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1.
Nano Lett ; 16(4): 2248-53, 2016 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-26910126

RESUMEN

Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is well suited for imaging, however, existing small molecule CEST agents suffer from low sensitivity. We have developed salicylic acid conjugated dendrimers as a versatile, high performance nanoplatform. In particular, we have prepared nanocarriers based on generation 5-poly(amidoamine) (PAMAM) dendrimers with salicylic acid covalently attached to their surface. The resulting conjugates produce strong CEST contrast 9.4 ppm from water with the proton exchange tunable from ∼1000 s(-1) to ∼4500 s(-1) making these dendrimers well suited for sensitive detection. Furthermore, we demonstrate that these conjugates can be used for monitoring convection enhanced delivery into U87 glioblastoma bearing mice, with the contrast produced by these nanoparticles persisting for over 1.5 h and distributed over ∼50% of the tumors. Our results demonstrate that SA modified dendrimers present a promising new nanoplatform for medical applications.


Asunto(s)
Neoplasias Encefálicas/diagnóstico por imagen , Medios de Contraste , Dendrímeros , Glioblastoma/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Ácido Salicílico , Animales , Medios de Contraste/química , Medios de Contraste/farmacología , Dendrímeros/química , Dendrímeros/farmacología , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Ácido Salicílico/química , Ácido Salicílico/farmacología
2.
Chemistry ; 22(1): 264-71, 2016 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-26616530

RESUMEN

Diamagnetic chemical exchange saturation transfer (CEST) contrast agents offer an alternative to Gd(3+) -based contrast agents for MRI. They are characterized by containing protons that can rapidly exchange with water and it is advantageous to have these protons resonate in a spectral window that is far removed from water. Herein, we report the first results of DFT calculations of the (1) H nuclear magnetic shieldings in 41 CEST agents, finding that the experimental shifts can be well predicted (R(2) =0.882). We tested a subset of compounds with the best MRI properties for toxicity and for activity as uncouplers, then obtained mice kidney CEST MRI images for three of the most promising leads finding 16 (2,4-dihydroxybenzoic acid) to be one of the most promising CEST MRI contrast agents to date. Overall, the results are of interest since they show that (1) H NMR shifts for CEST agents-charged species-can be well predicted, and that several leads have low toxicity and yield good in vivo MR images.


Asunto(s)
Medios de Contraste/química , Gadolinio/química , Hidroxibenzoatos/química , Imagen por Resonancia Magnética/métodos , Animales , Espectroscopía de Resonancia Magnética , Ratones , Teoría Cuántica
3.
Nanomedicine ; 11(2): 401-5, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25461289

RESUMEN

Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging ("theranostics"). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥7 mol% PEG diffused only ~10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7 mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3mol%-PEG liposomes. However, increasing PEG content to ~12 mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7 mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. FROM THE CLINICAL EDITOR: This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.


Asunto(s)
Moco del Cuello Uterino/diagnóstico por imagen , Sistemas de Liberación de Medicamentos , Imagen por Resonancia Magnética , Membrana Mucosa/diagnóstico por imagen , Barbitúricos/química , Moco del Cuello Uterino/efectos de los fármacos , Medios de Contraste , Humanos , Liposomas , Membrana Mucosa/patología , Nanopartículas/química , Polietilenglicoles/química , Radiografía
4.
Biochim Biophys Acta ; 1828(5): 1365-73, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23357356

RESUMEN

Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160nm. Such nanoparticles have a negative surface charge (-11 to -18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.


Asunto(s)
Péptidos de Penetración Celular/química , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Ácidos Nucleicos/genética , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Cricetinae , Cricetulus , Vectores Genéticos/química , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Luciferasas/genética , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/ultraestructura , Oligonucleótidos/genética , Plásmidos/química , Plásmidos/genética , ARN Interferente Pequeño/genética , Ácidos Esteáricos/química , Transfección/métodos
5.
J Biol Chem ; 287(20): 16880-9, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22437827

RESUMEN

Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. The elevated Ca(2+) concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca(2+) and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca(2+) if its level is not drastically elevated.


Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Exocitosis/efectos de los fármacos , Lisosomas/metabolismo , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Células HeLa , Humanos , Células Jurkat
6.
Neuroimage ; 77: 114-24, 2013 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-23567889

RESUMEN

Chemical exchange saturation transfer (CEST) is a magnetization transfer (MT) technique to indirectly detect pools of exchangeable protons through the water signal. CEST MRI has focused predominantly on signals from exchangeable protons downfield (higher frequency) from water in the CEST spectrum. Low power radiofrequency (RF) pulses can slowly saturate protons with minimal interference of conventional semi-solid based MT contrast (MTC). When doing so, saturation-transfer signals are revealed upfield from water, which is the frequency range of non-exchangeable aliphatic and olefinic protons. The visibility of such signals indicates the presence of a relayed transfer mechanism to the water signal, while their finite width reflects that these signals are likely due to mobile solutes. It is shown here in protein phantoms and the human brain that these signals build up slower than conventional CEST, at a rate typical for intramolecular nuclear Overhauser enhancement (NOE) effects in mobile macromolecules such as proteins/peptides and lipids. These NOE-based saturation transfer signals show a pH dependence, suggesting that this process is the inverse of the well-known exchange-relayed NOEs in high resolution NMR protein studies, thus a relayed-NOE CEST process. When studying 6 normal volunteers with a low-power pulsed CEST approach, the relayed-NOE CEST effect was about twice as large as the CEST effects downfield and larger in white matter than gray matter. This NOE contrast upfield from water provides a way to study mobile macromolecules in tissue. First data on a tumor patient show reduction in both relayed NOE and CEST amide proton signals leading to an increase in magnetization transfer ratio asymmetry, providing insight into previously reported amide proton transfer (APT) effects in tumors.


Asunto(s)
Mapeo Encefálico/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Adulto , Algoritmos , Encéfalo , Simulación por Computador , Humanos , Masculino , Protones , Ondas de Radio
7.
Bioconjug Chem ; 24(3): 305-13, 2013 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-23350661

RESUMEN

Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using in vitro phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where in vitro phage display has yielded an efficient, in vivo working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems.


Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Péptidos de Penetración Celular/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Vectores Genéticos/administración & dosificación , Glioma/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Péptidos de Penetración Celular/genética , Femenino , Vectores Genéticos/genética , Glioma/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
8.
Nucleic Acids Res ; 39(9): 3972-87, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21245043

RESUMEN

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.


Asunto(s)
Péptidos de Penetración Celular/química , Lipopéptidos/química , Quinolinas/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/toxicidad , Células Cultivadas , Endosomas/metabolismo , Humanos , Indicadores y Reactivos , Mediadores de Inflamación/metabolismo , Lípidos , Lipopéptidos/metabolismo , Ratones , Nanopartículas/química , Nanopartículas/toxicidad , Quinolinas/metabolismo
9.
Mol Ther ; 19(8): 1457-67, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21343913

RESUMEN

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.


Asunto(s)
Péptidos de Penetración Celular/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Plásmidos/metabolismo , Transfección/métodos , Animales , Transporte Biológico , Línea Celular , Cricetinae , Cricetulus , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Terapia Genética/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Ácidos Nucleicos/metabolismo
10.
Bioconjug Chem ; 22(11): 2255-62, 2011 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-21978260

RESUMEN

Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1ß, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 µM and 5 µM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.


Asunto(s)
Péptidos de Penetración Celular/inmunología , Péptidos de Penetración Celular/toxicidad , Portadores de Fármacos/toxicidad , Galanina/inmunología , Galanina/toxicidad , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad , Venenos de Avispas/inmunología , Venenos de Avispas/toxicidad , Animales , Caspasas/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética , Células Cultivadas , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Galanina/química , Humanos , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopéptidos/química , Lipopéptidos/inmunología , Lipopéptidos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/química , Transfección , Factor de Necrosis Tumoral alfa/inmunología , Venenos de Avispas/química
11.
Methods Mol Biol ; 2355: 117-129, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34386955

RESUMEN

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.


Asunto(s)
Glioma , Animales , Línea Celular Tumoral , Péptidos de Penetración Celular , Sistemas de Liberación de Medicamentos , Glioma/tratamiento farmacológico , Humanos , Ratones , Ratones Desnudos , Preparaciones Farmacéuticas
12.
Biochim Biophys Acta ; 1747(2): 261-6, 2005 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-15698961

RESUMEN

Mutants of L-type pyruvate kinase with modified peptide sequence around the Ser-12 phosphorylation site were prepared and kinetics of their phosphorylation by protein kinase A was studied. The profile of substrate specificity obtained for these proteins was compared with the kinetic data of phosphorylation of short peptide substrates. Alterations made in protein structure caused weaker effects than the corresponding alterations made in peptides, while the amino acid preferences and the overall specificity pattern remained similar in the both cases. Thus, similar consensus motif holds for both protein and peptide substrates, but is less critical for recognition of proteins if compared with short peptides.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos/metabolismo , Catálisis , Electroforesis en Gel de Poliacrilamida , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/síntesis química , Fosforilación , Piruvato Quinasa/metabolismo , Especificidad por Sustrato
13.
J Biomol Screen ; 10(4): 320-8, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15964933

RESUMEN

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.


Asunto(s)
Proteína Quinasa C/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Inhibidores Enzimáticos/farmacología , Cinética , Datos de Secuencia Molecular , Biblioteca de Péptidos , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Especificidad por Sustrato
14.
ACS Macro Lett ; 4(1): 34-38, 2015 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-25642384

RESUMEN

The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect chromosomal DNA in germ cells by packing it densely using electrostatic interactions. Human protamine-1 (hPRM1) has been developed as a magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) reporter gene, based on a sequence that is approximately 50% arginine, which has a side chain with rapidly exchanging protons. In this study, we have synthesized hPRM1 and determined how its CEST MRI contrast varies as a function of pH, phosphorylation state, and upon noncovalent interaction with nucleic acids and heparin (as antagonist). CEST contrast was found to be highly sensitive to phosphorylation on serine residues, intra- and intermolecular disulfide bridge formation, and the binding of negatively charged nucleotides and heparin. In addition, the nucleotide binding constants (Keq) for the protamines were determined through plotting the molar concentration of heparin versus CEST contrast and compared between hPRM1 and salmon protamine. Taken together, these findings are important for explaining the CEST contrast of existing arginine-rich probes as well as serving as a guideline for designing new genetic or synthetic probes.

15.
Acta Neurobiol Exp (Wars) ; 74(2): 188-96, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24993628

RESUMEN

For centuries, inflammatory/foreign body reactions have plagued the attempts of clinicians to use metals for tissue and bone reconstructions. Since corrosion contributes to the rejection of metal by the body, an extremely bioinert metal - tantalum - has been successfully used in medicine. The outstanding biocompatibility and flexibility of tantalum established the basis for a growing cadre of clinical applications. One important application which benefited from the introduction of powder (particle) metallurgy is use of tantalum as bone implants. Porous materials have re-shaped the landscape of bone implants, as they allow for bone ingrowth and biological fixation, and eliminate implant loosening and related treatment failures. The unique bone-mimicking properties of porous tantalum enabled the use of tantalum as a material for bulk implants, and not only for coatings, as is the case with other porous metals. Moreover, porous tantalum also facilitates the ingrowth of soft tissue, including the formation of blood vessels that were found to assemble on the surface and within the structure of the porous tantalum. Also, since tantalum is strongly radiopaque due its high atomic number, this property is widely employed for marking in orthopedics and in endovascular medical devices. Another important development was the production of nanoparticles based on tantalum. These particles have been shown to be superior to iodinated contrast agents for blood pool imaging applications due to their longer circulation time. Their properties are similar to gold nanoparticles, but are far more cost-effective, and thus, well-positioned to replace gold in regenerative medicine for labeling and tracking of cell grafts through x-ray-based imaging. However, the amount of tantalum nanoparticles that can be taken up by stem cells is not enough to make individual cells visible in x-ray images. Thus, alternative strategies are needed, such as hydrogel or nanofiber scaffolds, which can be loaded with higher concentrations of nanoparticles, to increase the precision of cell deposition and allow tracking under x-ray guidance.


Asunto(s)
Óxidos/uso terapéutico , Regeneración/efectos de los fármacos , Tantalio/uso terapéutico , Animales , Humanos , Nanopartículas/uso terapéutico
16.
ACS Chem Biol ; 9(1): 134-8, 2014 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-24138139

RESUMEN

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.


Asunto(s)
Medios de Contraste/química , Genes Reporteros , Imagen por Resonancia Magnética/métodos , Protaminas/química , Protaminas/genética , Secuencia de Aminoácidos , Clonación Molecular , Medios de Contraste/metabolismo , Escherichia coli/genética , Expresión Génica , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular
17.
PLoS One ; 8(7): e69659, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23861978

RESUMEN

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.


Asunto(s)
Péptidos de Penetración Celular/metabolismo , ADN Viral/metabolismo , Vectores Genéticos/metabolismo , Lipopéptidos/metabolismo , Quinolinas/metabolismo , ARN Viral/metabolismo , Virus de los Bosques Semliki/genética , Transfección/métodos , Infecciones por Alphavirus/virología , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Lípidos , Luciferasas/metabolismo , Orgánulos/metabolismo , ARN Interferente Pequeño/metabolismo , Replicón/genética , Virus de los Bosques Semliki/fisiología , Replicación Viral
18.
J Control Release ; 141(1): 42-51, 2010 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-19744531

RESUMEN

In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)(4) peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)(4) peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine 2000. We show that stearyl-(RxR)(4) mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)(4) or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine 2000, we show that stearyl-(RxR)(4) is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)(4) complexed with 2'-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)(4) promotes dose-dependent splice correction in parity with (RxR)(4)-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)(4) an intriguing vector for future in vivo experiments.


Asunto(s)
Portadores de Fármacos/química , Ácidos Nucleicos/administración & dosificación , Péptidos/química , Oligonucleótidos Fosforotioatos/administración & dosificación , Ácidos Esteáricos/química , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Células CHO , Permeabilidad de la Membrana Celular , Proliferación Celular , Cricetinae , Cricetulus , Citometría de Flujo , Expresión Génica , Células HeLa , Humanos , Lípidos , Luciferasas/genética , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Oligonucleótidos Fosforotioatos/química , Oligonucleótidos Fosforotioatos/genética , Plásmidos , Transfección
19.
J Control Release ; 134(3): 221-7, 2009 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-19105971

RESUMEN

Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.


Asunto(s)
Empalme Alternativo/efectos de los fármacos , Galanina/química , Técnicas de Transferencia de Gen , Oligonucleótidos Fosforotioatos/administración & dosificación , Proteínas Recombinantes de Fusión/química , Ácidos Esteáricos/química , Venenos de Avispas/química , Empalme Alternativo/genética , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Medios de Cultivo , Células HeLa , Humanos , Lípidos/química , Oligonucleótidos Fosforotioatos/genética , Transfección
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