Evaluation of polymeric nanocarriers for RNA delivery

Therapeutic RNAs, especially siRNAs, are a promising approach for treating diseases like cancer, neurodegenerative disorders and viral infections. Their application, however, is limited due to a lack of safe and efficient delivery systems. Nanosized carriers with the ability to either complex or entrap RNA species are a promising option. rn rn rnSuch a carrier has to meet a lot of requirements, some of which are even partly contradictive. Understanding and controlling the interplay between the different demands would advance a strategic design at an early stage of therapeutic development. rn rn This work is centered around a systematic evaluation of polyplexes, such carriers that are able to complex siRNA due to electrostatic interactions. Six structurally and chemically diverse candidates, poly-L-lysine brushes, block copolymers, cationic peptides, cationic lipids, nanohydrogels, and manganese oxide particles, were tested in a simultaneous fashion. The assays, mostly based on fluorescently labeled siRNA, ranged from the evaluation of polyplex formation and stability to in vitro parameters like cellular uptake and knockdown capability. The analysis from several perspectives offered insight into the interplay between the specifications of one polyplex. Assessing the different carriers under exactly the same experimental conditions also allowed conclusions about favourable traits and starting points for further optimization. This comparative approach also revealed weaknesses of some of the conventional protocols, which were therefore contrasted with alternative methods. In addition, in vitro knockdown assays were optimized and the impact of fluorescently labeled siRNA on knockdown efficiency was assessed. rn rn rn A second class of carriers, which share the ability to entrap siRNA inside their matrix, are briefly addressed. Nanocapsules, dextran particles and liposomes were assessed for basic features like siRNA encapsulation and knockdown capability. rn rn rn rn In an approach towards targeted delivery of RNA, liposomes were endowed with mitochondriotropic tags. Despite successful functionalization, no colocalization between the liposomal cargo and mitochondria was so far observed, which makes further optimization necessary.

Novel magnetic iron oxide nanoparticles coated with poly(ethylene imine)-g-poly(ethylene glycol) for potential biomedical application: synthesis, stability, cytotoxicity and MR imaging

Magnetic iron oxide nanoparticles have found application as contrast agents for magnetic resonance imaging (MRI) and as switchable drug delivery vehicles. Their stabilization as colloidal carriers remains a challenge. The potential of poly(ethylene imine)-g-poly(ethylene glycol) (PEGPEI) as stabilizer for iron oxide (γ-Fe₂O₃) nanoparticles was studied in comparison to branched poly(ethylene imine) (PEI). Carrier systems consisting of γ-Fe₂O₃-PEI and γ-Fe₂O₃-PEGPEI were prepared and characterized regarding their physicochemical properties including magnetic resonance relaxometry. Colloidal stability of the formulations was tested in several media and cytotoxic effects in adenocarcinomic epithelial cells were investigated. Synthesized γ-Fe₂O₃ cores showed superparamagnetism and high degree of crystallinity. Diameters of polymer-coated nanoparticles γ-Fe₂O₃-PEI and γ-Fe₂O₃-PEGPEI were found to be 38.7 ± 1.0 nm and 40.4 ± 1.6 nm, respectively. No aggregation tendency was observable for γ-Fe₂O₃-PEGPEI over 12 h even in high ionic strength media. Furthermore, IC₅₀ values were significantly increased by more than 10-fold when compared to γ-Fe₂O₃-PEI. Formulations exhibited r₂ relaxivities of high numerical value, namely around 160 mM⁻¹ s⁻¹. In summary, novel carrier systems composed of γ-Fe₂O₃-PEGPEI meet key quality requirements rendering them promising for biomedical applications, e.g. as MRI contrast agents.

Drug-induced respiratory depression: an integrated model of drug effects on the hypercapnic and hypoxic drive

Drug-induced respiratory depression is a common side effect of the agents used in anesthesia practice to provide analgesia and sedation. Depression of the ventilatory drive in the spontaneously breathing patient can lead to severe cardiorespiratory events and it is considered a primary cause of morbidity. Reliable predictions of respiratory inhibition in the clinical setting would therefore provide a valuable means to improve the safety of drug delivery. Although multiple studies investigated the regulation of breathing in man both in the presence and absence of ventilatory depressant drugs, a unified description of respiratory pharmacodynamics is not available. This study proposes a mathematical model of human metabolism and cardiorespiratory regulation integrating several isolated physiological and pharmacological aspects of acute drug-induced ventilatory depression into a single theoretical framework. The description of respiratory regulation has a parsimonious yet comprehensive structure with substantial predictive capability. Simulations relative to the synergistic interaction of the hypercarbic and hypoxic respiratory drive and the global effect of drugs on the control of breathing are in good agreement with published experimental data. Besides providing clinically relevant predictions of respiratory depression, the model can also serve as a test bed to investigate issues of drug tolerability and dose finding/control under non-steady-state conditions.

On the modeling of drug induced respiratory depression in the non-steady-state

The ability of anesthetic agents to provide adequate analgesia and sedation is limited by the ventilatory depression associated with overdosing in spontaneously breathing patients. Therefore, quantitation of drug induced ventilatory depression is a pharmacokinetic-pharmacodynamic problem relevant to the practice of anesthesia. Although several studies describe the effect of respiratory depressant drugs on isolated endpoints, an integrated description of drug induced respiratory depression with parameters identifiable from clinically available data is not available. This study proposes a physiological model of CO2 disposition, ventilatory regulation, and the effects of anesthetic agents on the control of breathing. The predictive performance of the model is evaluated through simulations aimed at reproducing experimental observations of drug induced hypercarbia and hypoventilation associated with intravenous administration of a fast-onset, highly potent anesthetic mu agonist (including previously unpublished experimental data determined after administration of 1 mg alfentanil bolus). The proposed model structure has substantial descriptive capability and can provide clinically relevant predictions of respiratory inhibition in the non-steady-state to enhance safety of drug delivery in the anesthetic practice.

Quantum modeling of bioconjugated nanomaterials

In recent years, the bio-conjugated nanostructured materials have emerged as a new class of materials for the bio-sensing and medical diagnostics applications. In spite of their multi-directional applications, interfacing nanomaterials with bio-molecules has been a challenge due to somewhat limited knowledge about the underlying physics and chemistry behind these interactions and also for the complexity of biomolecules. The main objective of this dissertation is to provide such a detailed knowledge on bioconjugated nanomaterials toward their applications in designing the next generation of sensing devices. Specifically, we investigate the changes in the electronic properties of a boron nitride nanotube (BNNT) due to the adsorption of different bio-molecules, ranging from neutral (DNA/RNA nucleobases) to polar (amino acid molecules). BNNT is a typical member of III-V compounds semiconductors with morphology similar to that of carbon nanotubes (CNTs) but with its own distinct properties. More specifically, the natural affinity of BNNTs toward living cells with no apparent toxicity instigates the applications of BNNTs in drug delivery and cell therapy. Our results predict that the adsorption of DNA/RNA nucleobases on BNNTs amounts to different degrees of modulation in the band gap of BNNTs, which can be exploited for distinguishing these nucleobases from each other. Interestingly, for the polar amino acid molecules, the nature of interaction appeared to vary ranging from Coulombic, van der Waals and covalent depending on the polarity of the individual molecules, each with a different binding strength and amount of charge transfer involved in the interaction. The strong binding of amino acid molecules on the BNNTs explains the observed protein wrapping onto BNNTs without any linkers, unlike carbon nanotubes (CNTs). Additionally, the widely varying binding energies corresponding to different amino acid molecules toward BNNTs indicate to the suitability of BNNTs for the biosensing applications, as compared to the metallic CNTs. The calculated I-V characteristics in these bioconjugated nanotubes predict notable changes in the conductivity of BNNTs due to the physisorption of DNA/RNA nucleobases. This is not the case with metallic CNTs whose transport properties remained unaltered in their conjugated systems with the nucleobases. Collectively, the bioconjugated BNNTs are found to be an excellent system for the next generation sensing devices.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Osteoinductive implants: the mise-en-scène for drug-bearing biomimetic coatings

In orthopaedic and dental implantology, novel tools and techniques are being sought to improve the regeneration of bone tissue. Numerous attempts have been made to enhance the osteoconductivity of titanium prostheses, including modifications in their surface properties and coating with layers of calcium phosphate. The technique whereby such layers are produced has recently undergone a revolutionary change, which has had profound consequences for their potential to serve as drug-carrier systems. Hitherto, calcium phosphate layers were deposited upon the surfaces of metal implants under highly unphysiological physical conditions, which precluded the incorporation of proteinaceous osteoinductive drugs. These agents could only be adsorbed, superficially, upon preformed layers. Such superficially adsorbed molecules are released too rapidly within a biological milieu to be effective in their osteoinductive capacity. Now, it is possible to deposit calcium phosphate layers under physiological conditions of temperature and pH by the so-called biomimetic process, during which bioactive agents can be coprecipitated. Since these molecules are integrated into the inorganic latticework, they are released gradually in vivo as the layer undergoes degradation. This feature enhances the capacity of these coatings to act as a carrier system for osteogenic agents.

A compact and portable deposition chamber to study nanoparticles in air-exposed tissue

BACKGROUND Epidemiological studies show that elevated levels of particulate matter in ambient air are highly correlated with respiratory and cardiovascular diseases. Atmospheric particles originate from a large number of sources and have a highly complex and variable composition. An assessment of their potential health risks and the identification of the most toxic particle sources would require a large number of investigations. Due to ethical and economic reasons, it is desirable to reduce the number of in vivo studies and to develop suitable in vitro systems for the investigation of cell-particle interactions. METHODS We present the design of a new particle deposition chamber in which aerosol particles are deposited onto cell cultures out of a continuous air flow. The chamber allows for a simultaneous exposure of 12 cell cultures. RESULTS Physiological conditions within the deposition chamber can be sustained constantly at 36-37°C and 90-95% relative humidity. Particle deposition within the chamber and especially on the cell cultures was determined in detail, showing that during a deposition time of 2 hr 8.4% (24% relative standard deviation) of particles with a mean diameter of 50 nm [mass median diameter of 100 nm (geometric standard deviation 1.7)] are deposited on the cell cultures, which is equal to 24-34% of all charged particles. The average well-to-well variability of particles deposited simultaneously in the 12 cell cultures during an experiment is 15.6% (24.7% relative standard deviation). CONCLUSIONS This particle deposition chamber is a new in vitro system to investigate realistic cell-particle interactions at physiological conditions, minimizing stress on the cell cultures other than from deposited particles. A detailed knowledge of particle deposition characteristics on the cell cultures allows evaluating reliable dose-response relationships. The compact and portable design of the deposition chamber allows for measurements at any particle sources of interest.

Novel Use of Dual Anti-Inflammatory Therapy to Overcome Drug Resistance and Improve Functional Recovery Following Spinal Cord Injury

Over 1.2 million Americans are currently living with a traumatic spinal cord injury (SCI). Despite the need for effective therapies, there are currently no proven effective treatments that can improve recovery of function in SCI patients. Many therapeutic compounds have shown promise in preclinical models of SCI, but all of these have fallen short in clinical trials. P-glycoprotein (Pgp) is an active transporter expressed on capillary endothelial cell membranes at the blood-spinal cord barrier (BSCB). Pgp limits passive diffusion of blood-borne drugs into the CNS, by actively extruding drugs from the endothelial cell membrane. Pgp can become pathologically up-regulated, thus greatly impeding therapeutic drug delivery (‘multidrug resistance’). Importantly, many drugs that have been evaluated for the treatment of SCI are Pgp substrates. We hypothesized that Pgp-mediated drug resistance diminishes the delivery and efficacy of neuroprotective drugs following SCI. We observed a progressive, spatial spread of Pgp overexpression within the injured spinal cord. To assess Pgp function, we examined spinal cord uptake of systemically-delivered riluzole, a drug that is currently being evaluated in clinical trials as an SCI intervention. Blood-to-spinal cord riluzole penetration was reduced following SCI in wild-type but not Pgp-null rats, highlighting a critical role for Pgp in mediating spinal cord drug resistance after injury. Others have shown that pro-inflammatory signaling drives Pgp up-regulation in cancer and epilepsy. We have detected inflammation in both acutely- and chronically-injured spinal cord tissue. We therefore evaluated the ability of the dual COX-/5-LOX inhibitor licofelone to attenuate Pgp-mediated drug resistance following SCI. Licofelone treatment both reduced spinal cord Pgp levels and enhanced spinal cord riluzole bioavailability following SCI. Thus, we propose that licofelone may offer a new combinatorial treatment strategy to enhance spinal cord drug delivery following SCI. Additionally, we assessed the ability of licofelone, riluzole, or both to enhance recovery of locomotor function following SCI. We found that licofelone treatment conferred a significant improvement in hindlimb function that was sustained through the end of the study. In contrast, riluzole did not improve functional outcome. We therefore conclude that licofelone holds promise as a potential neuroprotective intervention for SCI.

Perceptual distortion modeling for side-by-side 3D video delivery

A frame-level distortion model based on perceptual features of the human visual system is proposed to improve the performance of unequal error protection strategies and provide better quality of experience to users in Side-by-Side 3D video delivery systems.

Design of a new neutron delivery system for high flux source

La construcción en la actualidad de nuevas fuentes para el uso de haces de neutrones así como los programas de renovación en curso en algunas de las instalaciones experimentales existentes han evidenciado la necesidad urgente de desarrollar la tecnología empleada para la construcción de guías de neutrones con objeto de hacerlas mas eficientes y duraderas. Esto viene motivado por el hecho de que varias instalaciones de experimentación con haces de neutrones han reportado un número de incidentes mecánicos con tales guías, lo que hace urgente el progresar en nuestro conocimiento de los susbtratos vítreos sobre los cuales se depositan los espejos que permiten la reflexión total de los neutrones y como aquellos se degradan con la radiación. La presente tesis se inscribe en un acuerdo de colaboración establecido entre el Institut Max von Laue - Paul Langevin (ILL) de Grenoble y el Consorcio ESS-Bilbao con objeto de mejorar el rendimiento y sostenibilidad de los sistemas futuros de guiado de neutrones. El caso de la Fuente Europea de Espalación en construcción en Lund sirve como ejemplo ya que se contempla la instalación de guías de neutrones de más de 100 metros en algunos de los instrumentos. Por otro lado, instalaciones como el ILL prevén también dentro del programa Endurance de rejuvenecimiento la reconstrucción de varias líneas de transporte de haz. Para el presente estudio se seleccionaron cuatro tipos de vidrios borosilicatados que fueron el Borofloat, N-ZK7, N-BK7 y SBSL7. Los tres primeros son bien conocidos por los especialistas en instrumentación neutrónica ya que se han empleado en la construcción de varias instalaciones mientras que el último es un candidato potencial en la fabricación de substratos para espejos neutrónicos en un futuro. Los cuatro vidrios tiene un contenido en óxido de Boro muy similar, approximadamente un 10 mol.%. Tal hecho que obedece a las regulaciones para la fabricación de estos dispositivos hace que tales substratos operen como protección radiológica absorbiendo los neutrones transmitidos a través del espejo de neutrones. Como contrapartida a tal beneficio, la reacción de captura 10B(n,_)7Li puede degradar el substrato vítreo debido a los 2.5 MeV de energía cinética depositados por la partícula _ y los núcleos en retroceso y de hecho la fragilidad de tales vidrios bajo radiación ha sido atribuida desde hace ya tiempo a los efectos de esta reacción. La metodología empleada en esta tesis se ha centrado en el estudio de la estructura de estos vidrios borosilicatados y como esta se comporta bajo condiciones de radiación. Los materiales en cuestión presentan estructuras que dependen de su composición química y en particular del ratio entre formadores y modificadores de la red iono-covalente. Para ello se han empleado un conjunto de técnicas de caracterización tanto macro- como microscópicas tales como estudios de dureza, TEM, Raman, SANS etc. que se han empleado también para determinar el comportamiento de estos materiales bajo radiación. En particular, algunas propiedades macroscópicas relacionadas con la resistencia de estos vidrios como elementos estructurales de las guías de neutrones han sido estudiadas así como también los cambios en la estructura vítrea consecuencia de la radiación. Para este propósito se ha diseñado y fabricado por el ILL un aparato para irradiación de muestras con neutrones térmicos en el reactor del ILL que permite controlar la temperatura alcanzada por la muestra a menos de 100 °C. Tal equipo en comparación con otros ya existences permite en cuestión de dias acumular las dosis recibidas por una guía en operación a lo largo de varios años. El uso conjunto de varias técnicas de caracterización ha llevado a revelar que los vidrios aqui estudiados son significativamente diferentes en cuanto a su estructura y que tales diferencias afectan a sus propiedades macroscópicas asi como a su comportamiento bajo radiación. Tal resultado ha sido sorprendente ya que, como se ha mencionado antes, algunos de estos vidrios eran bien conocidos por los fabricantes de guías de neutrones y hasta el momento eran considerados prácticamente similares debido a su contenido comparable en óxido de Boro. Sin embargo, los materiales N-BK7 and S-BSL7 muetran gran homogeneidad a todas las escalas de longitud, y más específicamente, a escalas nanométricas las subredes de Sílice y óxido de Boro se mezclan dando logar a estructuras locales que recuerdan a la del cristal de Reedmergnerita. Por el contrario, N-ZK7 y Borofloat muestran dominios separados ricos en Sílice o Boro. Como era de esperar, las importantes diferencias arriba mencionadas se traducen en comportamientos dispares de estos materiales bajo un haz de neutrones térmicos. Los resultados muestran que el N-BK7 y el S-BSL7 son los más estables bajo radiación, lo que macroscópicamente hace que estos materiales muestren un comportamiento similar expandiéndose lentamente en función de la dosis recibida. Por el contario, los otros dos materiales muestran un comportamiento mucho más reactivo, que hace que inicialmente se compacten con la dosis recibida lo que hace que las redes de Silicio y Boro se mezclen resultando en un incremento en densidad hasta alcanzar un valor límite, seguido por un proceso de expansión lenta que resulta comparable al observado para N-BK7 y SBSL7. Estos resultados nos han permitido explicar el origen de las notorias diferencias observadas en cuanto a las dosis límite a partir de las cuales estos materiales desarrollan procesos de fragmentación en superficie. ABSTRACT The building of new experimental neutron beam facilities as well as the renewal programmes under development at some of the already existing installations have pinpointed the urgent need to develop the neutron guide technology in order to make such neutron transport devices more efficient and durable. In fact, a number of mechanical failures of neutron guides have been reported by several research centres. It is therefore important to understand the behaviour of the glass substrates on top of which the neutron optics mirrors are deposited and how these materials degrade under radiation conditions. The case of the European Spallation Source (ESS) at present under construction at Lund is a good example. It previews the deployment of neutron guides having more than 100 metres of length for most of the instruments. Also, the future renovation programme of the ILL, called Endurance, foresees the refurbishment of several beam lines. This Ph.D. thesis was the result of a collaboration agreement between the ILL and ESS-Bilbao aiming to improve the performance and sustainability of future neutron delivery systems. Four different industrially produced alkali-borosilicate glasses were selected for this study: Borofloat, N-ZK7, N-BK7 and SBSL7. The first three are well known within the neutron instrumentation community as they have already been used in several installations whereas the last one is at present considered as a candidate for making future mirror substrates. All four glasses have a comparable content of boron oxide of about 10 mol.%. The presence of such a strong neutron absorption element is in fact a mandatory component for the manufacturing of neutron guides because it provides a radiological shielding for the environment. This benefit is however somewhat counterbalanced since the resulting 10B(n,_)7Li reactions degrade the glass due to the deposited energy of 2.5 MeV by the _ particle and the recoil nuclei. In fact, the brittleness of some of these materials has been ascribed to this reaction. The methodology employed by this study consisted in understanding the general structure of borosilicates and how they behave under irradiation. Such materials have a microscopic structure strongly dependent upon their chemical content and particularly on the ratios between network formers and modifiers. The materials have been characterized by a suite of macroscopic and structural techniques such as hardness, TEM, Raman, SANS, etc. and their behaviour under irradiation was analysed. Some macroscopic properties related to their resistance when used as guide structural elements were monitored. Also, changes in the vitreous structure due to radiation were observed by means of several experimental tools. For such a purpose, an irradiation apparatus has been designed and manufactured to enable irradiation with thermal neutrons within the ILL reactor while keeping the samples below 100 °C. The main advantage of this equipment if compared to others previously available was that it allowed to reach in just some days an equivalent neutron dose to that accumulated by guides after several years of use. The concurrent use of complementary characterization techniques lead to the discovery that the studied glasses were deeply different in terms of their glass network. This had a strong impact on their macroscopic properties and their behaviour under irradiation. This result was a surprise since, as stated above, some of these materials were well known by the neutron guide manufacturers, and were considered to be almost equivalent because of their similar boron oxide content. The N-BK7 and S-BSL7 materials appear to be fairly homogeneous glasses at different length scales. More specifically, at nanometre scales, silicon and boron oxide units seem to mix and generate larger structures somewhat resembling crystalline Reedmergnerite. In contrast, N-ZK7 and Borofloat are characterized by either silicon or boron rich domains. As one could expect, these drastic differences lead to their behaviour under thermal neutron flux. The results show that N-BK7 and S-BSL7 are structurally the most stable under radiation. Macroscopically, such stability results in the fact that these two materials show very slow swelling as a function or radiation dose. In contrast, the two other glasses are much more reactive. The whole glass structure compacts upon radiation. Specifically, the silica network, and the boron units tend to blend leading to an increase in density up to some saturation, followed by a very slow expansion which comes to be of the same order than that shown by N-BK7 and S-BSL7. Such findings allowed us to explain the drastic differences in the radiation limits for macroscopic surface splintering for these materials when they are used in neutron guides.

Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (TM4) or 38.5°C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette–Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

A mouse model for adenovirus gene delivery

The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Vector-mediated delivery of 125I-labeled beta-amyloid peptide A beta 1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the A beta 1-40/vector complex.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue beta-amyloid peptide A beta 1-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Transport of 125I-labeled A beta 1-40 (125I-A beta 1-40) through the BBB was found to be negligible by experiments with both an intravenous injection technique and an internal carotid artery perfusion method in anesthetized rats. In addition, 125I-A beta 1-40 was rapidly metabolized after either intravenous injection or internal carotid artery perfusion. BBB transport was increased and peripheral metabolism was decreased by conjugation of monobiotinylated 125I-A beta 1-40 to a vector-mediated drug delivery system, which consisted of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the 125I,bio-A beta 1-40/SA-OX26 conjugate was 0.15 +/- 0.01, a level that is 2-fold greater than the brain uptake of morphine. The binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was demonstrated by both film and emulsion autoradiography performed on frozen sections of AD brain. Binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was completely inhibited by high concentrations of unlabeled A beta 1-40. In conclusion, these studies show that BBB transport and access to amyloid within brain may be achieved by conjugation of A beta 1-40 to a vector-mediated BBB drug delivery system.