In Situ Bioconjugation of a Maleimide-Functionalized Ruthenium-Based Photosensitizer to Albumin for Photodynamic Therapy.

Maleimide-containing prodrugs can quickly and selectively react with circulating serum albumin following their injection in the bloodstream. The drug-albumin complex then benefits from longer blood circulation times and better tumor accumulation. Herein, we have applied this strategy to a previously reported highly phototoxic Ru polypyridyl complex-based photosensitizer to increase its accumulation at the tumor, reduce off-target cytotoxicity, and therefore improve its pharmacological profile. Specifically, two complexes were synthesized bearing a maleimide group: one complex with the maleimide directly incorporated into the bipyridyl ligand, and the other has a hydrophilic linker between the ligand and the maleimide group. Their interaction with albumin was studied in-depth, revealing their ability to efficiently bind both covalently and noncovalently to the plasma protein. A crucial finding is that the maleimide-functionalized complexes exhibited significantly lower cytotoxicity in noncancerous cells under dark conditions compared to the nonfunctionalized complex, which is a highly desirable property for a photosensitizer. The binding to albumin also led to a decrease in the phototoxicity of the Ru bioconjugates in comparison to the nonfunctionalized complex, probably due to a decreased cellular uptake. Unfortunately, this decrease in phototoxicity was not compensated by a dramatic increase in tumor accumulation, as was demonstrated in a tumor-bearing mouse model using inductively coupled plasma mass spectrometry (ICP-MS) studies. Consequently, this study provides valuable insight into the future design of in situ albumin-binding complexes for photodynamic therapy in order to maximize their effectiveness and realize their full potential.

Influence of Liposomes' and Lipoplexes' Physicochemical Characteristics on Their Uptake Rate and Mechanisms by the Placenta.

Pregnant women are still considered as drug orphans. Developing new medications for pregnancy complications is an urgent need. Nanomedicines seem to be a promising approach to control the biodistribution of drugs to ensure both the mother's and the fetus' safety. Understanding the interaction between nanoparticles and the placental barrier is a key factor to the success of the development of nanomedicines for pregnant women. In this study, we evaluated the behavior of fluorescent PEGylated liposomes and lipoplexes in human placental tissue using in vitro and ex vivo models, BeWo cell culture and suspended villous placental explants, respectively. Fluorescent based analytical tools such as Fluorescence activated cells sorting (FACS), confocal microscopy and HPLC coupled to fluorescence detection were used to assess liposomes penetration and their endocytosis mechanisms in the placenta. First, no influence of the PEGylation density was observed on the cellular internalization of liposomal formulations using both models. The comparison between neutral and cationic liposomes exhibits a significant higher internalization of the cationic formulation compared to the neutral ones. In addition, the HPLC quantification of the fluorescent liposomes in human villous explants demonstrated an increase of cationic liposomes uptake with increasing incubation concentrations. Similar uptake of cationic liposomes and lipoplexes, containing the same cationic lipid, the DMAPAP but with an overall neutral surface charge, was observed and evidenced the higher effect of composition than charge surface on trophoblast penetration. Moreover, both cationic liposomes and lipoplexes exhibited an endocytosis mechanism of internalization via pathways implicating dynamin. These data highlight the key role of the liposome's lipid composition and the possibility to modulate their internalization in the placenta by adjusting their design.

Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics.

Lactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.

Novel Perfluorinated Triblock Amphiphilic Copolymers for Lipid-Shelled Microbubble Stabilization.

Amphiphilic triblock (Atri) copolymers made of perfluorinated alkyl chain linked to hydrocarbon chain and methoxy-poly(ethylene glycol) of three different molecular weights were synthesized. In vitro evaluation demonstrated that these new compounds were noncytotoxic. Characterization and interaction of each triblock copolymer with a branched polyamine myristoyl lipid (2-{3[bis-(3-amino-propyl)-amino]-propylamino}- N-ditetradecyl carbamoyl methyl-acetamide, DMAPAP) were studied by the Langmuir film method and thermal analysis. The triblock copolymer/cationic lipids (1:10, w/w) were mixed with perfluorobutane gas to form microbubbles (MBs). The latter were characterized by optical microscopy to get the microbubble size and concentration by densimetry to determine the amount of encapsulated gas and by ultrasound to assess oscillation properties. Atri with the lowest and intermediate weights were shown to interact with the cationic lipid DMAPAP and stabilize the Langmuir film. In that case, monodisperse microbubbles ranging from 2.3 ± 0.1 to 2.8 ± 0.1 µm were obtained. The proportion of encapsulated gas within the MB shell increased up to 3 times after the incorporation of the copolymer with the lowest and intermediate weights. Moreover, the acoustic response of the microbubbles was maintained in the presence of the copolymers.

The in vivo activation of persistent nanophosphors for optical imaging of vascularization, tumours and grafted cells.

Optical imaging for biological applications requires more sensitive tools. Near-infrared persistent luminescence nanoparticles enable highly sensitive in vivo optical detection and complete avoidance of tissue autofluorescence. However, the actual generation of persistent luminescence nanoparticles necessitates ex vivo activation before systemic administration, which prevents long-term imaging in living animals. Here, we introduce a new generation of optical nanoprobes, based on chromium-doped zinc gallate, whose persistent luminescence can be activated in vivo through living tissues using highly penetrating low-energy red photons. Surface functionalization of this photonic probe can be adjusted to favour multiple biomedical applications such as tumour targeting. Notably, we show that cells can endocytose these nanoparticles in vitro and that, after intravenous injection, we can track labelled cells in vivo and follow their biodistribution by a simple whole animal optical detection, opening new perspectives for cell therapy research and for a variety of diagnosis applications.

Preparation and Evaluation of Multiple Nanoemulsions Containing Gadolinium (III) Chelate as a Potential Magnetic Resonance Imaging (MRI) Contrast Agent.

PURPOSE: The objective was to develop, characterize and assess the potentiality of W1/O/W2 self-emulsifying multiple nanoemulsions to enhance signal/noise ratio for Magnetic Resonance Imaging (MRI). METHODS: For this purpose, a new formulation, was designed for encapsulation efficiency and stability. Various methods were used to characterize encapsulation efficiency ,in particular calorimetric methods (Differential Scanning Calorimetry (DSC), thermogravimetry analysis) and ultrafiltration. MRI in vitro relaxivities were assessed on loaded DTPA-Gd multiple nanoemulsions. RESULTS: Characterization of the formulation, in particular of encapsulation efficiency was a challenge due to interactions found with ultrafiltration method. Thanks to the specifically developed DSC protocol, we were able to confirm the formation of multiple nanoemulsions, differentiate loaded from unloaded nanoemulsions and measure the encapsulation efficiency which was found to be quite high with a 68% of drug loaded. Relaxivity studies showed that the self-emulsifying W/O/W nanoemulsions were positive contrast agents, exhibiting higher relaxivities than those of the DTPA-Gd solution taken as a reference. CONCLUSION: New self-emulsifying multiple nanoemulsions that were able to load satisfactory amounts of contrasting agent were successfully developed as potential MRI contrasting agents. A specific DSC protocol was needed to be developed to characterize these complex systems as it would be useful to develop these self-formation formulations.

Radiofrequency Combined with Intratumoral Immunotherapy: Preclinical Results and Safety in Metastatic Colorectal Carcinoma.

Radiofrequency ablation (RFA) of cancer induces an anti-tumor immunity, which is insufficient to prevent recurrences. In mice, RFA-intratumoral immunotherapy by granulocyte-macrophage colony-stimulating factor (GM-CSF) and Bacillus Calmette-Guerin resulted in complete metastases regression. Infectious risk in human needs replacement of live vaccines. Intratumoral purified protein derivatives (PPD) have never been tested in digestive cancers, and the safety of intratumoral immunotherapy after RFA has not yet been validated in human models. We investigated the therapeutic efficacy of combined radiofrequency ablation (RFA) and intratumoral immunotherapy (ITI) using an immune-muco-adherent thermogel (IMT) in a mouse model of metastatic colorectal cancer (CRC) and the safety of this approach in a pig model. Intratumoral stability of the immunogel was assessed using magnetic resonance imaging (MRI) and bioluminescent imaging. Seventy-four CT26 tumor-bearing female BALB/c mice were treated with RFA either alone or in combination with intratumoral IMT. Regression of distant metastasis and survival were monitored for 60 days. Six pigs that received liver radiofrequency and intralesional IMT injections were followed for 15 days. Experimental gel embolisms were treated using an intravascular approach. Pertinent rheology of IMT was confirmed in tumors, by the signal stability during 3 days in MRI and 7 days in bioluminescence imaging. In mice, the abscopal effect of RFA-intratumoral immunotherapy resulted in regression of distant lesions completed at day 16 vs. a volume of 350 ± 99.3 mm3 in the RFA group at day 25 and a 10-fold survival rate at 60 days. In pigs, injection of immunogel in the liver RFA area was safe after volume adjustment without clinical, hematological, and liver biology disorder. Flow cytometry showed an early increase in CD3 TCRγδ+T cells at D7 (p < 0.05) and a late decrease in CD29+-CD8 T cells at D15 (p < 0.05), reflecting the inflammation status changes. Systemic GM-CSF release was not detectable. Experimental caval and pulmonary thermogel embolisms were treated by percutaneous catheterism and cold serum infusion. RFA-intratumoral immunotherapy as efficient and safe mini-invasive interventional oncology is able to improve ablative treatment of colorectal liver metastases.

Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model.

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Three in One: In Vitro and In Vivo Evaluation of Anticancer Activity of a Theranostic Agent that Combines Magnetic Resonance Imaging, Optical Bioimaging, and Photodynamic Therapy Capabilities.

Cancer treatment is a crucial area of research and development, as current chemotherapeutic treatments can have severe side effects or poor outcomes. In the constant search for new strategies that are localized and minimally invasive and produce minimal side effects, photodynamic therapy (PDT) is an exciting therapeutic modality that has been gaining attention. The use of theranostics, which combine diagnostic and therapeutic capabilities, can further improve treatment monitoring through image guidance. This study explores the potential of a theranostic agent consisting of four Gd(III) DTTA complexes (DTTA: diethylenetriamine-N,N,N″,N″-tetraacetate) grafted to a meso-tetraphenylporphyrin core for PDT, fluorescence, and magnetic resonance imaging (MRI). The agent was first tested in vitro on both nonmalignant TIB-75 and MRC-5 and tumoral CT26 and HT-29 cell lines and subsequently evaluated in vivo in a preclinical colorectal tumor model. Advanced MRI and optical imaging techniques were employed with engineered quantitative in vivo molecular imaging based on dynamic acquisition sequences to track the biodistribution of agents in the body. With 3D quantitative volume computed by MRI and tumoral cell function assessed by bioluminescence imaging, we could demonstrate a significant impact of the molecular agent on tumor growth following light application. Further exhaustive histological analysis confirmed these promising results, making this theranostic agent a potential drug candidate for cancer.

Colon tumor growth and antivascular treatment in mice: complementary assessment with MR elastography and diffusion-weighted MR imaging.

PURPOSE: To investigate the potential value of magnetic resonance (MR) elastography and diffusion-weighted (DW) MR imaging in the detection of microstructural changes of murine colon tumors during growth and antivascular treatment. MATERIALS AND METHODS: The study was approved by the regional ethics committee for animal care. Sixty Balb-C mice, bearing ectopic and orthotopic colon tumors, were monitored for 3 weeks with high-resolution T2-weighted MR imaging, three-dimensional steady-state MR elastography, and DW MR imaging at 7 T. The same imaging protocol was performed 24 hours after injection of combretastatin A4 phosphate (CA4P) in 12 mice. The absolute value of the complex shear modulus (|G*|) and the apparent diffusion coefficient (ADC) were measured in the viable zones of tumors and compared with microvessel density (MVD), cellularity, and micronecrosis by using the Pearson correlation coefficient. RESULTS: During tumor growth, |G*| increase was correlated with MVD (r = 0.70 [P = .08] and r = 0.78 [P = .002], for both the ectopic and orthotopic models, respectively). Moreover, the ectopic tumors displayed decreased ADC, which correlated with increased cellularity (r = 0.77, P = .04), whereas no changes in ADC and cellularity were observed in orthotopic tumors. After CA4P administration, |G*| decreased in the ectopic model (P < .0001), similar to the MVD evolution (P = .03), whereas no significant changes in |G*| (P = .7) and MVD (P = .6) were observed in the orthotopic model. ADC increased in both models (P = .047 and P = .01 for the ectopic and the orthotopic models, respectively) in relation to increased micronecrosis. CONCLUSION: Imaging of mechanical properties and diffusivity provide complementary information during tumor growth and regression that are respectively linked to vascularity and tumor cell alterations, including cellularity and micronecrosis.

In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles.

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.

Vascular density and endothelial cell expression of integrin alpha v beta 3 and E-selectin in murine tumours.

The endothelial cell adhesion molecules, including the integrin alpha v beta 3 (αvß3) and E-selectin, are involved in the process of angiogenesis required for tumour growth, cell migration and metastasis. The purpose of this study was to assess and compare widely used tumour models to select the ones most suitable for angiogenesis research. Fifteen murine tumours were selected including melanoma (B16), colon (C26, C38, C51), mammary (MA13, MA16, MA16/Adr, MA17, MA17/Adr, MA25, MA44), pancreatic (PO2, PO3), Glasgow osteogenic sarcoma (GOS) and Lewis lung carcinoma (LLC). The tumour vascular density, assessed using the platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31) immunostaining, revealed that B16 melanoma was poorly vascularized (<5 %), whereas the colon and mammary tumours were well vascularized (5-15 %). The most vascularized tumours (>15 %) were the pancreatic tumours (PO2 and PO3), the sarcoma (GOS) and the lung tumour (LLC). The integrin αvß3 and E-selectin, evaluated by immunohistology, showed that 7/15 tumours expressed the αvß3 integrin which was homogeneously distributed on all tumour sections (B16, C26, MA17/Adr, MA25, MA44, PO2, LLC). E-selectin was expressed in 4/15 tumours and its expression was restricted to the tumour periphery. Only 2/15 tumours (B16 and C26) were shown to express both integrin αvß3 and E-selectin. In conclusion, these data not only contribute to a better understanding of the tumour biology of murine tumours but can also guide the choice of appropriate models for antiangiogenic therapy, for selective drug delivery to tumours and the validation of tumour imaging modalities targeting these endothelial cell adhesion molecules.

Co-encapsulation of flavonoids with anti-cancer drugs: A challenge ahead.

Flavonoids have been considered as promising molecules for cancer treatment due to their pleiotropic properties such as anti-carcinogenic, anti-angiogenic or efflux proteins inhibition. However, due to their lipophilic properties and their chemical instability, vectorization seems compulsory to administer flavonoids. Flavonoids have been co-encapsulated with other anti-cancer agents in a broad range of nanocarriers aiming to i) achieve a synergistic/additive effect at the tumor site, ii) delay drug resistance apparition by combining agents with different action mechanisms or iii) administer a lower dose of the anti-cancer drug, reducing its toxicity. However, co-encapsulation could lead to a change in the nanoparticles' diameter and drug-loading, as well as a decrease in their stability during storage. The preparation process should also take into accounts the physico-chemical properties of both the flavonoid and the anti-cancer agent. Moreover, the co-encapsulation could affect the release and activity of each drug. This review aims to study the formulation, preparation and characterization strategies of these co-loaded nanomedicines, as well as their stability. The in vitro assays to predict the nanomedicines' behavior in biological fluids, as well as their in vivo efficacy, are also discussed. A special focus concerns the evaluation of their synergistic effect on tumor treatment.

In vivo ultrasound modulated optical tomography with a persistent spectral hole burning filter.

We present in vivo ultrasound modulated optical tomography (UOT) results on mice, using the persistent spectral hole burning (PSHB) effect in a Tm3+:YAG crystal. Indocyanine green (ICG) solution was injected as an optical absorber and was clearly identified on the PSHB-UOT images, both in the muscle (following an intramuscular injection) and in the liver (following an intravenous injection). This demonstration also validates an experimental setup with an improved level of performance combined with an increased technological maturity compared to previous demonstrations.

Persistent luminescence nanoparticles functionalized by polymers bearing phosphonic acid anchors: synthesis, characterization, and in vivo behaviour.

Optical in vivo imaging has become a widely used technique and is still under development for clinical diagnostics and treatment applications. For further development of the field, researchers have put much effort into the development of inorganic nanoparticles (NPs) as imaging probes. In this trend, our laboratory developed ZnGa1.995O4Cr0.005 (ZGO) nanoparticles, which can emit a bright persistent luminescence signal through the tissue transparency window for dozens of minutes and can be activated in vivo with visible irradiation. These properties endow them with unique features, allowing us to recover information over a long-time study with in vivo imaging without any background. To target tissues of interest, ZGO must circulate long enough in the blood stream, a phenomenon which is limited by the mononuclear phagocyte system (MPS). Depending on their size, charge and coating, the NPs are sooner or later opsonized and stored into the main organs of the MPS (liver, spleen, and lungs). The NPs therefore have to be coated with a hydrophilic polymer to avoid this limitation. To this end, a new functionalization method using two different polyethylene glycol phosphonic acid polymers (a linear one, later named lpPEG and a branched one, later named pPEG) has been studied in this article. The coating has been optimized and characterized in various aqueous media. The behaviour of the newly functionalized NPs has been investigated in the presence of plasmatic proteins, and an in vivo biodistribution study has been performed. Among them ZGOpPEG exhibits a long circulation time, corresponding to low protein adsorption, while presenting an effective one-step process in aqueous medium with a low hydrodynamic diameter increase. This new method is much more advantageous than another strategy we reported previously that used a two-step PEG silane coating performed in an organic solvent (dimethylformamide) for which the final hydrodynamic diameter was twice the initial diameter.

Fate and biological impact of persistent luminescence nanoparticles after injection in mice: a one-year follow-up.

Persistent luminescence nanoparticles (PLNPs) are attracting growing interest for non-invasive optical imaging of tissues with a high signal to noise ratio. PLNPs can emit a persistent luminescence signal through the tissue transparency window for several minutes, after UV light excitation before systemic administration or directly in vivo through visible irradiation, allowing us to get rid of the autofluorescence signal of tissues. PLNPs constitute a promising alternative to the commercially available optical near infrared probes thanks to their versatile functionalization capabilities for improvement of the circulation time in the blood stream. Nevertheless, while biodistribution for a short time is well known, the long-term fate and toxicity of the PLNP's inorganic core after injection have not been dealt with in depth. Here we extend the current knowledge on ZnGa1.995O4Cr0.005 NPs (or ZGO) with a one-year follow-up of their fate after a single systemic administration in mice. We investigated the organ tissue uptake of ZGO with two different coatings and determined their intracellular processing up to one year after injection. The biopersistence of ZGO was assessed, with a long-term retention, quantified by ICP-MS, mostly in the liver and spleen, parallel with a loss of their luminescence properties. The analysis of the toxicity related to combining an animal's weight, key hematological and metabolic markers, histological observations of liver tissues and quantification of the expression of 31 genes linked to different metabolic reactions did not reveal any signs of noxiousness, from the macro scale to the molecular level. Therefore, the ZGO imaging probe has been proven to be a safe and relevant candidate for preclinical studies, allowing its long term use without any in vivo disturbance of the general metabolism.

Combination of thermal ablation by focused ultrasound, pFAR4-IL-12 transfection and lipidic adjuvant provide a distal immune response.

Aim: Gene-based immunotherapy against cancer is limited by low gene transfer efficiency. In the literature, interleukin-12 (IL-12) encoding plasmid associated with sonoporation has been shown to enhance antitumoral activity. Moreover, non-viral carriers and high-frequency ultrasound have both been shown to promote immune response activation. Here, IL-12 encoding plasmid, non-viral carrier stimulating the immune response and focused ultrasound were combined in order to improve the antitumoral efficiency. Methods: In order to enhance a gene-based antitumoral immune response, home-made lipids Toll-like receptor 2 (TLR2) agonists and plasmid free of antibiotic resistance version 4 (pFAR4), a mini-plasmid, encoding the IL-12 cytokine were combined with high-intensity focused ultrasound (HIFU). The lipid composition and the combination conditions were selected following in vitro and in vivo preliminary studies. The expression of IL-12 from our plasmid construct was measured in vitro and in vivo. The combination strategy was evaluated in mice bearing colon carcinoma cells (CT26) tumors following their weight, tumor volume, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels in the serum and produced by splenocytes exposed to CT26 tumor cells. Results: Lipid-mediated cell transfection and intratumoral injection into CT26 tumor mice using pFAR4-IL-12 led to the secretion of the IL-12 cytokine into cell supernatant and mice sera, respectively. Conditions of thermal deposition using HIFU were optimized. The plasmid encoding pFAR4-IL-12 or TLR2 agonist alone had no impact on tumor growth compared with control mice, whereas the complete treatment consisting of pFAR4-IL-12, TLR2 lipid agonist, and HIFU limited tumor growth. Moreover, only the complete treatment increased significantly mice survival and provided an abscopal effect on a metastatic CT26 model. Conclusions: The HIFU condition was highly efficient to stop tumor growth. The combined therapy was the most efficient in terms of IL-12 and IFN-γ production and mice survival. The study showed the feasibility and the limits of this combined therapy which has the potential to be improved.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy.

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at -80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Muscle transfection and permeabilization induced by electrotransfer or pluronic L64: Paired study by optical imaging and MRI.

BACKGROUND: Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein. METHODS: Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed. RESULTS: Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7 days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection. GENERAL SIGNIFICANCE: It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.

Combination of tumor cell anti-adhesion and anti-tumor effect to prevent recurrence after cytoreductive surgery in a mice model.

One of the main problems of colorectal cancer is not the treatment of the primary tumor but the metastatic stage. Means of metastatic spread is the invasion of the peritoneal cavity which leads to peritoneal metastasis (PM). PM cannot be easily cured, and the current treatments is rather heavy, combining cytoreductive surgery with intravenous and intraperitoneal chemotherapy. This therapeutic procedure is associated with significant morbidity, altered patient quality of life and poor prognosis. We postulated that development of a prophylactic treatment could be of high interest in this context. In this study, we formulated an anti-adhesive thermogel which contains chemotherapeutics to play a role of a barrier against tumor cells implantation, avoiding their adhesion and treating the remaining tumor cells with chemotherapy intraperitoneally in a mice model of PM. The bioavailability of the thermogel was tested intraperitoneally in mice. No sign of toxicity was observed in terms of change in body weight, anatomopathology and blood biomarkers. In vitro experiments proved that the thermogel induced limited adhesion of the tumor cells. Loading of oxaliplatin (Ox) and 5-Fluorouracil (5-FU) into the thermogel were able to significantly decreased peritoneal carcinomatosis index (PCI) (-58%) and ascites (-70%) in a murine model of peritoneal metastases. These pre-clinical results confirmed that smart thermogel associated with standard chemotherapy 5-FU and Ox could be a good candidate to decrease the risk of tumor cell implantation during cytoreductive surgery and prevent future metastatic process.