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In the original publication [...].
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For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.
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Técnicas de Cultura de Células em Três Dimensões , Nucleosídeos , Materiais Biocompatíveis , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Peso Molecular , Nucleosídeos/farmacologiaRESUMO
Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to apply this strategy by using a novel nanoformulation consisting of DOTAU, a nucleolipid carrier, in an attempt to simultaneously deliver antibiotic and anti-resistance oligonucleotides. Ceftriaxone, a third-generation cephalosporin, was formulated with DOTAU to form an ion pair, and was then nanoprecipitated. The obtained solid nanocapsules were characterized using FT-IR, XRD, HPLC, TEM and DLS techniques and further functionalized by the anti-resistance ONα sequence. To obtain an optimal anti-resistance activity and encapsulation yield, both the formulation protocol and the concentration of ONα were optimized. As a result, monodispersed negatively charged nanoparticles of CFX-DOTAU-ONα with a molar ratio of 10:24:1 were obtained. The minimum inhibitory concentration of these nanoparticles on the resistant Escherichia coli strain was significantly reduced (by 75%) in comparison with that of non-vectorized ONα. All aforementioned results reveal that our nanoformulation can be considered as an efficient and relevant strategy for oligonucleotide intrabacterial delivery in the fight against antibiotic resistance.
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Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.
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Eletroforese Capilar , Oligonucleotídeos , Cromatografia em Gel , Lipídeos , MicelasRESUMO
Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.
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Técnicas Biossensoriais , Fracionamento por Campo e Fluxo , Glioblastoma , Movimento Celular , Separação Celular , Humanos , Células-Tronco NeoplásicasRESUMO
Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system's components.
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Biocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleoside-derived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent.
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A green analytical chemistry strategy is described to develop a reversed-phase high-performance liquid chromatography method for amodiaquine and artesunate analysis using ethanol-based mobile phases. This method development was particularly challenging due to the basicity of amodiaquine and low UV absorption of artesunate, leading to peak asymmetry and detection issues, respectively. UV detection concern was even more challenging due to the baseline drift observed with ethanol in gradient mode. Several green pH modifiers were selected for their ecofriendly character and their impact on peak shape and detection was investigated. The screening of various stationary phases (19 columns) appeared as a relevant and necessary approach to reach satisfactory peak shape of basic compounds. To support the results of this study, some additional compounds related to artesunate and amodiaquine structures were included. Methods were optimized and validated using total error approach with a mobile phase composed of ethanol and 10 mM formic acid using three different stationary phases from different manufacturers, providing flexibility of the quality control approach. Method greenness was assessed using the National Environmental Methods Index, the Green Analytical Procedure Index, and the Analytical Eco-Scale. Finally, artesunate and amodiaquine were successfully analyzed in fixed dose combination tablets.
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Amodiaquina/análise , Artesunato/análise , Química Verde , Cromatografia Líquida de Alta Pressão , HumanosRESUMO
Lipid-oligonucleotide (LONs) based bioconjugates represent an emerging class of therapeutic agents, allowing the delivery of therapeutic oligonucleotide sequences. The LON development requests accurate and efficient analytical methods. In this contribution, LON analysis methods were developed in cyclodextrin-modified capillary zone electrophoresis (CD-CZE). The LONs selected in this study feature different structures, including i) the oligonucleotide length (from 10 to 20 nucleotides), ii) the inter-nucleotide linkage chemistry (phosphodiester PDE or phosphorothioate PTO), and iii) the lipidic part: single- (LONsc) or double-chain (LONdc) lipids. In CD-CZE, the effect of several parameters on the electrophoretic peaks was investigated (buffer, CD, and capillary temperature). The binding interaction between LON and Me-ß-CD was studied in affinity capillary electrophoresis and revealed a 1:1 LON:CD complex. Non-linear regression and three usual linearization methods (y-reciprocal, x-reciprocal, and double-reciprocal) were used to determine the binding constants (K values of 2.5.104 M-1 and 2.0.104 M-1 for LON PDE and LON PTO, respectively). Quantitative methods with good performances and analysis time lower than 5 min were achieved. Importantly, the developed analysis allows a separation between the i) full-length sequence LONs and their truncated sequences, (n-1), (n-2), and (n-4)-mers and ii) LONsc, LONdc and their corresponding unconjugated oligonucleotides. This work highlights the interest of CD-CZE methods for LON analysis.
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Ciclodextrinas , Eletroforese Capilar , Lipídeos , Oligonucleotídeos , TemperaturaRESUMO
Greening analytical methods has become of great interest in the field of pharmaceutical analysis to protect both the operators' health and the environment. In this work, an innovative methodology combining Quality-by-Design (QbD) and Green Chemistry principles was followed to develop a single, green and robust RP-HPLC method for the quantitative analysis of impurities of both artesunate and amodiaquine drugs. Ethanol was selected as the best ecofriendly alternative solvent in substitution to the commonly used organic solvents such as acetonitrile and methanol. To achieve method objectives, resolutions between the 10 peaks were chosen as critical method attributes (CMAs) to be optimized through QbD approach. Based on a quality risk assessment, pH, temperature, and gradient slope were then selected as critical method parameters (CMPs) and a three level full factorial design was used to model the CMAs as function of the CMPs. Response surface methodology associated to Monte Carlo simulations allowed to determine the method operable domain region (MODR), i.e., the multidimensional combination of CMPs where CMAs simultaneously satisfied specifications (Rs ≥ 1.5) with a probability at least equal to 95 %. Inside the MODR, the working point was chosen based on green criteria, involving a mobile phase composed of ethanol and 10 mM acetic acid only as pH modifier. The method was successfully validated for all impurities using accuracy profile methodology, which was fully compliant with the ICH Q2(R1) requirements. Finally, the method was applied to the analysis of amodiaquine and artesunate impurities in raw materials and formulations.
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Amodiaquina , Artesunato , Cromatografia Líquida de Alta Pressão , Amodiaquina/análise , Artesunato/análise , Método de Monte Carlo , SolventesRESUMO
Two green analytical approaches have been developed for the analysis of antimalarial fixed dose tablets of artemether and lumefantrine for quality control. The first approach consisted of investigating the qualitative performance of a low-cost handheld near-infrared spectrometer in combination with the principal component analysis as an exploratory tool to identify trends, similarities, and differences between pharmaceutical samples, before applying the data driven soft independent modeling of class analogy (DD-SIMCA) as a one-class classifier for proper drug falsification detection with 100% of both sensitivity and specificity in the studied cases. Despite its limited spectral range and low resolution, the handheld device allowed detecting falsified drugs with no active pharmaceutical ingredient and identifying specifically a pharmaceutical tablet brand name. The second approach was the quantitative analysis based on the green and fast RP-HPLC technique using ethanol as a green organic solvent and acetic acid as a green pH modifier. The optimal separation was achieved in 7 min using a mobile phase composed of ethanol 96% and 10 mM of acetic acid pH 3.35 (63:37, v/v). The developed method was validated according to the total error approach based on an accuracy profile, was applied to the analysis of tablets, and allowed confirming falsified drugs detected by spectroscopy.
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Antimaláricos/análise , Combinação Arteméter e Lumefantrina/análise , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos Falsificados/análise , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Contaminação de Medicamentos/prevenção & controle , Controle de Qualidade , Comprimidos/químicaRESUMO
The increase of fungal resistance to drugs, such as azole family, gave rise to the development of new antifungals. In this context, echinocandins emerged as a promising alternative for antifungal therapies. Following the commercialization of caspofungin in 2001, echinocandins became the first-line therapy for invasive candidiasis in different patient populations. The quantification of these drugs has gained importance since pharmacokinetic/pharmacodynamic and resistance studies are a paramount concern. This fact has led us to exhaustively examine the methodologies used for the analysis of echinocandins in biological fluids, which are mainly based on LC coupled to different detection techniques. In this review, we summarize the analytical methods for the quantification of echinocandins focusing on sample treatment, chromatographic separation and detection methods.
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Antifúngicos/análise , Cromatografia/métodos , Testes de Química Clínica/métodos , Equinocandinas/análise , Métodos Analíticos de Preparação de Amostras , Animais , Antifúngicos/química , Antifúngicos/farmacocinética , Equinocandinas/farmacocinética , Equinocandinas/farmacologia , HumanosRESUMO
Current pediatric antibiotic therapies often use oral and parenteral routes of administration. Neither are suitable for treating very sick neonates who cannot take oral medication and may be several hours away from hospital in developing countries. Here, we report on the development of rectal forms of ceftriaxone, a third-generation cephalosporin. Rectodispersible tablets and capsules were developed and successfully passed 6-month accelerated stability tests. Rabbit bioavailability showed plasma concentrations above the minimal inhibitory concentrations for 3 formulations of rectodispersible tablets and 2 formulations of hard capsules. Clinical batches are currently being prepared for human evaluation with the prospect of offering therapeutic alternatives for treating critically ill neonates. This proof of concept for efficient rectal delivery of antibiotics could help the development of other rectal antibiotic treatments and increase options for noninvasive drug development for pediatric patients.
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Antibacterianos/administração & dosagem , Ceftriaxona/administração & dosagem , Sepse Neonatal/tratamento farmacológico , Animais , Antibacterianos/sangue , Disponibilidade Biológica , Cápsulas , Ceftriaxona/sangue , Composição de Medicamentos , Humanos , Lactente , Recém-Nascido , Masculino , Coelhos , Supositórios , ComprimidosRESUMO
A novel hybrid bioinspired amphiphile featuring a cytosine moiety, which self-assembles into liposomes can be used to detect silver ions in aqueous media. The coordination of Ag+ ions by the nucleotide moiety increases membrane rigidity, which enhances the fluorescence of a common reporter, Thioflavin T. Ag+ can be sensed even at trace concentrations (3 ppb) with great specificity over other metals ions. These nucleotide based supramolecular structures can be used to detect silver ions in drinking water, demonstrating the robustness of this approach.
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Desoxicitidina Monofosfato/análogos & derivados , Glicerofosfolipídeos/química , Lipossomos/química , Prata/análise , Benzotiazóis/química , Água Potável/análise , Corantes Fluorescentes/química , Espectrometria de Fluorescência , Tensoativos/química , Poluentes Químicos da Água/análiseRESUMO
Nucleolipid supramolecular assemblies are promising Drug Delivery Systems (DDS), particularly for nucleic acids. Studies based on negatively and positively charged nucleolipids (diC16dT and DOTAU, respectively) demonstrated appropriate stability, safety, and purity profile to be used as DDS. Methylene Blue (MB) remains a good antimalarial drug candidate, and could be considered for the treatment of uncomplicated or severe malaria. However, the development of MB as an antimalarial drug has been hampered by a high dose regimen required to obtain a proper effect, and a short plasmatic half life. We demonstrated that nanoparticles formed by nucleolipid encapsulation of MB using diC16dT and DOTAU (MB-NPs) is an interesting approach to improve drug stability and delivery. MB-NPs displayed sizes, PDI, zeta values, and colloidal stability allowing a possible use in intravenous formulations. Nanoparticles partially protected MB from oxido-reduction reactions, thus preventing early degradation during storage, and allowing prolongated pharmacokinetic in plasma. MB-NPs' efficacy, tested in vitro on sensitive or multidrug resistant strains of Plasmodium falciparum, was statistically similar to MB alone, with a slightly lower IC50. This nucleolipid-based approach to protect drugs against degradation represents a new alternative tool to be considered for malaria treatment.
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Neonatal sepsis is a major cause of infant mortality in developing countries because of delayed injectable treatment, making it urgent to develop noninjectable formulations that can reduce treatment delays in resource-limited settings. Ceftriaxone, available only for injection, needs absorption enhancers to achieve adequate bioavailability via nonparenteral administration. This article presents all available data on the nonparenteral absorption of ceftriaxone in humans and animals, including unpublished work carried out by F. Hoffmann-La Roche (Roche) in the 1980s and new data from preclinical studies with rabbits, and discusses the importance of these data for the development of noninjectable formulations for noninvasive treatment. The combined results indicate that the rectal absorption of ceftriaxone is feasible and likely to lead to a bioavailable formulation that can reduce treatment delays in neonatal sepsis. A bile salt, chenodeoxycholate sodium salt (Na-CDC), used as an absorption enhancer at a 125-mg dose, together with a 500-mg dose of ceftriaxone provided 24% rectal absorption of ceftriaxone and a maximal plasma concentration of 21 µg/ml with good tolerance in human subjects. The rabbit model developed can also be used to screen for the bioavailability of other formulations before assessment in humans.
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Antibacterianos/farmacocinética , Ceftriaxona/farmacocinética , Ácido Quenodesoxicólico/administração & dosagem , Absorção Intestinal/efeitos dos fármacos , Triglicerídeos/administração & dosagem , Administração Retal , Adulto , Animais , Antibacterianos/sangue , Disponibilidade Biológica , Ceftriaxona/sangue , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Feminino , Voluntários Saudáveis , Humanos , Recém-Nascido , Masculino , Sepse Neonatal/tratamento farmacológico , Sepse Neonatal/prevenção & controle , Papio , CoelhosRESUMO
Today, one of the most popular strategies in drug delivery is the encapsulation of therapeutic agents in supramolecular nanosystems formed from amphiphilic molecules. Synthetic nucleoside-lipids, composed of one nucleoside and lipidic chains, constitute promising new amphiphilic excipients under research in the field of pharmaceutical and biomedical applications. The aim of this work was to study the chromatographic behavior of these nucleoside-lipids in reversed-phase HPLC to establish appropriate chromatographic conditions for their analysis in drug delivery systems. The effect of the stationary phase, the organic solvent, the pH* values, and pH modifier nature of the mobile phase were studied on retention, peak shape, and detection. Good chromatographic performance was achieved on both Syncronis® C18 and Acquity® BEH C18 with mobile phases composed of MeOH/water, 95:5 (v/v) mixtures at apparent pH above 5. Dual detection by diode array detection (DAD) and charged aerosol detection (CAD) was investigated. CAD signal was found to be dependent on the type of pH modifiers added to the mobile phase. In isocratic elution, the same order of magnitude of CAD responses was obtained for the tested nucleoside-lipids. This study led to suitable chromatographic conditions for purity and stability studies of nucleoside-lipids. The purity of the synthetized molecules was established to be superior to 98%. Different stability in organic solvents was noticed depending on nucleoside-lipid structure. This first study will allow quantitative applications to establish loading ratio and encapsulation yield in future drug delivery systems composed of nucleoside-lipid-based assemblies.
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Lipídeos/química , Nucleosídeos/química , Cromatografia Líquida de Alta Pressão/métodos , Lasers SemicondutoresRESUMO
The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol) with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.
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Cromatografia de Fase Reversa/métodos , Etanol/química , Preparações Farmacêuticas/química , Solventes/química , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 µM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 µM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.