells. In contrast to treatment with anticancer drugs, NPs/PDT with pTHPP-loaded nanoparticles resulted in equal photocytotoxic effect in A549RT-eto and parental cells. Intracellular pTHPP accumulation and light-induced superoxide anion generation were observed at similar levels in the two cell lines. The NPs/PDT killed A549RT-eto and parental cells through apoptosis as revealed by flow cytometry. In the metastasis-associated MDR model, A549 floating cells exhibited resistance to Etoposide (11.6-fold) and Paclitaxel (57.8-fold) compared to A549 attached cells, but the floating cells failed to show resistance against the photocytotoxic effect of the NPs/PDT. The MDR overcoming activity of NPs/PDT is mainly due to delivery ability of the PLGA-lipid hybrid nanoparticles. In conclusion, this work suggests that PLGA-lipid hybrid nanoparticles have potential in delivering photosensitizer or chemotherapeutic drug for treating both drug-selected and metastasis-associated MDR lung cancer cells.

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.