RESUMEN
In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P79-98) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine ⢠oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3' end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P79-98 -pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene.
Asunto(s)
ADN , Oligonucleótidos , Ratones , Animales , Oligonucleótidos/genética , Oligonucleótidos/química , ADN/genética , Plásmidos/genética , Transfección , Microtúbulos/metabolismo , Péptidos/genéticaRESUMEN
Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5-12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine ⢠oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.
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Fibrosis Quística , Distrofia Muscular de Duchenne , Animales , Transporte Activo de Núcleo Celular , Fibrosis Quística/genética , Fibrosis Quística/terapia , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , ADN , Distrofina/genética , Distrofina/metabolismo , Genes Reporteros , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Plásmidos/genéticaRESUMEN
BACKGROUND: The clinical general practitioner (GP) workforce is decreasing. Many studies have analysed the negative aspects of the profession but, few examine the positive aspects and job satisfaction. A European collaborative group including 8 participating countries recently conducted a qualitative study to analyse the positive factors and found 31 job satisfaction factors. OBJECTIVES: To determine which of these 31 factors are important and applicable to future policies to improve family medicine attractiveness, recruitment, and retention in France. METHOD: The Delphi consensus method was chosen. Two Delphi rounds were conducted in March-April 2017 and retained satisfaction factors with at least 70% of scores ≥7. The Nominal Group Technique (NGT) was used to rank these retained factors. Participants assigned 5 points to the factor they considered most important, 3 points to the second, and 1 point to the third. Factors receiving at least 5% (10 points) of the total points (198 points) were included in the final list. The expert panel included GPs and non-GPs. RESULTS: Twenty-nine experts began the procedure and 22 completed it. Thirty factors were retained after the 2 Delphi rounds. The NGT resulted in 8 factors: (i) Engage in family medicine to take care of the patients; (ii) Care coordination, patient advocacy; (iii) Flexibility in work; (iv) Trying to be a person-centred doctor; (v) Involvement in healthcare organization; (vi) Benefiting from a well-managed practice; (vii) Being a teacher, a trainer; (viii) Efficient professional collaboration. CONCLUSION: These 8 job satisfaction factors are important to consider and apply to future policy development.
In Europe, general practitioner (GP) numbers are falling. Policies considering GP job satisfaction could be a solution. GPs with higher job satisfaction have lower levels of stress and burnout, are more interested in their job, and stay in their job for longer. Recently, a European study found 31 GP factors that influence job satisfaction. However, it is not clear which of these 31 factors policy makers could use to improve attractiveness, recruitment, and retention in family medicine in France. A panel of experts consisting of GPs and non-GPs used the Delphi consensus method to agree on which satisfaction factors were relevant and important. These factors were then ranked in order of importance. The experts agreed upon thirty satisfaction factors. From these, 8 were ranked as most important: (i) Engage in family medicine to take care of patients; (ii) Care coordination, patient advocacy; (iii) Flexibility in work; (iv) Trying to be a person-centred doctor; (v) Involvement in the healthcare organization; (vi) Benefiting from a well-managed practice; (vii) Being a teacher, a trainer; (viii) Efficient professional collaboration. These should be considered and applied to future policy development.
RESUMEN
A convergent synthesis of cationic amphiphilic compounds is reported here with the use of the phosphonodithioester-amine coupling (PAC) reaction. This versatile reaction occurs at room temperature without any catalyst, allowing binding of the lipid moiety to a polar head group. This strategy is illustrated with the use of two lipid units featuring either two oleyl chains or two-branched saturated lipid chains. The final cationic amphiphiles were evaluated as carriers for plasmid DNA delivery in four cell lines (A549, Calu3, CFBE and 16HBE) and were compared to standards (BSV36 and KLN47). These new amphiphilic derivatives, which were formulated with DOPE or DOPE-cholesterol as helper lipids, feature high transfection efficacies when associated with DOPE. The highest transfection efficacies were observed in the four cell lines at low charge ratios (CR = 0.7, 1 or 2). At these CRs, no toxic effects were detected. Altogether, this new synthesis scheme using the PAC reaction opens up new possibilities for investigating the effects of lipid or polar head groups on transfection efficacies.
Asunto(s)
Aminas/química , Diseño de Fármacos , Ésteres/química , Técnicas de Transferencia de Gen , Compuestos de Sulfhidrilo/química , Tensoactivos/química , Cationes/síntesis química , Cationes/química , Línea Celular Tumoral , Humanos , Lípidos/química , Tensoactivos/síntesis químicaRESUMEN
Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well-tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non-viral gene transfer following hydrodynamic limb vein injection.
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Técnicas de Transferencia de Gen , Terapia Genética/métodos , Inyecciones Intravenosas/métodos , Animales , Humanos , Hidrodinámica , Músculo Esquelético , Distrofia Muscular de Duchenne/terapiaRESUMEN
A ramified lipid alcohol, 2-hexyldecanol, was used as a hydrophobic moiety to prepare cationic amphiphiles on a gram scale in 3 to 4 steps, featuring either a trimethylammonium 5, dimethylhydroxyethylammonium 6 or N-methylimidazolium 7 polar head group. Compression isotherms at the air-water interface reveal that all these cationic amphiphiles collapse at a relatively low pressure indicating a weak stabilization of the monolayer via hydrophobic interactions. Ellipsometry measurements point out the presence of a thin monolayer at low lateral pressure whereas thickening of the monolayer occurs at higher pressure with a high percentage of variation of the thickness, thus demonstrating an adaptability to the constraints. 31P NMR spectroscopy of the hydrated cationic amphiphiles clearly shows that these cationic amphiphiles self-assemble in water to form hexagonal phases, irrespective of the nature of their polar head group. Furthermore, a comparison of molecular structures suggests that compounds 5-7 self-organize into an inverted hexagonal phase (HII). These cationic amphiphiles, alone or in the presence of DOPE, were evaluated for the transfection of three human-derived cell lines (i.e. A549, 16HBE and HeLa). The three compounds demonstrated high transfection efficacies in every cell line tested, 7/DOPE being the most efficient.
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Técnicas de Transferencia de Gen , Lípidos/química , Tensoactivos/química , Liposomas Unilamelares , Cationes , Línea Celular , Alcoholes Grasos/química , Humanos , Lípidos/síntesis química , Fosfatidiletanolaminas , Tensoactivos/síntesis química , AguaRESUMEN
Inflammation is involved in the pathogenesis of many disorders. However, the underlying mechanisms are often unknown. Here, we test whether cystinosin, the protein involved in cystinosis, is a critical regulator of galectin-3, a member of the ß-galactosidase binding protein family, during inflammation. Cystinosis is a lysosomal storage disorder and, despite ubiquitous expression of cystinosin, the kidney is the primary organ impacted by the disease. Cystinosin was found to enhance lysosomal localization and degradation of galectin-3. In Ctns-/- mice, a mouse model of cystinosis, galectin-3 is overexpressed in the kidney. The absence of galectin-3 in cystinotic mice ameliorates pathologic renal function and structure and decreases macrophage/monocyte infiltration in the kidney of the Ctns-/-Gal3-/- mice compared to Ctns-/- mice. These data strongly suggest that galectin-3 mediates inflammation involved in kidney disease progression in cystinosis. Furthermore, galectin-3 was found to interact with the pro-inflammatory cytokine Monocyte Chemoattractant Protein-1, which stimulates the recruitment of monocytes/macrophages, and proved to be significantly increased in the serum of Ctns-/- mice and also patients with cystinosis. Thus, our findings highlight a new role for cystinosin and galectin-3 interaction in inflammation and provide an additional mechanistic explanation for the kidney disease of cystinosis. This may lead to the identification of new drug targets to delay cystinosis progression.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistinosis/complicaciones , Síndrome de Fanconi/inmunología , Galectina 3/metabolismo , Inflamación/inmunología , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Cistina/metabolismo , Cistinosis/inmunología , Cistinosis/metabolismo , Cistinosis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Síndrome de Fanconi/metabolismo , Síndrome de Fanconi/patología , Femenino , Galectina 3/genética , Humanos , Inflamación/metabolismo , Inflamación/patología , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/patología , Lisosomas/metabolismo , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Monocitos/inmunología , ProteolisisRESUMEN
Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.
RESUMEN
The hydrophobic moiety of cationic amphiphiles plays an important role in the transfection process because its structure has an impact on both the type of the supramolecular assembly and the dynamic properties of these assemblies. The latter have to exhibit a compromise between stability and instability to efficiently compact then deliver DNA into target cells. In the present work, we report the synthesis of new cationic amphiphiles featuring a thioether function at different positions of two 18-atom length lipid chains and we study their physicochemical properties (anisotropy of fluorescence and compression isotherms) with analogues possessing either oleyl (C18:1) or stearyl (C18:0) chains. We show that the fluidity of cationic lipids featuring a thioether function located close to the middle of each lipid chain is intermediate between that of oleyl- and stearyl-containing analogues. These properties are also supported by the compression isotherm assays. When used as carriers to deliver a plasmid DNA, thioether-containing cationic amphiphiles demonstrate a good ability to transfect human-derived cell lines, with those incorporating such a moiety in the middle of the chain being the most efficient. This work supports the use of a thioether function as a possible alternative to unsaturation in aliphatic lipid chains of cationic amphiphiles to modulate physicochemical behaviours and in turn biological activities such as gene delivery ability.
Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/química , Sulfuros/química , Tensoactivos/química , Cationes/química , Química Física , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.
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Membrana Celular/química , Membrana Celular/metabolismo , Ciclodextrinas/química , Polietilenglicoles/química , Polinucleótidos/metabolismo , Secuencia de Bases , Transporte Biológico , ADN/genética , ADN/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismoRESUMEN
Metastatic melanoma has been described as a highly aggressive cancer with low sensibility to chemotherapeutic agents. New types of drug, such as metal-based drugs (ferrocifens) have emerged and could represent an alternative for melanoma treatment since they show interesting anticancer potential. Furthermore, molecular analysis has evidenced the role of apoptosis in the low sensibility of melanomas and especially of the key regulator, Bcl-2. The objective of this study was to combine two strategies in the same lipid nanocapsules (LNCs): i) gene therapy to modulate anti-apoptotic proteins by the use of Bcl-2 siRNA, and ii) ferrocifens as a new type of anticancer agent. The efficient gene silencing with LNCs was verified by the specific extinction of Bcl-2 in melanoma cells. The cellular toxicity of ferrocifens (ferrociphenol (FcDiOH) or Ansa-FcDiOH) was demonstrated, showing higher efficacy than dacarbazine. Interestingly, the association of siBcl-2 LNCs with Ansa-FcDiOH demonstrated a significant effect on melanoma cell viability. Moreover, the co-encapsulation of siRNA and ferrocifens was successfully performed into LNCs for animal experiments. A reduction of tumor volume and mass was proved after siBcl-2 LNC treatment and Ansa-FcDiOH LNC treatment, individually (around 25%). Finally, the association of both components into the same LNCs increased the reduction of tumor volume to about 50% compared to the control group. In conclusion, LNCs appeared to provide a promising tool for the co-encapsulation of a metal-based drug and siRNA.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Ferrosos/farmacología , Lípidos/química , Melanoma/tratamiento farmacológico , Nanocápsulas/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Femenino , Terapia Genética/métodos , Humanos , Melanoma/metabolismo , Ratones , Ratones Desnudos , ARN Interferente Pequeño/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. METHODS: We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup containing one (compound 6) or two (compound 5) imidazole groups and two oleyl chains linked by an amide group. We studied their association with the aminoglycoside lipidic derivative dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphology and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. RESULTS: The phosphoramido linker in the helper lipid induces a major difference in terms of morphology and resistance to decomplexation at physical pH for DOSP/helper lipid complexes. CONCLUSIONS: This hybrid dioleyl linker composition of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker.
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Imidazoles/química , Lípidos/química , Liposomas/química , Fosforamidas/química , Transfección/métodos , Animales , Cationes/química , ADN/química , Endosomas/metabolismo , Células HEK293 , Hepatocitos , Humanos , Imidazoles/síntesis química , Lípidos/síntesis química , Ratones , Microscopía Electrónica de Transmisión , Mioblastos , Cultivo Primario de Células , RatasRESUMEN
The ramification of cationic amphiphiles on their unsaturated lipid chains is readily achieved by using the thiol-ene click reaction triggering the formation of an inverted hexagonal phase (HII). The new ramified cationic lipids exhibit different bio-activities (transfection, toxicity) including higher transfection efficacies on 16HBE 14o-cell lines.
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Alquenos/química , Portadores de Fármacos/química , Lípidos/química , Nanoestructuras/química , Compuestos de Sulfhidrilo/química , Transfección , Línea Celular , Química ClicRESUMEN
Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity.
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Amidas/química , Lípidos/química , Ácidos Fosfóricos/química , Transfección , Amidas/síntesis química , Espectroscopía de Resonancia Magnética , Ácidos Fosfóricos/síntesis química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The structure of the cationic moiety of amphiphiles is a key factor which directly influences their transfection efficacy. Accordingly, in the present work, we have synthesized three new lipophosphoramide-based amphiphilic compounds incorporating a methoxy 5, hydroxyl 6, or dihydroxyl 7 functional group in their cationic part. Gene delivery efficacies of these novel vectors were compared to our benchmark compound, the arsenolipophosphoramidate KLN47, and to its trimethylammonium (TMA) analogue 4. We next studied the characteristics (size, ζ potential) of the nanometric assemblies formed (liposomes and lipid/DNA complexes), and the DNA binding ability of the cationic liposomes was characterized at the physicochemical level. In vitro, all of the cationic lipids evaluated were efficient not only to condense plasmids but also to transfect two types of human airway epithelial cells. Interestingly, in vivo administration to mice (via simple tail vein injection) showed that compound 6 was the most efficient in transfecting the lungs when compared to that of the other cationic lipids studied, including compound KLN47. All of these results suggest that a hydroxyethyldimethylammonium (HE-DMA) polar head could be a valuable alternative to a trimethylarsonium (TMAs) polar head and that they also invite further evaluation of the in vivo potential of compound 6 using more clinically relevant delivery procedures.
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Cationes/química , Portadores de Fármacos/química , Lípidos/química , Técnicas de Transferencia de Gen , Hidroxilación , Liposomas/química , Compuestos de Amonio Cuaternario/químicaRESUMEN
Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.
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Butileno Glicoles/síntesis química , Poliaminas/síntesis química , Polímeros/síntesis química , Tensoactivos/síntesis química , Curcumina/química , Curcumina/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Células HEK293 , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , SolubilidadRESUMEN
In this work that aims to synthesize and evaluate new cationic lipids as vectors for gene delivery, we report the synthesis of a series of cationic lipids in which a phosphate functional group acts as a linker to assemble on a molecular scale, two lipid chains and one cationic polar head. The mono or dicationic moiety is connected to the phosphate group by an aryl spacer. In this work, two synthesis strategies were evaluated. The first used the Atherton-Todd coupling reaction to introduce a phenolic derivative to dioleylphosphite. The second strategy used a sequential addition of lipid alcohol and a phenolic derivative on POCl3. The two methods are efficient, but the latter allows larger yields. Different polar head groups were introduced, thus producing amphiphilic compounds possessing either one permanent (N-methyl-imidazolium, pyridinium, trimethylammonium) or two permanent cationic charges. All these cationic lipids were formulated as liposomal solutions and characterized (size and zeta potential). They formed stable liposomal solutions both in water (at pH 7.0) and in a weakly acidic medium (at pH 5.5). Finally, this new generation of cationic lipids was used to deliver DNA into various human-derived epithelial cells cultured in vitro. Compared with Lipofectamine used as a reference commercial lipofection reagent, some cationic dialkylarylphosphates were able to demonstrate potent gene transfer abilities, and noteworthily, monocationic derivatives were much more efficient than dicationic analogues.
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Materiales Biomiméticos/química , Portadores de Fármacos/química , Lípidos/química , Fosfatos/química , Transfección/métodos , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/toxicidad , Línea Celular , ADN/química , ADN/genética , Portadores de Fármacos/síntesis química , Portadores de Fármacos/toxicidad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/síntesis química , Lípidos/toxicidad , LiposomasRESUMEN
The objective of lung gene therapy is to reach the respiratory epithelial cells in order to deliver a functional nucleic acid sequence. To improve the synthetic carrier's efficacy, knowledge of their biodistribution and elimination pathways, as well as cellular barriers faced, depending on the administration route, is necessary. Indeed, the in vivo fate guides the adaptation of their chemical structure and formulation to increase their transfection capacity while maintaining their tolerance. With this goal, lipidic fluorescent probes were synthesized and formulated with cationic lipophosphoramidate KLN47 (KLN: Karine Le Ny). We found that such formulations present constant compaction properties and similar transfection results without inducing additional cytotoxicity. Next, biodistribution profiles of pegylated and unpegylated lipoplexes were compared after systemic injection in mice. Pegylation of complexes led to a prolonged circulation in the bloodstream, whereas their in vivo bioluminescent expression profiles were similar. Moreover, systemic administration of pegylated lipoplexes resulted in a transient liver toxicity. These results indicate that these new fluorescent compounds could be added into lipoplexes in small amounts without perturbing the transfection capacities of the formulations. Such additional properties allow exploration of the in vivo biodistribution profiles of synthetic carriers as well as the expression intensity of the reporter gene.
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Amidas/administración & dosificación , Amidas/farmacocinética , Colorantes Fluorescentes , Técnicas de Transferencia de Gen , Ácidos Fosfóricos/administración & dosificación , Ácidos Fosfóricos/farmacocinética , Amidas/química , Amidas/toxicidad , Animales , Línea Celular , Supervivencia Celular , ADN/química , Colorantes Fluorescentes/química , Humanos , Liposomas , Mediciones Luminiscentes/métodos , Ratones , Imagen Molecular , Estructura Molecular , Ácidos Fosfóricos/química , Ácidos Fosfóricos/toxicidad , Plásmidos/química , Distribución Tisular , TransfecciónRESUMEN
Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multimodular nonviral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/neutralization/relaxation abilities, and colloidal stability in the presence of serum. In addition, gene transfer experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration route. This original colipid appears thus as an innovative modular platform endowed with properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.
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Archaea/química , Cationes/química , Éteres/química , Lípidos/química , Tensoactivos/química , Animales , ADN/administración & dosificación , ADN/química , Femenino , Técnicas de Transferencia de Gen , Liposomas/química , Ratones , Transfección/métodosRESUMEN
Cationic lipids constitute a family of synthetic vectors commonly used for nucleic acids delivery. We herein report the results of a systematic study that aimed to compare the transfection efficacies of cationic lipophosphoramidates possessing either two identical lipid chains (termed symmetric cationic lipids) or two different lipid chains (non-symmetric cationic lipids). In addition, we also compared the transfection results of such a 'molecular approach' (the two different lipid chains being included in the same molecule) with those of a 'supramolecular approach' in which two types of symmetrical cationic lipids were mixed in one liposomal formulation. Thus, the present work allowed us first to optimize the methods used to synthesize non-symmetric cationic lipophosphoramidates. In addition, we could also identify two non-symmetric cationic lipids exhibiting high transfection efficiencies with a series of mammalian cell lines, both vectors being characterized by a single phytanyl chain and either an oleyl or a lauryl lipid chain.