RESUMO
Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA--each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Psi0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Psi0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Psi0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Psi0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA-/L+ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.
Assuntos
DNA/química , Ácidos Graxos Monoinsaturados/química , Lipídeos/química , Lipossomos/química , Compostos de Amônio Quaternário/química , Espermina/análogos & derivados , Cátions/química , Difenilexatrieno/análogos & derivados , Difenilexatrieno/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Luz , Modelos Moleculares , Espalhamento de Radiação , Cloreto de Sódio/química , Espermina/química , Eletricidade EstáticaRESUMO
Gene delivery using self-assembled polyplexes, formed between negatively charged nucleic acids and cationic polymers, have several drawbacks including low transgene expression and toxicity effects related to their positive charge. Recently, a novel cationic polymer based on dextran-spermine (D-SPM) was developed for gene delivery. This polymer showed systemic biodistribution upon local administration (intramuscular (i.m.) and intranasal (i.n.)) followed by transgene expression in organs remote from the site of injection (liver and lungs). Polyplexes based on D-SPM were administered both i.m. and i.n. to BALB/c female mice. LacZ expression in the liver, lungs, and muscles was assessed using whole-mount 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-gal) staining and paraffin sectioning. The local toxicity in these organs was evaluated from hematoxylin and eosin stained sections. The systemic toxicity of the polymer and polyplexes was estimated by comparing the mice total weight, major organ weights, blood counts (primarily white blood cells (WBC) and platelets), and serum transaminases, to those of control animals (which received PBS). Transgene expression using D-SPM polyplexes was dependent upon the dosage and the polyplexes (+/-) charge ratio. Using the i.m. and i.n. routes of administration the transfection occurred primarily in the bronchial epithelial cells, pneumocytes, and bronchial alveoli of the lungs; in the muscle's fibrocytes; and in the liver's hepatocytes. Histopathological assays revealed mild toxicity in muscle and no abnormal findings in liver and lung. No systemic toxicity was obtained, as we did not find decrease in WBC count or platelet and no increase in serum transaminases. In addition, mice body weights and major organ weights were similar to the control group at both 2 and 28 days post-administration. This study demonstrates systemic transfection efficacy upon local administration of D-SPM complexes with good tolerability and low toxicity.
Assuntos
Materiais Biocompatíveis , Dextranos , Sistemas de Liberação de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Espermina , Transgenes , Animais , Materiais Biocompatíveis/toxicidade , Dextranos/toxicidade , Avaliação Pré-Clínica de Medicamentos , Feminino , Genes Reporter , Vetores Genéticos , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Especificidade de Órgãos/genética , Espermina/toxicidade , TransfecçãoRESUMO
Biodegradable water-soluble polysaccharide-spermine (SPM) polycation conjugates for nucleic acid delivery were synthesized by oxidizing polysaccharides using potassium periodate, followed by SPM conjugation. The polycations differ in their polysaccharide type, arabinogalactan (AG) or dextran (D), and/or in the IO(4)- /saccharide mole ratio used for polysaccharide oxidation (1:1, 1:3, or 1:5), resulting in either D(1:1)-SPM, AG(1:1)-SPM, D(1:3)-SPM, AG(1:3)-SPM, or AG(1:5)-SPM. Chemical structure of the conjugates was characterized for total nitrogen and primary amino groups. Surface pH and electrical surface potential were determined by means of spectral changes of covalently attached 7-hydroxycoumarin (HC, a pH- and electrical surface potential-sensitive fluorophore). The binding and the electrostatic neutralization of the polycations by plasmid DNA, as well as the relationship between chemical structure, physical parameters, and transfection of NIH3T3 cells, were also studied. D(1:1)-SPM, the only polycation that showed efficient cell transfection in culture, was shown to have: (1) high SPM content (2000 nmol/mg); (2) high levels of cross-linked SPM (39-51%); (3) at DNA P-/NH3+ ratio of 2.0, a plateau in neutralization of cationic groups (+48 mV, as determined by HC-labeled D(1:1)-SPM titration with DNA), and a drop in zeta-potential from +42 mV for the polymer alone to 0 mV for the polyplex, suggesting that some of the charges are hidden from the DNA; (4) pH(surface) value of 9.2, suggesting that at physiological bulk pH the polymer is only partially ionized, and therefore can act as a "proton sponge" in the endosome; and (5) high sensitivity to serum-rich growth medium. An oleyl derivative, N-oleyl-dextran-spermine (ODS), was synthesized and demonstrated improved transfection efficiency in serum-rich medium.
Assuntos
Materiais Biocompatíveis , Polissacarídeos , Espermina , Transfecção , Animais , Materiais Biocompatíveis/síntese química , DNA , Cinética , Camundongos , Células NIH 3T3 , Plasmídeos , Poliaminas/síntese química , Polieletrólitos , Polissacarídeos/síntese química , Espermina/síntese química , Eletricidade Estática , TermodinâmicaRESUMO
This work describes a versatile and universal polycation system based on oligoamines grafted on natural polysaccharides that is capable of complexing various plasmids and administering them into various cells in high yield to produce a desired protein. These polycations are expected to better meet the requirements for effective complexation and delivery of plasmid or an antisense and to biodegrade into nontoxic components at a controlled rate. The developed biodegradable polycations are based on spermine, a natural tetramine, conjugated to dextran or arabinogalactan. These polycations were prepared by reductive amination of oxidized polysaccharides with the desired oligoamines. The Schiff base conjugates thus obtained were reduced to the stable amine conjugates by sodium borohydride. Over 300 different polycations were prepared starting from various polysaccharides and oligoamines, mainly oligoamines of two to four amino groups. Although most of these conjugates formed stable complexes with various plasmids as determined by turbidity experiments, only a few polycations were found to be active in transfecting cells. This work indicates that the structure of the polycation plays a significant role in the transfection activity of polycations.
Assuntos
Técnicas de Transferência de Genes , Poliaminas/química , Polissacarídeos/química , Cátions , Linhagem Celular , Genes Reporter , Proteínas de Fluorescência Verde , Hormônio do Crescimento Humano/genética , Humanos , Luciferases/genética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Peso Molecular , Plasmídeos , TransfecçãoRESUMO
Dextran polysaccharide was grafted by reductive-amination with mixtures of spermine and other natural/synthetic oligoamines of two to four amine groups. The transfection efficiencies of the polycations thus obtained were assessed in various cell lines, and found to depend on the spermine contents. Higher spermine ratios of grafted oligoamines resulted in high gene expression, whereas low to negligible expressions were obtained with lower spermine contents. The effect was explained by spermine residues which exhibit altered buffering capacity in comparison to other substituted oligoamines. Hydrophobization of dextran-spermine (D-SPM) was achieved by treating the polymer with N-hydroxysuccinimide derivatives of cholesterol and fatty acids in a mixture of water/THF. The degree of hydrophobization was in the range of 1-30% mol/mol (hydrophobic moieties/primary amine) and the coupling yields were >95% as determined by (1)H-NMR. The oleate-modified D-SPM remarkably enhanced the gene expression in serum rich media, in marked contrast to unmodified D-SPM which resulted with a drastic decrease in the transfection yields. Modified D-SPM derivatives of other fatty acids and cholesterol showed improved transfection yields in comparison to unmodified D-SPM, but to a lower extent when compared to oleate modification. The improvement in cell transfection was attributed to oleate residues which probably play a role in increasing stability and uptake of polycation-DNA complexes.
Assuntos
Dextranos/química , Portadores de Fármacos/química , Espermina/química , Transfecção/métodos , Aminas/química , Animais , Sequência de Carboidratos , Colesterol/química , DNA/administração & dosagem , Ácidos Graxos/química , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luciferases/biossíntese , Luciferases/genética , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Succinimidas/química , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Polycations are effective nonviral carriers for gene delivery systems. These carriers vary in molecular weight, polymer structure, polymer:DNA ratio, molecular architecture, and the ability to introduce target-specific moieties. Polycations are capable of complexing various plasmids and transfecting them into different cells to produce a high yield of a desired protein. Cationic polysaccharides are attractive candidates for gene delivery. They are natural or semi-natural, nontoxic, biodegradable, and biocompatible materials that can be modified for improved physicochemical properties. Cationic polysaccharides are synthesized by conjugation of various oligoamines to oxidized polysaccharides via reductive amination. These conjugates have been rigorously tested for gene delivery in cultured cells and in animals. From more than 300 polysaccharide-oligoamine derivatives tested, only dextran-spermine (D-SPM) was found to be highly effective in gene transfection, both in vitro and in vivo. This protocol describes the synthesis of cationic polysaccharides and their use for DNA transfection in vitro.
Assuntos
Poliaminas/síntese química , Poliaminas/metabolismo , Polissacarídeos/síntese química , Polissacarídeos/metabolismo , Transfecção/métodos , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , PolieletrólitosRESUMO
We recently showed that lipid assemblies comprised of a novel polycationic sphingolipid (ceramide carbamoyl-spermine, CCS) are an effective adjuvant/carrier when complexed with cholesterol (CCS/C) for influenza and other vaccines administered parenterally and intranasally (i.n.) in mice. Here we expand these studies to ferrets, an established model of influenza infection. We also address the question of why the CCS/C-based liposomal vaccine (also known as VaxiSome™) in mice is superior to vaccines based on liposomes of other lipid compositions (neutral, anionic or cationic). Ferrets immunized i.n. with CCS/C-influenza vaccine produced significantly higher hemagglutination inhibition (HI) antibody titers compared to ferrets immunized intramuscularly with the unadjuvanted influenza vaccine, indicating that the CCS/C-based vaccine is very immunogenic. Furthermore, the i.n. adjuvanted vaccine was shown to significantly reduce the severity of influenza virus infection in ferrets following homologous viral challenge as determined by weight loss, temperature rise and viral titer. No adverse reactions were observed. Pharmacokinetic and biodistribution studies following i.n. administration in mice of CCS/C-based vaccine showed that both the lipids and antigens are retained in the nose and lung for at least 24h, and it appears that this retention correlates with the superior immunogenicity elicited by the adjuvanted vaccine formulation. The CCS lipid also increases production of cytokines (mainly IFN gamma, IL-2 and IL-12) and co-stimulatory molecules' expression, which might further explain the robust adjuvantation of this liposome-based vaccine.
Assuntos
Ceramidas/administração & dosagem , Vacinas contra Influenza/imunologia , Lipídeos/administração & dosagem , Espermina/administração & dosagem , Adjuvantes Imunológicos , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Temperatura Corporal , Peso Corporal , Ceramidas/efeitos adversos , Citocinas/metabolismo , Feminino , Furões , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Lipídeos/efeitos adversos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Espermina/efeitos adversos , Carga ViralRESUMO
Although most pathogens use the mucosal routes for invasion, the majority of currently available vaccines are administered parenterally. Injectable vaccines induce good systemic immunity but often unsatisfactory mucosal immunity. A non-injectable mucosal vaccine, which can be self-administered intranasally, may provide both effective systemic and mucosal immunity and can be used for vaccination of large populations within a short period of time in case of a sudden epidemic. Here, we report on a new intranasal (i.n.) influenza vaccine, based on a novel polycationic sphingolipid, N-palmitoyl D-erythro-sphingosyl carbamoyl-spermine (ceramide carbamoyl-spermine = CCS), having combined carrier and adjuvant activities, which elicits, in mice, strong systemic (serum) and local (lung and nasal) humoral and cellular responses, and provides protective immunity. In a comparative study, we show that both unmodified commercial vaccine and vaccine formulated with neutral or anionic liposomes were poorly immunogenic upon i.n. administration. Of five vaccine formulations based on well-established monocationic lipids in the form of unsized liposomes, three (DC-Chol, DDAB, and DSTAP-based) resulted in low serum and local responses, while two others (DMTAP and DOTAP-based vaccines) induced both systemic and local vigorous Th1+Th2 immune responses. However, only the vaccine formulated with CCS was equivalent or superior to the commercial vaccine co-administered with cholera toxin as an adjuvant. Furthermore, the CCS-based influenza vaccine was highly efficacious following a single or a repeated (x2) i.n. or a single i.m. administration, without an added adjuvant, in both young (2 months) and old (18 months) mice. It elicited high titers of strain cross-reactive hemagglutination inhibition (HI) antibodies, and the high antibody titers and protective immunity persisted for at least 9 months. No systemic adverse effects, and only a mild local inflammatory response, were observed in mice and rabbits vaccinated i.n. with the CCS vaccine formulation. A similar approach may prove efficacious for i.n. vaccination against other pathogens.