RESUMEN
UVB irradiation of human telomeric d(GGGTTA)3 GGG sequences in potassium ion solution crosslinks the first and third TTA segments through anti cyclobutane pyrimidine dimer (CPD) formation. The photocrosslinking reaction was first proposed to occur through a form 3 two-tetrad G-quadruplex in which the lateral four-nucleotide GTTA loop can interact with an adjacent TTA loop. Curiously, the reaction does not occur with sodium ion, which was explained by the formation of a basket structure which only has three-nucleotide TTA loops that cannot interact. Sequences known or expected to favor the two-tetrad basket did not show enhanced photocrosslinking, suggesting that some other structure was the reactive intermediate. Herein, we report that anti CPDs form in human telomeric DNA sequences with lithium ion that is known to disfavor G-quadruplex formation, as well as with potassium ion when the bases are modified to interfere with G-quartet formation. We also show that anti CPDs form in sequences containing A's in place of G's that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions.
Asunto(s)
Reactivos de Enlaces Cruzados/química , ADN/química , G-Cuádruplex , Conformación de Ácido Nucleico , Procesos Fotoquímicos , Telómero/genética , Emparejamiento Base , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Humanos , Cinética , Litio/química , Electroforesis en Gel de Poliacrilamida Nativa , Potasio/química , Dímeros de Pirimidina/química , Sodio/química , TemperaturaRESUMEN
Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5Î-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation.
Asunto(s)
ADN Circular/química , Histonas/química , Nucleosomas/efectos de la radiación , Dímeros de Pirimidina/agonistas , Timina/química , Animales , Pollos , ADN Circular/aislamiento & purificación , Desaminación , Eritrocitos/química , Histonas/metabolismo , Humanos , Mutagénesis , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/metabolismo , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismo , Timina/metabolismo , Rayos UltravioletaRESUMEN
The direct synthesis of an acid-labile polyphosphoramidate by organobase-catalyzed ring-opening polymerization and an overall two-step preparation of polyphosphodiester ionomers (PPEI) by acid-assisted cleavage of the phosphoramidate bonds along the backbone of the polyphosphoramidate were developed in this study. The ultrafast organobase-catalyzed ring-opening polymerization of a cyclic phospholane methoxyethyl amidate monomer initiated by benzyl alcohol allowed for the preparation of well-defined polyphosphoramidates (PPA) with predictable molecular weights, narrow molecular weight distributions (PDI<1.10), and well-defined chain ends. Cleavage of the acid-labile phosphoramidate bonds on the polyphosphoramidate repeat units was evaluated under acidic conditions over a pH range of 1-5, and the complete hydrolysis produced polyphosphodiesters. The thermal properties of the resulting polyphosphoester ionomer acid and polyphosphoester ionomer sodium salt exhibited significant thermal stability. The parent PPA and both forms of the PPEIs showed low cytotoxicities toward HeLa cells and RAW 264.7 mouse macrophage cells. The synthetic methodology developed here has enriched the family of water-soluble polymers prepared by rapid and convenient organobase-catalyzed ring-opening polymerizations and straightforward chemical medication reactions, which are designed to be hydrolytically degradable and have promise for numerous biomedical and other applications.
RESUMEN
Gold nanoparticles have attracted much interest as a platform for development of multifunctional imaging and therapeutic agents. Multifunctionalized gold nanoparticles are generally constructed by covalent assembly of a gold core with thiolated ligands. In this study, we have assembled multifunctionalized gold nanoparticles in one step by nucleic acid hybridization of ODN (oligodeoxynucleotide)-derivatized gold nanoparticles with a library of pre-functionalized complementary PNAs (peptide nucleic acids). The PNAs were functionalized by conjugation with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for chelating 64Cu for PET imaging, PEG (polyethylene glycol) for conferring stealth properties, and Cy5 for fluorescent imaging. The resulting nanoparticles showed good stability both in vitro and in vivo showing biodistribution behavior in a mouse that would be expected for a PEGylated gold nanoparticle rather than that for the radiolabelled PNA used in its assembly.
RESUMEN
A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nanopartículas/química , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Polímeros/química , Polímeros/farmacología , Animales , Transporte Biológico , Línea Celular , Inhibidores Enzimáticos/metabolismo , Ésteres , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Óxido Nítrico/biosíntesis , Polímeros/metabolismoRESUMEN
Detailed analyses of the electron spin resonance (ESR) spectra, cell viability, and DNA degradation studies are presented for the photolyzed Type I phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates. The ESR studies provided evidence that copious free radicals can be generated from these N-H, N-S, and S-O containing compounds upon photoirradiation with UV/visible light. The analyses of spectral data allowed us to identify the free radical species. The cell viability studies showed that these agents after exposure to light exert cytotoxicity to kill cancer cells (U937 leukemia cell lines HTC11, KB, and HT29 cell lines) in a dosage- and time-dependent manner. We examined a possible pathway of cell death via DNA degradation by a plasmid cleavage assay for several compounds. The effects of photosensitization with benzophenone in the presence of oxygen were examined. The studies indicate that planar tricyclic amines and sulfenamides tend to form π-electron delocalized aminyl radicals, whereas nonplanar ones tend to yield nitroxide radicals resulting from the recombination of aminyl radicals with oxygen. The ESR studies coupled with the results of cell viability measurements and DNA degradation reveal that planar N-centered radicals can provide higher potency in cell death and allow us to provide some insights on the reaction mechanisms. We also found the formation of azatropylium cations possessing high aromaticity derived from azepines can facilitate secondary electron transfer to form toxic O2(â¢-) radicals, which can further exert oxidative stress and cause cell death.
Asunto(s)
Aminas/farmacología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Ácidos Sulfénicos/farmacología , Aminas/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Radicales Libres/farmacología , Células HT29 , Humanos , Células KB , Estructura Molecular , Fármacos Fotosensibilizantes/química , Relación Estructura-Actividad , Ácidos Sulfénicos/química , Factores de Tiempo , Células U937RESUMEN
Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.
Asunto(s)
Cationes/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , ARN Interferente Pequeño , Cationes/administración & dosificación , Regulación de la Expresión Génica , Células HeLa , Humanos , Nanopartículas/administración & dosificación , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Acute lung injury (ALI) is a complex syndrome with many aetiologies, resulting in the upregulation of inflammatory mediators in the host, followed by dyspnoea, hypoxemia and pulmonary oedema. A central mediator is inducible nitric oxide synthase (iNOS) that drives the production of NO and continued inflammation. Thus, it is useful to have diagnostic and therapeutic agents for targeting iNOS expression. One general approach is to target the precursor iNOS mRNA with antisense nucleic acids. Peptide nucleic acids (PNAs) have many advantages that make them an ideal platform for development of antisense theranostic agents. Their membrane impermeability, however, limits biological applications. Here, we report the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabelled antisense PNA-YR9 · oligodeoxynucleotide (ODN) hybrid and a cationic shell-cross-linked knedel-like nanoparticle (cSCK). The Y (tyrosine) residue was used for (123)I radiolabelling, whereas the R9 (arginine9) peptide was included to facilitate cell exit of untargeted PNA. Complete binding of the antisense PNA-YR9 · ODN hybrid to the cSCK was achieved at an 8 : 1 cSCK amine to ODN phosphate (N/P) ratio by a gel retardation assay. The antisense PNA-YR9 · ODN · cSCK nanocomplexes efficiently entered RAW264.7 cells, whereas the PNA-YR9 · ODN alone was not taken up. Low concentrations of (123)I-labelled antisense PNA-YR9 · ODN complexed with cSCK showed significantly higher retention of radioactivity when iNOS was induced in lipopolysaccharide+interferon-γ-activated RAW264.7 cells when compared with a mismatched PNA. Moreover, statistically, greater retention of radioactivity from the antisense complex was also observed in vivo in an iNOS-induced mouse lung after intratracheal administration of the nanocomplexes. This study demonstrates the specificity and sensitivity by which the radiolabelled nanocomplexes can detect iNOS mRNA in vitro and in vivo and their potential for early diagnosis of ALI.
RESUMEN
In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Nanopartículas , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/química , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Humanos , Espectroscopía de Resonancia Magnética , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/genéticaRESUMEN
Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of CâT mutations, but occurs with a half life of â¼50 000 years. In contrast, cytosine within sunlight induced cyclobutane dipyrimidine dimers (CPD's), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlate with CâT mutation hotspots in skin cancers. MeCP2 binds to mCG sites and acts as a transcriptional regulator and chromatin modifier affecting thousands of genes, but its effect on CPD formation and deamination is unknown. We report that the methyl CpG binding domain of MeCP2 (MBD) greatly enhances C=mC CPD formation at a TCmCG site in duplex DNA and binds with equal or better affinity to the CPD-containing duplex compared with the undamaged duplex. In comparison, MBD does not enhance T=mC CPD formation at a TTmCG site, but instead increases CPD formation at the adjacent TT site. MBD was also found to completely suppress deamination of the T=mCG CPD, suggesting that MeCP2 may have the capability to both suppress UV mutagenesis at PymCpG sites as well as enhance it.
Asunto(s)
Islas de CpG , Proteína 2 de Unión a Metil-CpG/metabolismo , Dímeros de Pirimidina/química , Islas de CpG/efectos de la radiación , Huella de ADN , Metilación de ADN , Desaminación , Dimerización , Proteína 2 de Unión a Metil-CpG/química , Unión Proteica , Tirosina/química , Rayos UltravioletaRESUMEN
The UV portion of sunlight is mutagenic and can modify DNA by producing various photoproducts. UV photodamage often occurs at dipyrimidine sites, to give cyclobutane, pyrimidine-(6-4)-pyrimidone (6-4), and pyrimidine-(6-4)-Dewar pyrimidone (Dewar) photoproducts, and at TA and AA sites. There is no reported evidence, however, of UV photoproduct formation between C or 5-methylC ((m)C) and A. Irradiation of d(GTAT(m)CATGAGGTGC) with UVB light at physiological pH gives an unexpected photoproduct that undergoes fast thermal deamination but does not revert to its original structure under UVC irradiation. Evidence from nuclease P1 digestion coupled with electrospray ionization (ESI)-MS/MS is in accord with product formation between (m)C and A. HPLC analysis indicates that deamination gives a T<>A photoproduct that coelutes on reverse-phase chromatography with the well-known TA* photoproduct, formed from an initial [2 + 2] reaction between C5-C6 and C6-C5 of the adjacent thymine and adenine [as shown by Zhao , X. , et al. ( 1996 ) Nucleic Acids Res. 24 , 1554 - 1560 and Davies , R. J. , et al. ( 2007 ) Nucleic Acids Res. 35 , 1048 - 1053 ]. Furthermore, the deamination product of the unknown (m)C<>A photoproduct and the TA* photoproduct undergo nearly identical fragmentation in tandem MS. The evidence, taken together, indicates that the deamination product of the unknown (m)CA photoproduct has the same chemical structure as the TA* photoproduct. Therefore, the unknown photoproduct is referred to as the (m)CA* photoproduct, which, upon deamination, gives the TA* photoproduct.
Asunto(s)
5-Metilcitosina/química , Adenina/química , Cromatografía Líquida de Alta Presión , ADN/química , Espectrometría de Masas , Secuencia de Bases , Fotoquímica , Rayos UltravioletaRESUMEN
Cationic nanoparticles are a promising class of transfection agents for oligonucleotide and gene delivery, but vary greatly in their effectiveness and cytotoxicity. Recently, we developed a new class of cationic transfection agents based on cationic shell-crosslinked knedel-like nanoparticles (cSCKs) that efficiently transfect mammalian cells with both oligonucleotides and plasmid DNA. In an effort to further improve transfection efficiency without increasing cytotoxicity, we examined the effects of the composition of primary amine (pa), tertiary amine (ta) and carboxylic acid (ca) groups in the shell of these nanoparticles. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) or phosphorothioate 2'-OMe oliogonucleotides (ps-MeON) were prepared over a broad range of amine to phosphate ratios (N/P ratio) of 4:1-40:1. The sizes of the complexes and the ability of the nanoparticles to completely bind ODNs were found to depend on the cSCK amine to DNA phosphate (N/P) ratio and the cSCK buffering capacity. The cSCKs were then evaluated for their ability to transfect cells with plasmid DNA by monitoring fluorescence from an encoded EGFP, and for delivery of ps-MeON by monitoring luminescence from luciferase resulting from ps-MeON-mediated splicing correction. Whereas the cationic cSCK-pa(25)-ta(75) was found to be best for transfecting plasmid DNA into HeLa cells at an N/P ratio of 20:1, cSCK-pa(50)-ta(50), at an N/P ratio 10:1 was best for ps-MeON delivery. We also found that increasing the proportion of tertiary relative to primary amine reduced the cytotoxicity. These results demonstrate that a dramatic improvement in gene and oligonucleotide delivery efficiency with decreased cytotoxicity in HeLa cells can be achieved by incorporation of tertiary amines into the shells of cSCKs.
Asunto(s)
Cationes/química , Reactivos de Enlaces Cruzados/farmacología , Nanopartículas/química , Transfección , Tampones (Química) , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , ADN/ultraestructura , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Células HeLa , Humanos , Luciferasas/metabolismo , Microscopía Confocal , Nanopartículas/ultraestructura , Oligonucleótidos/química , Oligonucleótidos/farmacología , Plásmidos/genética , Relación Estructura-ActividadRESUMEN
This mini-review highlights developments that have been made over the past year to advance the construction of well-defined nanoscale objects to serve as devices for cell transfection. Design of the nanoscale objects originated from biomimicry concepts, using histones as the model, to afford cationic shell crosslinked knedel-like (cSCK) nanoparticles. Packaging and delivery of plasmid DNA, oligonucleotides, and peptide nucleic acids were studied by dynamic light scattering, transmission electron microscopy, gel electrophoresis, biological activity assays, RT-PCR measurements, flow cytometry, and confocal fluorescence microscopy. With the demonstration of more efficient cell transfection in vitro than that achieved using commercially-available transfection agents, together with the other features offered by the robust nanostructural framework, work continues toward the application of these cSCKs for in vivo molecular recognition of genetic material, for imaging and therapy targeted specifically to pulmonary injury and disease.
Asunto(s)
ADN , Nanopartículas , Ácidos Nucleicos de Péptidos , Transfección/métodos , ADN/genética , ADN/uso terapéutico , Humanos , Nanopartículas/química , Nanopartículas/uso terapéutico , Ácidos Nucleicos de Péptidos/genéticaRESUMEN
Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes, and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein, we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs) and compare the effect of noncleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1-3 microM, the activity of LP-PNA-R9 continues to increase from 4-6 microM, while the activity of L-PNA-R9 remains constant and that of LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 dramatically increased in the presence of chloroquine, as expected for an endocytotic entry mechanism. The constructs were also found to have CMC values of 1.0 and 4.5 microM, respectively, in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis.
Asunto(s)
Portadores de Fármacos/síntesis química , Sistemas de Liberación de Medicamentos , Ácidos Nucleicos de Péptidos/química , Fosfolípidos/química , Animales , Muerte Celular , Línea Celular , Permeabilidad de la Membrana Celular , Cloroquina/farmacología , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/farmacocinética , Humanos , Micelas , Ácidos Nucleicos de Péptidos/farmacocinética , Fosfolípidos/farmacocinética , Relación Estructura-ActividadRESUMEN
The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)(3)], forms a basket structure in the presence of Na(+) and may form multiple equilibrating structures in the presence of K(+) with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)(3)] in the presence of Na(+) results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K(+), however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na(+). Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.
Asunto(s)
G-Cuádruplex , Dímeros de Pirimidina/química , Telómero , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/farmacología , Humanos , Fotoquímica , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Polymer vesicles with diameters of ca. 100-600 nm and bearing benzaldehyde functionalities within the vesicular walls were constructed through self-assembly of an amphiphilic block copolymer PEO(45)-b-PVBA(26) in water. The reactivity of the benzaldehyde functionalities was verified by cross-linking the polymersomes and also by a one-pot cross-linking and functionalization approach to further render the vesicles fluorescent, each via reductive amination. In vitro studies found these labeled nanostructures to undergo cell association.
Asunto(s)
Benzaldehídos/química , Nanoestructuras/química , Polímeros/química , Animales , Células CHO , Cricetinae , Cricetulus , Reactivos de Enlaces Cruzados/química , Células HeLa , Humanos , Agua/químicaRESUMEN
Peptide nucleic acids have a number of features that make them an ideal platform for the development of in vitro biological probes and tools. Unfortunately, their inability to pass through membranes has limited their in vivo application as diagnostic and therapeutic agents. Herein, we describe the development of cationic shell-cross-linked knedel-like (cSCK) nanoparticles as highly efficient vehicles for the delivery of PNAs into cells, either through electrostatic complexation with a PNA * ODN hybrid, or through a bioreductively cleavable disulfide linkage to a PNA. These delivery systems are better than the standard Lipofectamine/ODN-mediated method and much better than the Arg(g)-mediated method for PNA delivery in HeLa cells, showing lower toxicity and higher bioactivity. The cSCKs were also found to facilitate both endocytosis and endosomal release of the PNAs, while themselves remaining trapped in the endosomes.
Asunto(s)
Cationes/química , Sistemas de Liberación de Medicamentos , Nanopartículas , Oligonucleótidos/administración & dosificación , Ácidos Nucleicos de Péptidos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Endocitosis , Endosomas , Células HeLa , Humanos , Lípidos , Luciferasas/genética , Luciferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.
Asunto(s)
Nanopartículas/química , Transfección/métodos , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , ADN/química , ADN/metabolismo , Citometría de Flujo , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Nanopartículas/efectos adversos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Polímeros/químicaRESUMEN
UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.
Asunto(s)
G-Cuádruplex/efectos de la radiación , Oligodesoxirribonucleótidos/efectos de la radiación , Dímeros de Pirimidina/síntesis química , Rayos Ultravioleta , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Isomerismo , Espectroscopía de Resonancia Magnética , Modelos Químicos , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/químicaRESUMEN
In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective cross-linking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese hamster ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.