Step-By-Step Standardization of the Bottom-Up Semi-Automated Nanocrystallization of Pharmaceuticals: A Quality By Design and Design of Experiments Joint Approach.

Nanosized drug crystals have been reported with enhanced apparent solubility, bioavailability, and therapeutic efficacy compared to microcrystal materials, which are not suitable for parenteral administration. However, nanocrystal design and development by bottom-up approaches are challenging, especially considering the non-standardized process parameters in the injection step. This work aims to present a systematic step-by-step approach through Quality-by-Design (QbD) and Design of Experiments (DoE) for synthesizing drug nanocrystals by a semi-automated nanoprecipitation method. Curcumin is used as a drug model due to its well-known poor water solubility (0.6 µg mL-1, 25 °C). Formal and informal risk assessment tools allow identifying the critical factors. A fractional factorial 24-1 screening design evaluates their impact on the average size and polydispersity of nanocrystals. The optimization of significant factors is done by a Central Composite Design. This response surface methodology supports the rational design of the nanocrystals, identifying and exploring the design space. The proposed joint approach leads to a reproducible, robust, and stable nanocrystalline preparation of 316 nm with a PdI of 0.217 in compliance with the quality profile. An orthogonal approach for particle size and polydispersity characterization allows discarding the formation of aggregates. Overall, the synergy between advanced data analysis and semi-automated standardized nanocrystallization of drugs is highlighted.

Understanding the Impact of Polyunsaturated Fatty Acids on Age-Related Macular Degeneration: A Review.

Age-related Macular Degeneration (AMD) is a multifactorial ocular pathology that destroys the photoreceptors of the macula. Two forms are distinguished, dry and wet AMD, with different pathophysiological mechanisms. Although treatments were shown to be effective in wet AMD, they remain a heavy burden for patients and caregivers, resulting in a lack of patient compliance. For dry AMD, no real effective treatment is available in Europe. It is, therefore, essential to look for new approaches. Recently, the use of long-chain and very long-chain polyunsaturated fatty acids was identified as an interesting new therapeutic alternative. Indeed, the levels of these fatty acids, core components of photoreceptors, are significantly decreased in AMD patients. To better understand this pathology and to evaluate the efficacy of various molecules, in vitro and in vivo models reproducing the mechanisms of both types of AMD were developed. This article reviews the anatomy and the physiological aging of the retina and summarizes the clinical aspects, pathophysiological mechanisms of AMD and potential treatment strategies. In vitro and in vivo models of AMD are also presented. Finally, this manuscript focuses on the application of omega-3 fatty acids for the prevention and treatment of both types of AMD.

H2 O2 -Induced Persistent Luminescence Signal Enhancement Applied to Biosensing.

Persistent luminescence nanoparticles (PLNPs) are innovative materials able to emit light for a long time after the end of their excitation. Thanks to this property, their detection can be separated in time from the excitation, making it possible to obtain images with a high signal-to-noise ratio. This optical property can be of particular interest for the development of in vitro biosensors. Here, we report the unexpected effect of hydrogen peroxide (H2 O2 ) on the signal intensity of ZnGa2 O4 :Cr3+ (ZGO) nanoparticles. In the presence of H2 O2 , the signal intensity of ZGO can be amplified. This signal amplification can be used to detect and quantify H2 O2 in various media, using non-functionalized ZGO nanoparticles. This small molecule can be produced by several oxidases when they react with their substrate. Indeed, the quantification of glucose, lactic acid, and uric acid is possible. The limit of detection could be lowered by modifying the nanoparticles synthesis route. These optimized nanoparticles can also be used as new biosensor to detect larger molecules such as antigen, using the appropriate antibody. This unique property, i.e., persistent luminescence signal enhancement induced by H2 O2 , represents a new way to detect biomolecules which could lead to a very large number of bioassay applications.

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.

Modeling study of long-term stability of the monoclonal antibody infliximab and biosimilars using liquid-chromatography-tandem mass spectrometry and size-exclusion chromatography-multi-angle light scattering.

Monoclonal antibodies (mAbs) represent a dynamic class of biopharmaceutical products, as evidenced by an increasing number of market authorizations for mAb innovator and biosimilar products. Stability studies are commonly performed during product development, for instance, to exclude unstable molecules, optimize the formulation or determine the storage limit. Such studies are time-consuming, especially for mAbs, because of their structural complexity which requires multiple analytical techniques to achieve a detailed characterization. We report the implementation of a novel methodology based on the accelerated stability assessment program (ASAP) in order to model the long-term stability of mAbs in relation to different structural aspects. Stability studies of innovator infliximab and two different biosimilars were performed using forced degradation conditions alongside in-use administration conditions in order to investigate their similarity regarding stability. Thus, characterization of post-translational modifications was achieved using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the formation of aggregates and free chain fragments was characterized using size-exclusion chromatography-multi-angle light scattering (SEC-MALS-UV/RI) analysis. Consequently, ASAP models were investigated with regard to free chain fragmentation of mAbs concomitantly with N57 deamidation, located in the hypervariable region. Comparison of ASAP models and the long-term stability data from samples stored in intravenous bags demonstrated a relevant correlation, indicating the stability of the mAbs. The developed methodology highlighted the particularities of ASAP modeling for mAbs and demonstrated the possibility to independently consider the different types of degradation pathways in order to provide accurate and appropriate prediction of the long-term stability of this type of biomolecule.

Structural identification and absolute quantification of monoclonal antibodies in suspected counterfeits using capillary electrophoresis and liquid chromatography-tandem mass spectrometry.

Monoclonal antibodies (mAbs) represent a major category of biopharmaceutical products which due to their success as therapeutics have recently experienced the emergence of mAbs originating from different types of trafficking. We report the development of an analytical strategy which enables the structural identification of mAbs in addition to comprehensive characterization and quantification in samples in potentially counterfeit samples. The strategy is based on the concomitant use of capillary zone electrophoresis analysis (CZE-UV), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). This analytical strategy was applied to the investigation of different samples having unknown origins seized by the authorities, and potentially incorporating an IgG 4 or an IgG 1. The results achieved from the different techniques demonstrated to provide orthogonal and complementary information regarding the nature and the structure of the different mAbs. Therefore, they allowed to conclude unequivocally on the identification of the mAbs in the potentially counterfeit samples. Finally, a LC-MS/MS quantification method was developed which specificity was to incorporate a different mAbs labeled with stable isotopes as internal standard. The LC-MS/MS quantification method was validated and thus demonstrated the possibility to use common peptides with the considered IgG in order to achieve limit of quantification as low as 41.4 nM. The quantification method was used to estimate the concentration in the investigated samples using a single type of internal standard and experimental conditions, even in the case of mAbs with no stable isotope labeled homologues available.

Stability of calcium levofolinate, 5-fluorouracil and oxaliplatin (FOLFOX) mixture.

FOLFOX is the most common chemotherapy combination prescribed in colorectal cancer. It is composed of calcium levofolinate, 5-fluorouracil and oxaliplatin which demonstrated synergistic outcome. Nowadays, the lack of all-in-one formulation is due to the chemical composition of the pharmaceutical products and the highly pH-dependent stability of each drug. Herein, we aimed to investigate the stability of a ternary mixture of 5-fluorouracil, oxaliplatin and calcium levofolinate, knowing that coadministering these drugs would improve their efficacy. The effect of three pHs (5.0, 6.0 and 7.5) and two drug concentrations (8/3/6 and 1/1/1 mg/ml for 5-fluorouracil, oxaliplatin and calcium levofolinate, respectively) were examined. A high-performance liquid chromatography method was developed to separate and quantify the three drugs in one run. At higher concentrations, the ternary mixture was unstable regardless of pH. By reducing concentration, drug stability and compatibility in the mixture was improved at pH 5.0 for up to 3 days at +5°C ± 3 °C. In addition, binary mixtures provided stable properties at defined pHs. 5-fluorouracil/oxaliplatin mixture was stable at pH 5.0 over 48 hours while 5-fluorouracil/calcium levofolinate mixture was stable at pHs 6.0 and 7.5 up to 7 days.

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.

Electrokinetic elucidation of the interactions between persistent luminescent nanoprobes and the binary apolipoprotein-E/albumin protein system.

The affinity between functional nanoparticles (NPs) and proteins could determine the efficacy of nanoprobes, nanosensors, nanocarriers, and many other devices for biomedical applications. Therefore, it is necessary to develop analytical strategies to accurately evaluate the magnitude of these protein corona interactions in physiological media. In this work, different electrokinetic strategies were implemented to accurately determine the interactions between PEGylated ZnGa1.995Cr0.005O4 persistent luminescent NPs (ZGO-PEG) and two important serum proteins: human serum albumin (HSA), the most abundant serum protein, and apolipoprotein-E (ApoE), associated with the active transport of NPs through the blood-brain barrier. Firstly, the injection of ZGO-PEG in a background electrolyte (BGE) containing individual proteins allowed an affinity study to separately characterize each NP-protein system. Then, the same procedure was applied in a buffer containing a mixture of the two proteins at different molar ratios. Finally, the NPs were pre-incubated with one protein and thereafter electrokinetically separated in a BGE containing the second protein. These analytical strategies revealed the mechanisms (comparative, cooperative or competitive systems) and the magnitude of their interactions, resulting in all cases in notably higher affinity and stability between ZGO-PEG and ApoE (Ka = 1.96 ± 0.25 × 1010 M-M) compared to HSA (Ka = 4.60 ± 0.41 × 106 M-M). For the first time, the inter-protein ApoE/HSA interactions with ZGO-PEG were also demonstrated, highlighting the formation of a ternary ZGO-PEG/ApoE/HSA nanocomplex. These results open the way for a deeper understanding of the protein corona formation, and the development of versatile optical imaging applications for ZGO-PEG and other systemically delivered nanoprobes ideally vectorized to the brain.

Comprehensive and quantitative stability study of ascorbic acid using capillary zone electrophoresis with ultraviolet detection and high-resolution tandem mass spectrometry.

Ascorbic acid is a powerful antioxidant compound involved in many biological functions, and a chronic deficiency is at the origin of scurvy disease. A simple, rapid, and cost-effective capillary electrophoresis method was developed for the separation and simultaneous quantification of ascorbic acid and the major degradation products: dehydroascorbic acid, furfural, and furoic acid. Systematic optimization of the conditions was performed that enabled baseline separation of the compounds in less than 10 min. In addition to simultaneous quantification of ascorbic acid alongside to the degradation products, stability studies demonstrated the possibility using capillary electrophoresis to separate and identify the major degradation products. Thus, high-resolution tandem mass spectrometry experiments were conducted in order to identify an unknown degradation product separated by capillary electrophoresis and significantly present in degraded samples. Comparison of mass spectrometry data and capillary electrophoresis electropherograms allowed to identify unambiguously trihydroxy-keto-valeraldehyde. Finally, capillary electrophoresis was successfully applied to evaluate the composition of different pharmaceutical preparation of ascorbic acid. Results showed the excellent performance of the capillary electrophoresis method due to the separation of excipients from the compounds of interest, which demonstrated the relevance of using an electrophoretic separation in order to perform comprehensive stability studies of ascorbic acid.

In vitro distinction between proinflammatory and antiinflammatory macrophages with gadolinium-liposomes and ultrasmall superparamagnetic iron oxide particles at 3.0T.

BACKGROUND: Inflammation involves a heterogeneous macrophage population, for which there is no readily available MR assessment method. PURPOSE: To assess the feasibility of distinguishing proinflammatory M1 and antiinflammatory M2 macrophages at MRI enhanced with gadolinium liposomes or ultrasmall superparamagnetic iron oxide particles. STUDY TYPE: In vitro. SPECIMEN: We employed cultured RAW macrophages. M0 macrophages were polarized with lipopolysaccharide (LPS) or interleukin-4 (IL-4), resulting in M1 or M2 macrophages. The macrophages were incubated with gadolinium (±rhodamine) liposomes or iron oxide particles and cell pellets were prepared for MRI. FIELD STRENGTH/SEQUENCE: Transverse relaxation rates and quantitative susceptibility were obtained at 3.0T with multiecho turbo spin echo and spoiled gradient echo sequences. ASSESSMENT: MRI results were compared with confocal microscopy, flow cytometry, and expression of endocytosis, M1 and M2 genes. STATISTICAL TESTS: Mann-Whitney and Kruskal-Wallis tests were performed. RESULTS: Higher transverse relaxation rates and susceptibility were observed in M1 than in M2 and M0 macrophages (P < 0.01 both with liposomes and USPIO) and significantly different susceptibility in M2 and M0 macrophages (P < 0.01 both with liposomes and USPIO). These MRI results were confirmed at confocal microscopy and flow cytometry. LPS macrophages displayed M1 gene expression, whereas IL-4 macrophages showed M2 polarization and lower endocytosis gene expression rates. DATA CONCLUSION: These in vitro results show that it is feasible to distinguish between proinflammatory M1 and antiinflammatory M2 macrophages according to their level of contrast agent uptake at MRI. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:1166-1173.

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.

Liposomes as Gene Delivery Vectors for Human Placental Cells.

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

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.

Thymoquinone-loaded self-nano-emulsifying drug delivery system against ischemia/reperfusion injury.

In the present study, a self-nano-emulsifying drug delivery system (SNEDDS) was developed to evaluate the efficiency of thymoquinone (TQ) in hepatic ischemia/reperfusion. SNEDDS was pharmaceutically characterized to evaluate droplet size, morphology, zeta potential, thermodynamic stability, and dissolution/diffusion capacity. Animals were orally pre-treated during 10 days with TQ-loaded SNEDDS. Biochemical analyses, hematoxylin-eosin staining, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR) were carried out to assess cell injury, oxidative stress, inflammation, and apoptosis. The TQ formulation showed good in vitro characteristics, including stable nanoparticle structure and size with high drug release rate. In vivo determinations revealed that TQ-loaded SNEDDS pre-treatment of rats maintained cellular integrity by decreasing transaminase (ALT and AST) release and preserving the histological characteristics of their liver. The antioxidant ability of the formulation was proven by increased SOD activity, reduced MDA concentration, and iNOS protein expression. In addition, this formulation exerted an anti-inflammatory effect evidenced by reduced plasma CRP concentration, MPO activity, and gene expressions of TLR-4, TNF-α, NF-κB, and IL-6. Finally, the TQ-loaded SNEDDS formulation promoted cell survival by enhancing the Bcl-2/Bax ratio. In conclusion, our results indicate that TQ encapsulated in SNEDDS significantly protects rat liver from I/R injury.

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.

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.

Investigating the Wound-Healing Potential of a Nanoemulsion-Gel Formulation of Pituranthos tortuosus Essential Oil.

This study explores a nanoemulsion (NE)-based gel incorporating Tunisian Pituranthos tortuosus essential oil, with a focus on its wound-healing potential. The essential oil, extracted via hydrodistillation, underwent GC-MS analysis for compositional verification. The physicochemical characterization included dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential measurement, pH, and viscosity. The gelification of the NE facilitated topical application. The results revealed an average extraction yield of 0.45% and identified 38 compounds in the essential oil. The NE exhibited a particle size of 27 ± 0.4 nm, a polydispersity index (PDI) of 0.3, and a zeta potential of -22.8 ± 1.4 mV. The stability of the gelified preparation was confirmed through thermodynamic stability studies, TEM observations, and zeta and size results. In vivo experiments confirmed significant wound-healing effects, highlighting the promising role of the NE-based gel in healthcare advancements. This research underscores the potential of novel phyto-based delivery systems in wound care.

Liposome Biodistribution via Europium Complexes.

Vector biodistribution is a requirement prior pharmaceutical development. Radioactive tracers allow the most sensitive and quantitative assessment of biodistribution, and conventional fluorophores are widely used in academic laboratories. We propose here to use europium complexes as a label for nanoparticles or biotherapeutics taking liposomes as models. Time-resolved fluorimetry (TRF) has the tremendous advantage of taking into accounts the fluorescence decay time of the lanthanide chelates, resulting in an improved sensitivity in biological media. The work described aimed following liposome biodistribution by TRF. An octadecyl-DTPA.Eu compound has been prepared and incorporated into liposomes without altering its fluorescence signal. The method has been validated through a comparison with fluorophore-labeled liposomes. The way to proceed when using this method for liposome biodistribution assessment is detailed. It could obviously be applied to other nanosystems, such as lipid nanoparticles.