We present a low-cost, portable microfluidic platform that uses laminated polymethylmethacrylate chips, peristaltic micropumps and LEGO® Mindstorms components for the generation of magnetoliposomes that does not require extrusion steps. Mixtures of lipids reconstituted in ethanol and an aqueous phase were injected independently in order to generate a combination of laminar flows in such a way that we could effectively achieve four hydrodynamic focused nanovesicle generation streams. Monodisperse magnetoliposomes with characteristics comparable to those obtained by traditional methods have been obtained. The magnetoliposomes are responsive to external magnetic field gradients, a result that suggests that the nanovesicles can be used in research and applications in nanomedicine.
To explore the potential of magnetofection in delivering pDNA to primary mouse embryonic fibroblasts (PMEFs) and porcine fetal fibroblasts (PFFs) and investigate an effect of magnetic cell labeling on transfection efficacy.
Targeted gene or drug delivery aims to locally accumulate the active agent and achieve the maximum local therapeutic effect at the target-site while reducing unwanted effects at nontarget sites. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected magnetic nanoparticles (MNPs), phospholipids, and nucleic acid are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres or microbubbles. This manuscript describes the protocols for preparation of magnetic lipospheres and microbubbles for nucleic acid delivery, and it also describes the procedures for labeling the components of the bubbles (lipids, MNPs, and nucleic acids) for the visualization of the vectors and their characterization, such as magnetic responsiveness and ultrasound contrast effects. Protocols are given for the transfection procedure in adherent cells, evaluation of the association of the magnetic vectors with the cells, reporter gene expression analysis, and cell viability assessment.
Nucleic acid delivery to cells to make them produce a desired protein or to shut down the expression of endogenous genes opens unique possibilities for research and therapy. During the last decade, to realize the potential of this approach, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as Magnetofection. Our research group at the Institute of Experimental Oncology and Therapy Research, located at the University Hospital Klinikum rechts der Isar in the center of Munich, Germany, develops new magnetic nanomaterials and, their formulations with gene-delivery vectors and technologies to allow localized and efficient gene delivery in vitro and in vivo for a variety of research, diagnostic and therapeutic applications.
Intrauterine growth restriction is associated with impaired lung function in adulthood. It is unknown whether such impairment of lung function is linked to the transforming growth factor (TGF)-? system in the lung. Therefore, we investigated the effects of IUGR on lung function, expression of extracellular matrix (ECM) components and TGF-? signaling in rats. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 70. Pulmonary activity of the TGF-? system was determined at P1 and P70. TGF-? signaling was blocked in vitro using adenovirus-delivered Smad7. At P70, respiratory airway compliance was significantly impaired after IUGR. These changes were accompanied by decreased expression of TGF-?1 at P1 and P70 and a consistently dampened phosphorylation of Smad2 and Smad3. Furthermore, the mRNA expression levels of inhibitors of TGF-? signaling (Smad7 and Smurf2) were reduced, and the expression of TGF-?-regulated ECM components (e.g. collagen I) was decreased in the lungs of IUGR animals at P1; whereas elastin and tenascin N expression was significantly upregulated. In vitro inhibition of TGF-? signaling in NIH/3T3, MLE 12 and endothelial cells by adenovirus-delivered Smad7 demonstrated a direct effect on the expression of ECM components. Taken together, these data demonstrate a significant impact of IUGR on lung development and function and suggest that attenuated TGF-? signaling may contribute to the pathological processes of IUGR-associated lung disease.
Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.
Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an optimal stimulating CpG motif in horses with regard to species specificity on equine bronchoalveolar lavage (BAL) cells, in terms of a possible specific immunomodulation effect (Th2/Th1 shift) by used CpG-ODN. Accordingly, GNPs were evaluated as a delivery system to improve CpG-ODN immunostimulation in equine BAL cells. BAL fluid (BALF) was obtained from seven horses with moderate RAO and from four healthy horses and was subsequently incubated with five different CpG-ODN sequences (from A-, B- and C-class) and one ODN without any CpG motif. Release of three key cytokines (IL-4, IL-10 and IFN-?) was quantified by ELISA to detect an allergy mediated Th2 immunoresponse (IL-4) as well as a proinflammatory Th1 response (IFN-?). Due to its specific anti-inflammatory and anti-allergic effects, IL-10 was considered as a beneficial agent in pathophysiology of RAO. Results showed a significant upregulation of IL-10 and IFN-? on the one hand and a downregulation of IL-4 on the other hand in RAO affected horses. Cell cultures from healthy horses had a significantly stronger response in cytokine release to all the applied stimuli in contrast to RAO derived cells. Comparing all five CpG sequences, A-class 2216 significantly showed the highest immunomodulatory effects on equine BALF cells and, hence, was chosen for follow-up preliminary clinical studies.
Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-? and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life.
Bioactive implants are promising tools in regenerative medicine. Here we describe a versatile procedure for preparing a gene-activated matrix on titanium. Lyophilized copolymer-protected gene vectors (COPROGs) suspended in poly(d,l-lactide) (PDLLA) solutions in ethyl acetate were used to varnish solid surfaces. The gene-activated PDLLA surfaces were first established on polypropylene 96-well plates. Vector release from these surfaces in aqueous buffer, cell viability and gene transfer efficiency to NIH 3T3 fibroblasts was strongly dependent on the vector dose and its ratio to PDLLA film thickness. A detailed analysis of these relationships allowed establishing correlations which can be used to calculate suitable combinations of COPROGs and PDLLA yielding optimal gene transfer efficiency. This was verified with COPROG-activated PDLLA coatings on titanium foils. HEK 293 and mesenchymal stem cells expressed the BMP-2 gene comprised in the gene-activated surface in a manner that was consistent with the predicted dose-response and toxicity profiles found in NIH 3T3 cells. The systematic procedure presented here for identifying optimal coating compositions can be applied to any combination of vector type and coating material.
Intrauterine growth restriction (IUGR) is associated with an increased risk of renal diseases in adulthood. However, while low-birth-weight-infants often undergo accelerated postnatal growth, the impact of postnatal environmental factors such as nutrition and early postnatal stressors on renal development and function remains unclear. In this context, Neuropeptide Y (NPY) may act as a critical factor. NPY is a sympathetic coneurotransmitter involved in blood pressure regulation and tubular function. Yet, little is known about the expression and function of endogenous NPY in the kidney and the functional relevance for the transmission of persistent postnatal-induced effects.
Research applications and cell therapies involving genetically modified cells require reliable, standardized, and cost-effective methods for cell manipulation. We report a novel nanomagnetic method for integrated cell separation and gene delivery. Gene vectors associated with magnetic nanoparticles are used to transfect/transduce target cells while being passaged and separated through a high gradient magnetic field cell separation column. The integrated method yields excellent target cell purity and recovery. Nonviral and lentiviral magselectofection is efficient and highly specific for the target cell population as demonstrated with a K562/Jurkat T-cell mixture. Both mouse and human enriched hematopoietic stem cell pools were effectively transduced by lentiviral magselectofection, which did not affect the hematopoietic progenitor cell number determined by in vitro colony assays. Highly effective reconstitution of T and B lymphocytes was achieved by magselectofected murine wild-type lineage-negative Sca-1(+) cells transplanted into Il2rg(-/-) mice, stably expressing GFP in erythroid, myeloid, T-, and B-cell lineages. Furthermore, nonviral, lentiviral, and adenoviral magselectofection yielded high transfection/transduction efficiency in human umbilical cord mesenchymal stem cells and was fully compatible with their differentiation potential. Upscaling to a clinically approved automated cell separation device was feasible. Hence, once optimized, validated, and approved, the method may greatly facilitate the generation of genetically engineered cells for cell therapies.
Results from large epidemiological studies suggest a clear relation between low birth weight and adverse renal outcome evident as early as during childhood. Such adverse outcomes may include glomerular disease, hypertension, and renal failure and contribute to a phenomenon called fetal programming. Other factors potentially leading to an adverse renal outcome following fetal programming are maternal diabetes mellitus, smoking, salt overload, and use of glucocorticoids during pregnancy. However, clinical data on the latter are scarce. Here, we discuss potential underlying mechanisms of fetal programming, including reduced nephron number via diminished nephrogenesis and other renal (e.g., via the intrarenal renin-angiotensin-aldosterone system) and non-renal (e.g., changes in endothelial function) alterations. It appears likely that the outcomes of fetal programming may be influenced or modified postnatally, for example, by the amount of nutrients given at critical times.
Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.
Low birth weight and intrauterine growth restriction (IUGR) can be caused by numerous different conditions. In many experimental settings, however, these different causes are not accounted for. This study aimed at comparing the impact of two frequent causes of IUGR (low utero-placental blood flow vs. malnutrition) on fetal programming of gene expression. We studied offspring of dams treated by uterine artery ligation or sham operation compared with untreated controls and offspring of dams that were fed either a low protein or normal protein diet. After Cesarean section at term, placental and fetal hepatic expression of key "metabolic" and "vasoregulative" genes was investigated by quantitative RT-PCR. Ligation neonates showed IUGR, reduced expression of placental leptin, placental and hepatic IGF-I, hepatic inducible nitric oxide synthase, and increased expression of placental IGF binding protein 1, hepatic IGF-II receptor and erythropoietin (EPO). Low protein offspring also showed IUGR but increased expression of placental leptin; IGF-I; placental and hepatic inducible nitric oxide synthase; hepatic insulin, IGF-I, and IGF-II receptors; and reduced expression of placental IGF binding protein 1, IGF-II, leptin-receptor type A, placental and hepatic leptin receptor type B, and EPO. Expression was independent of sex, birth weight, fetal intrauterine position, and EPO expression. In conclusion, the impact of IUGR on fetal and placental gene expression depends on the cause of low birth weight. Therefore, morbidity after IUGR should be analyzed referring to its pathophysiological cause rather than referring to low birth weight itself. Fetal hypoxia as estimated by hepatic EPO expression does not seem to be a key regulator of transcriptional activity in our models.
Lipospheres made from soy bean oil and a combination of the cationic lipid Metafectene and the helper lipid dioleoylphosphatidyl-ethanolamine were functionalized with magnetic nanoparticles (NPs) and small interfering RNA (siRNA). The resulting magnetic lipospheres loaded with siRNA are proven here as efficient nonviral vectors for gene silencing. Embedding magnetic NPs in the shell of lipospheres allows for magnetic force-assisted transfection (magnetofection) as well as magnetic targeting in both static and fluidic conditions mimicking the bloodstream.
The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo.
Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium.
A copolymer-protected gene vector (COPROG) is a three-component gene delivery system consisting of a preformed DNA and branched polyethylenimine (bPEI) complex subsequently modified by the addition of a copolymer (P6YE5C) incorporating both poly(ethylene glycol) (PEG) and anionic peptides. Using fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM), we characterized and compared the self-assembly of bPEI/DNA particles and COPROG complexes. In low salt buffer, both bPEI/DNA and COPROG formulations form stable nanoparticles with hydrodynamic radii between 60-120 nm. COPROG particles, as compared to bPEI/DNA, show greatly improved particle stability to both physiological salt as well as low pH conditions. Binding stoichiometry of the three-component COPROG system was investigated by dual-color fluorescence cross-correlation spectroscopy (FCCS). It was found that a significant fraction of P6YE5C copolymer aggregates with excess bPEI forming bPEI/P6YE5C "ghost complexes" with no DNA inside. The ratio of ghost particles to COPROG complexes is about 4:1. In addition, we find a large fraction of excess P6YE5C copolymer, which remains unbound in solution. We observe a 2-4-fold enhanced reporter gene expression with COPROG formulations at various equivalents as compared to bPEI-DNA alone. We believe that both complex stabilization as well as the capture of excess bPEI into ghost particles induced by the copolymer is responsible for the improvement in gene expression.
Intrauterine growth restriction (IUGR) is associated with systemic hypertension of the offspring later in life. The exact mechanisms are still incompletely understood. 11?-Hydroxysteroid dehydrogenase 2 (11?-HSD2) in the distal renal tubule protects the mineralocorticoid receptor from cortisol. As we did not find a suppression of 11?-HSD2 in total kidney of IUGR animals, our objective was to investigate whether a suppression of 11?-HSD2 could be detected on a more sophisticated level such as in situ protein and gene expression of 11?-HSD2 in mildly hypertensive IUGR offspring.
Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the oncolytic effect by magnetic force is mainly due to the improved internalization of magnetic virus complexes resulting in potentiated virus progeny formation. Upon intratumoral injection and application of a gradient magnetic field in a murine xenograft model, magnetic virus complexes exhibited a stronger oncolytic effect than adenovirus alone. We propose that this approach would be useful during in vivo administration to tumor-feeding blood vessels to boost the efficacy of the primary infection cycle within the tumor. For systemic application, further modification of magnetic adenovirus complexes for shielding and retargeting of the whole magnetic virus complex entity is needed.
Adrenomedullin (ADM) has antiproliferative effects on glomerular mesangial cells. The study was performed to determine changes in glomerular gene expression of the ADM system by ADM treatment in anti-Thy1 glomerulonephritis (GN).
Low birth weight is a risk factor for the development of a more severe course of secondary renal diseases. We tested the hypothesis that experimental mesangioproliferative glomerulonephritis (GN) shows an aggravated course in rats inflicted with experimental uteroplacental insufficiency during gestation.
Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 +/- 2 nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cytotoxicity occurred at approximately 10-fold higher drug concentrations (maximal toxicity at 1-10 microM), was sensitive to mevalonate, and was significantly enhanced in the presence of NG-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition.
In a magnetofection procedure, self-assembling complexes of enhancers like cationic lipids with plasmid DNA or small interfering RNA (siRNA) are associated with magnetic nanoparticles and are then concentrated at the surface of cultured cells by applying a permanent inhomogeneous magnetic field. This process results in a considerable improvement in transfection efficiency compared to transfection carried out with nonmagnetic gene vectors. This article describes how to synthesize magnetic nanoparticles suitable for nucleic acid delivery by liposomal magnetofection and how to test the plasmid DNA and siRNA association with the magnetic components of the transfection complex. Protocols are provided for preparing magnetic lipoplexes, performing magnetofection in adherent and suspension cells, estimating the association/internalization of vectors with cells, performing reporter gene analysis, and assessing cell viability. The methods described here can be used to screen magnetic nanoparticles and formulations for the delivery of nucleic acids by liposomal magnetofection in any cell type.
Targeted delivery systems for anticancer drugs are urgently needed to achieve maximum therapeutic efficacy by site-specific accumulation and thereby minimizing adverse effects resulting from systemic distribution of many potent anticancer drugs. We have prepared folate receptor targeted magnetic liposomes loaded with doxorubicin, which are designed for tumor targeting through a combination of magnetic and biological targeting. Furthermore, these liposomes are designed for hyperthermia-induced drug release to be mediated by an alternating magnetic field and to be traceable by magnetic resonance imaging (MRI). Here, detailed preparation and relevant characterization techniques of targeted magnetic liposomes encapsulating doxorubicin are described.
Immunotherapy with whole cell cancer vaccines has been tested in various tumor types. This study investigated the safety profile and antitumor activity of an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant human interleukin-2 and human interferon-gamma. Thirty HLA-A*0201-matched patients with progressive, castration-resistant prostate cancer received four intradermal injections on days 1, 15, 29, and 92, and then every 90 days, as long as no tumor progression occurred. Three patients received a dose level of 7.5 million cells, and 27 patients received 15 million cells per injection. The primary study criteria were safety and the difference in prostate-specific antigen doubling time (PSA-DT), determined in the pretreatment phase (before the start of vaccination) and in the trial treatment phase (during vaccination). No dose-limiting or autoimmune toxicity was seen. During vaccination there was a significant prolongation of the PSA-DT compared with the prevaccination period (prolongation from 63 to 114 days; p < 0.01; intention to treat). In addition, results showed a period of PSA stabilization of at least 12 weeks, together with stable bone scans in 12 of 30 patients, and 3 patients sustained a >50% decrease in PSA versus baseline. The median overall survival time from first vaccination was 32 months (mean value, 34 months). Immune monitoring revealed T cell stimulation in the majority of patients. This vaccine strategy was found to be safe and well tolerated and was accompanied by prolongation of PSA-DT. The results of this trial warrant clinical development of this vaccine.
The magnetic labeling of living cells has become of major interest in the areas of cell therapy and tissue engineering. Magnetically labeled cells have been reported to allow increased and controlled seeding, tracking, and targeting of cells. In this work, we comprehensively characterize magnetic nanoparticles (MNPs) possessing a magnetite core of about 11 nm, and which are coated with the fluorinated surfactant F(CF2)nCH2CH2SCH2CH2C(O)OLi and 1,9-nonandithiol (NDT) for the nonspecific labeling of human pulmonary epithelial (H441) cells. We achieved a non-specific cell loading of 38 pg Fe/cell. In this work we combine magnetic cell labeling with subsequent genetic modification of the cells with non-viral transfection complexes associated with PEI-Mag2 magnetic nanoparticles upon gradient magnetic field application called magnetofection. The magnetic responsiveness and magnetic moment of the MNP-labeled cells and the magnetic transfection complexes were evaluated by measuring changes in the turbidity of prepared cells suspensions and complexes in a defined magnetic gradient field. The magnetic responsiveness of cells that were loaded with NDT-Mag1 MNPs (20-38 pg Fe/cell) was sufficient to engraft these labeled cells magnetically onto the luminal surface of a culture tube. This was achieved using a solenoid electromagnet that produced a radial magnetic field of 20-30 mT at the seeding area and an axial gradient field of approx. 4 T/m. The MNP-labeled cells were magnetofected in 2D arrays (well plates) and at the luminal surface of cell culture tube. The optimized magnetic pre-labeling of cells did not interfere with, or even increased, the efficiency of magnetofection in both culture systems without causing cell toxicity. Cell loading of 38 pg Fe/cell of NDT-Mag1 MNPs resulted in high transverse relaxivities r2*, thus allowing the MRI detection of cell concentrations that were equivalent to (or higher than) 1.2 microg Fe/ml. Multi-echo gradient echo imaging and R2* mapping detected as few as 1533 MNP-labeled H441 cells localized within a 50 microl fibrin clot and MNP-labeled cell monolayers that were engrafted on the luminal surface of a cell culture tube. Further loading of cells with MNPs did not increase either the magnetic responsiveness of the cells or the sensitivity of MR imaging. In summary, the NDT-Mag1 magnetic nanoparticles provided a high cell-loading efficiency, resulting in strong cell magnetic moments and a high sensitivity to MRI detection. The transfection ability of the labeled cells was also maintained, thereby increasing the magnetofection efficiency.
We describe folate receptor targeted thermosensitive magnetic liposomes, which are designed to combine features of biological and physical (magnetic) drug targeting for use in magnetic hyperthermia-triggered drug release. The optimized liposome formulation DPPC:cholesterol:DSPE-PEG(2000):DSPE-PEG(2000)-Folate at 80:20:4.5:0.5 molar ratio showed calcein release of about 70% both in PBS and in 50% FBS (fetal bovine serum) at 43 degrees C and less than 5% release at 37 degrees C following 1h incubation. Folate-targeted doxorubicin-containing magnetic liposomes of the above lipid composition (MagFolDox) showed encapsulation efficiencies of about 85% and 24% for doxorubicin and magnetic nanoparticles (mean crystallite size 10nm), respectively. This magnetic formulation displayed the desired temperature sensitivity with 52% doxorubicin release in 50% fetal bovine serum (FBS) following 1h incubation at 43 degrees C. MagFolDox, when physically targeted to tumor cells in culture by a permanent magnetic field yielded a substantial increase in cellular uptake of doxorubicin as compared to Caelyx (a commercially available liposomal doxorubicin preparation), non-magnetic folate-targeted liposomes (FolDox) and free doxorubicin in folate receptor expressing tumor cell lines (KB and HeLa cells). This resulted in a parallel increase in cytotoxicity over Caelyx and FolDox. Magnetic hyperthermia at 42.5 degrees C and 43.5 degrees C synergistically increased the cytotoxicity of MagFolDox. The results suggest that an integrated concept of biological and physical drug targeting, triggered drug release and hyperthermia based on magnetic field influence can be used advantageously for thermo-chemotherapy of cancers.
Hypoxia and insulin are known key players in the activation leptin transcription and translation in vivo and in vitro. These insulin- and hypoxia-dependent effects are leptin transcription are mediated via independent elements on the leptin-promotor, even more coincubation of the two stimuli in vitro results in a supraadditive effect on leptin transcription. The aim of this study was to examine whether hyperinsulinemia is able to interfere with the hypoxia-driven expression of leptin in adipose and extra-adipose tissue in vivo.
Large epidemiological studies suggest a clear relation between low birth weight and adverse renal outcomes evident as early as during childhood. Such adverse outcomes may include glomerular disease, hypertension, and renal failure. Data from autopsy material and from experimental models suggest that reduction in nephron number via diminished nephrogenesis may be a major mechanism, and factors that lead to this reduction are incompletely elucidated. Other mechanisms appear to be renal (e.g., via the intrarenal renin-angiotensin-aldosterone system) and nonrenal (e.g. changes in endothelial function). It also appears likely that the outcomes of fetal programming may be influenced postnatally, for example, by the amount of nutrients given at critical times.
This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.
Intrauterine growth restriction (IUGR) is associated with an increased prevalence of renal malfunction. Two principal pathogenetic pathways appear to be involved: on the one hand non-renal mechanisms such as hypertension and type 2 diabetes, both associated with previous IUGR, predispose to secondary renal damage; on the other hand, renal mechanisms are involved such as the reduced number of nephrons in low-birth-weight children, which is a risk factor for future renal failure. In addition, glomerular diseases show a severer course in IUGR children. The course of the nephrotic syndrome is less favourable, and IgA nephropathy is associated with a higher prevalence of glomerular sclerosis. Data from animal experiments suggest an increased susceptibility of glomeruli to inflammatory stimuli and reduced regenerative capacities. However, not only prenatal environment, but also postnatal hyperalimentation is responsible for the manifestation of renal disease after IUGR.
Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
Intrauterine growth restriction (IUGR) leads to low nephron number and higher incidence of renal disease. We hypothesized that IUGR induces early podocyte alterations based on a dysregulation of Wilms tumour suppressor gene 1 (WT1), a key player of nephrogenesis and mediator of podocyte integrity.
The Notch pathway is dysregulated and a potential target in glioblastoma multiforme (GBM). Currently available Notch inhibitors block ?-secretase, which is necessary for Notch processing. However, Notch is first cleaved by ?-secretase outside the plasma membrane, via a disintegrin and metalloproteinase-10 and -17. In this work, we used a potent ?-secretase inhibitor (ASI) to test inhibition of glioblastoma growth and inhibition of Notch and of both novel and known Notch targets. Featured in this study are luciferase reporter assays and immunoblot, microarray analysis, chromatin immunoprecipitation (ChIP), quantitative real-time PCR, cell number assay, bromodeoxyuridine incorporation, plasmid rescue, orthotopic xenograft model, and local delivery of treatment with convection-enhanced delivery using nanoparticles, as well as survival, MRI, and ex vivo luciferase assay. A CBF1-luciferase reporter assay as well as an immunoblot of endogenous Notch revealed Notch inhibition by the ASI. Microarray analysis, quantitative real-time PCR, and ChIP of ASI and ?-secretase inhibitor (GSI) treatment of GBM cells identified known Notch pathway targets, as well as novel Notch targets, including YKL-40 and leukemia inhibitory factor. Finally, we found that local nanoparticle delivery of ASIs but not GSIs increased survival time significantly in a GBM stem cell xenograft treatment model, and ASI treatment resulted in decreased tumor size and Notch activity. This work indicates ?-secretase as an alternative to ?-secretase for inhibition of Notch in GBM and possibly other cancers as well, and it identifies novel Notch targets with biologic relevance and potential as biomarkers.
The application of scaffolds in bone tissue engineering often comes along with side effects such as poor integrity, low regeneration rates of bone tissue with inadequate functionality, and, in case of non-degradable implants, the necessity of a second removal surgery after therapy. In this study, we coated a bioresorbable FDA-approved poly-(?-caprolactone)-scaffold for bone regeneration with a poly-(D,L-lactide) layer containing copolymer-protected gene vectors to locally provide bone morphogenetic protein-2 (BMP-2). Results show that the presence of such gene vectors did not affect the distribution and attachment of seeded cells on gene-activated surfaces. BMP-2 was released into cell culture supernatants and furthermore detected in homogenised scaffolds. Increased amounts of osteoblastic markers, such as osteocalcin, osteopontin and the activity of alkaline phosphatase, in gene-activated scaffolds in vitro suggest a transdifferentiation of myoblastic C2C12 cells into the osteoblastic phenotype. With this study we present a new technology to bioactivate implant surfaces with non-viral gene vectors. This tool allows the stimulation of tissue regeneration by a local release of therapeutic proteins in vivo.
Site specific vascular gene delivery for therapeutic implications is favorable because of reduction of possible side effects. Yet this technology faces numerous hurdles that result in low transfection rates because of suboptimal delivery. Combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer could make it possible to use the systemic vasculature as the route of application and to magnetically trap these compounds at the target of interest. In this study we show that magnetic nanoparticle-coated microbubbles bind plasmid DNA and successfully deliver it to endothelial cells in vitro and more importantly transport their cargo through the vascular system and specifically deliver it to the vascular wall in vivo at sites where microbubbles are retained by magnetic force and burst by local ultrasound application. This resulted in a significant enhancement in site specific gene delivery compared with the conventional microbubble technique. Thus, this technology may have promising therapeutic potential.
In the recent years, nanotechnology has boosted the development of potential drug delivery systems and material engineering on nanoscale basis in order to increase drug specificity and reduce side effects. A potential delivery system for immunostimulating agents such as cytosine-phosphate-guanine-oligodeoxynucleotides (CpG-ODN) needs to be developed to maximize the efficacy of immunotherapy against hypersensitivity. In this study, an aerosol formulation of biodegradable, biocompatible and nontoxic gelatin nanoparticle-bound CpG-ODN 2216 was used to treat equine recurrent airway obstruction in a clinical study.
Intrauterine growth restriction (IUGR) is a risk factor for impairment of lung function in adolescence and adulthood. Inflammatory and proliferative processes linking IUGR and perturbed extracellular matrix (ECM) as an underlying mechanism have not been addressed so far. Therefore, in this study, we aimed to investigate the developmental regulation of inflammatory and profibrotic processes in the lung subsequent to IUGR. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 28 and P70. Lungs were obtained at P1, P42, and P70 for assessment of mRNA, protein expression, immunohistochemistry, and gelatinolytic activity. Both respiratory system resistance and compliance were impaired subsequent to IUGR at P28 and this impairment was even more pronounced at P70. In line with these results, the expression of ECM components and metabolizing enzymes was deregulated. The deposition of collagen was increased at P70. In addition, the expression of inflammatory cytokines and both the activity and the expression of target genes of Stat3 signaling were dynamically regulated, with unaltered or decreased expression at P1 and significantly increased expression art P70. Taken together, these data give evidence for an age-dependent impairment of lung function as a result of a developmentally regulated increase in inflammatory and profibrotic processes subsequent to IUGR.
Early postnatal hyperalimentation has long-term implications for obesity and developing renal disease. Suppressor of cytokine signaling (SOCS) 3 inhibits phosphorylation of signal transducer and activator of transcription (STAT) 3 and ERK1/2 and thereby plays a pivotal role in mediating leptin resistance. In addition, SOCS-3 is induced by both leptin and inflammatory cytokines. However, little is known about the intrinsic-renal leptin synthesis and function. Therefore, this study aimed to elucidate the implications of early postnatal hyperalimentation on renal function and on the intrinsic-renal leptin signaling. Early postnatal hyperalimentation in Wistar rats during lactation was induced by litter size reduction at birth (LSR) either to LSR10 or LSR6, compared with home cage control male rats. Assessment of renal function at postnatal day 70 revealed decreased glomerular filtration rate and proteinuria after LSR6. In line with this impairment of renal function, renal inflammation and expression as well as deposition of extracellular matrix molecules, such as collagen I, were increased. Furthermore, renal expression of leptin and IL-6 was up-regulated subsequent to LSR6. Interestingly, the phosphorylation of Stat3 and ERK1/2 in the kidney, however, was decreased after LSR6, indicating postreceptor leptin resistance. In accordance, neuropeptide Y (NPY) gene expression was down-regulated; moreover, SOCS-3 protein expression, a mediator of postreceptor leptin resistance, was strongly elevated and colocalized with NPY. Thus, our findings not only demonstrate impaired renal function and profibrotic processes but also provide compelling evidence of a SOCS-3-mediated intrinsic renal leptin resistance and concomitant up-regulated NPY expression as an underlying mechanism.
To optimize silica-iron oxide magnetic nanoparticles with surface phosphonate groups decorated with 25-kD branched polyethylenimine (PEI) for gene delivery.
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