European Journal of Pharmaceutics and Biopharmaceutics (v.77, #3)
From alternative methods to a new toxicology
by Thomas Hartung (pp. 338-349).
Regulatory toxicology is currently undergoing a transformation from a descriptive analysis of animal experiments to a pathway-based paradigm of cellular and molecular mechanisms. The experience from the validation of alternative methods plays a key role here.Mechanistic toxicology has evolved by relying, to a large extent, on methodologies that substitute or complement traditional animal tests. The biotechnology and informatics revolutions of the last decades have made such technologies broadly available and useful, but regulatory toxicology has been slow to embrace these new approaches. Major validation efforts, however, have delivered the evidence that new approaches do not lower safety standards and can be integrated into regulatory safety assessments.Particularly in the EU, political pressures, such as the REACH legislation and the 7th Amendment to the cosmetic legislation, have prompted the need of new approaches. In the US, the NRC vision report calling for a toxicology for the 21st century (and its most recent adaptation by EPA for their toxicity testing strategy) have initiated a debate about how to create a novel approach based on human cell cultures, lower species, high-throughput testing, and modeling.Lessons learned from the development, validation, and acceptance of alternative methods support the creation of a new approach based on identified toxicity pathways. Conceptual steering and an objective assessment of current practices by evidence-based toxicology (EBT) are required. EBT is modeled on evidence-based medicine, which has demonstrated that rigorous systematic reviews of current practices and meta-analyses of studies provide powerful tools to provide health care professionals and patients with the current best scientific evidence. Similarly, a portal for high-quality reviews of toxicological approaches and tools for the quantitative meta-analyses of data promise to serve as door opener for a new regulatory toxicology.
Keywords: Alternative methods; Animal testing; Toxicology; Validation; Evidence-based medicine; Omics technologies
The influence of pulmonary surfactant on nanoparticulate drug delivery systems
by Carsten Schleh; Barbara Rothen-Rutishauser; Wolfgang G. Kreyling (pp. 350-352).
Surfactant protein D bind/adsorb to gold nanoparticles. This binding/adsorption leads to agglomeration of the gold nanoparticles.The pulmonary route is very attractive for drug delivery by inhalation. In this regard, nanoparticulate drug delivery systems, designed as multifunctional engineered nanoparticles, are very promising since they combine several opportunities like a rather uniform distribution of drug dose among all ventilated alveoli allowing for uniform cellular drug internalization. However, although the field of nanomedicine offers multiple opportunities, it still is in its infancy and the research has to proceed in order to obtain a specific targeting of the drug combined with minimum side effects. If inhaled nanoparticulate drug delivery systems are deposited on the pulmonary surfactant, they come into contact with phospholipids and surfactant proteins. It is highly likely that the interaction of nanoparticulate drug delivery systems with surfactant phospholipids and proteins will be able to mediate/modulate the further fate of this specific drug delivery system. In the present comment, we discuss the potential interactions of nanoparticulate drug delivery systems with pulmonary surfactant as well as the potential consequences of this interaction.
Keywords: Nanoparticulate drug delivery system; Engineered nanoparticles; Pulmonary surfactant; Surfactant protein; Phospholipids; Air-blood barrier
A shotgun proteomic study of the protein corona associated with cholesterol and atheronal-B surface-modified quantum dots
by Kanlaya Prapainop; Paul Wentworth Jr. (pp. 353-359).
The corona of sterol and oxysterol-coated quantum dots is rich in lipoproteins and their composite proteomes.As part of ongoing research in our group, we are keen to monitor the protein binding and movement of sterols and oxysterols in biological systems in real time. However, prior to performing these in vivo studies, we have herein studied how sterol and oxysterol surface modification of quantum dots affects their associated protein coronas. Thus, we have synthesized and analyzed cholesterol and atheronal-B surface-modified quantum dots (termed QD-chol and QD-ath-B, respectively). The fluorescence properties and aggregation propensities of QD-chol and QD-ath-B are unchanged relative to amino-functionalized quantum dots (QD-NH2) in aqueous buffers. Shotgun proteomic analyses of the protein coronas reveal that QD-ath-B and QD-chol are bound significantly higher to LDL, vLDL and HDL particles than QD-NH2. Thus, almost all the component proteins of the HDL and LDL proteomes are elevated in the protein coronas around the QD-chol and QD-ath-B nanomaterials. In addition, the reduced positive surface charge of the QD-chol and QD-ath-B materials, relative to QD-NH2, means that hydrophobic antibody light chain fragments and β-2-glycoprotein (apo H) bind them preferentially to QD-NH2.
Keywords: Keyword; Quantum dots; Protein corona; Oxysterols; Cholesterol; Nanoparticles; Proteomics
Internal benchmarking of a human blood–brain barrier cell model for screening of nanoparticle uptake and transcytosis
by Michelle Nic Ragnaill; Meredith Brown; Dong Ye; Mattia Bramini; Sean Callanan; Iseult Lynch; Kenneth A. Dawson (pp. 360-367).
Preliminary evidence of 50nm SiO2 nanoparticles uptake and transport through a human in vitro blood-brain barrier model as shown by transmission electron microscopy.Transport of drugs across the blood–brain barrier, which protects the brain from harmful agents, is considered the holy grail of targeted delivery, due to the extreme effectiveness of this barrier at preventing passage of non-essential molecules through to the brain. This has caused severe limitations for therapeutics for many brain-associated diseases, such as HIV and neurodegenerative diseases. Nanomaterials, as a result of their small size (in the order of many protein–lipid clusters routinely transported by cells) and their large surface area (which acts as a scaffold for proteins thereby rendering nanoparticles as biological entities) offer great promise for neuro-therapeutics. However, in parallel with developing neuro-therapeutic applications based on nanotechnology, it is essential to ensure their safety and long-term consequences upon reaching the brain. One approach to determining safe application of nanomaterials in biology is to obtain a deep mechanistic understanding of the interactions between nanomaterials and living systems (bionanointeractions). To this end, we report here on the establishment and internal round robin validation of a human cell model of the blood–brain barrier for use as a tool for screening nanoparticles interactions, and assessing the critical nanoscale parameters that determine transcytosis.
Keywords: Blood–brain barrier; Nanoparticles; Transcytosis; Bionanointeraction
Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited
by David O. Raemy; Ludwig K. Limbach; Barbara Rothen-Rutishauser; Robert N. Grass; Peter Gehr; Karin Birbaum; Christina Brandenberger; Detlef Günther; Wendelin J. Stark (pp. 368-375).
The uptake kinetics of airborne cerium oxide nanoparticles into air–liquid cultivated A549 cell cultures over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established aerosol exposure system.Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization.In this context, the uptake kinetics of representative airborne NPs over 30min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO2) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air–liquid interface.Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO2 could be detected intracellularly.On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO2 is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization.
Keywords: Abbreviations; NP; nanoparticle; CeO; 2; cerium oxide; ICP-MS; inductively coupled plasma mass spectrometry; LDH; lactate dehydrogenase; TEM; transmission electron microscope; OsO; 4; osmium tetroxide; XRD; X-ray powder diffraction; XDC; X-ray disc centrifugation; SSA; specific surface area; d; BET; Brunauer–Emmett–Teller (BET)-equivalent particle diameter; OD; optical density; d; day; ESI; electron spectroscopic imaging; M; massNanoparticle; Lung cell cultures; Uptake kinetics; Air–liquid interface; Cerium oxide
Interaction of metal oxide nanoparticles with lung surfactant protein A
by Christine Schulze; Ulrich F. Schaefer; Christian A. Ruge; Wendel Wohlleben; Claus-Michael Lehr (pp. 376-383).
SP-A as a component of the alveolar lining fluid interacts with inhaled nanoparticles.The alveolar lining fluid (ALF) covering the respiratory epithelium of the deep lung is the first biological barrier encountered by nanoparticles after inhalation. We here report for the first time significant differences for metal oxide nanoparticles to the binding of surfactant protein A (SP-A), the predominant protein component of ALF. SP-A is a physiologically most relevant protein and provides important biological signals. Also, it is involved in the lung’s immune defence, controlling e.g. particle binding, uptake or transcytosis by epithelial cells and macrophages. In our study, we could prove different particle–protein interaction for eight different nanoparticles, whereas particles of the same bulk material revealed different adsorption patterns. In contrast to other proteins as bovine serum albumin (BSA), SP-A does not seem to significantly deagglomerate large agglomerates of particles, indicating different adsorption mechanisms as in the well-investigated model protein BSA. These findings may have important consequences for biological fate and toxicological effects of inhaled nanomaterials.
Keywords: Abbreviations; ALF; alveolar lining fluid; SP-A; surfactant protein A; BSA; bovine serum albumin; pBALF; porcine broncheoalveolar lavage fluid; BCA assay; bicinchinoninic acid protein quantification assay; AUC; analytical ultracentrifugation; CRD; carbohydrate recognition domains; SDS–PAGE; sodiumdodecylsulfate–polyacryl-gelelectrophoresis; SPs; surfactant proteins; FCS; fetal calf serum; CNTs; carbonanotubesSurfactant protein A; Nanoparticles; Particle–protein interaction; Protein adsorption; Deagglomeration; BCA assay
Surfactant protein A (SP-A)-tacrolimus complexes have a greater anti-inflammatory effect than either SP-A or tacrolimus alone on human macrophage-like U937 cells
by Almudena López-Sánchez; Alejandra Sáenz; Cristina Casals (pp. 384-391).
SP-A binds to FK506 with high affinity and strengthens tacrolimus potency as an anti-inflammatory agent on macrophages by facilitating FK506 entrance into the cell, overcoming P-glycoprotein.Intratracheal administration of immunosuppressive agents to the lung is a novel treatment after lung transplantation. Nanoparticles of tacrolimus (FK506) might interact with human SP-A, which is the most abundant lipoprotein in the alveolar fluid. This study was undertaken to determine whether the formation of FK506/SP-A complexes interferes with FK506 immunosuppressive actions on stimulated human macrophage-like U937 cells. We found that SP-A was avidly bound to FK506 ( Kd=35±4nM), as determined by solid phase–binding assays and dynamic light scattering. Free FK506, at concentrations ⩽1μM, had no effect on the inflammatory response of LPS-stimulated U937 macrophages. However, coincubation of FK506 and SP-A, at concentrations where each component alone did not affect LPS-stimulated macrophage response, significantly inhibited LPS-induced NF- κB activation and TNF-alpha secretion. Free FK506, but not FK506/SP-A, functioned as substrate for the efflux transporter P-glycoprotein. FK506 bound to SP-A was delivered to macrophages by endocytosis, since several endocytosis inhibitors blocked FK506/SP-A anti-inflammatory effects. This process depended partly on SP-A binding to its receptor, SP-R210. These results indicate that FK506/SP-A complexes have a greater anti-inflammatory effect than either FK506 or SP-A alone and suggest that SP-A strengthened FK506 anti-inflammatory activity by facilitating FK506 entrance into the cell, overcoming P-glycoprotein.
Keywords: Lung; Tacrolimus; Surfactant protein A; Inflammation; Macrophages; P-glycoproteinAbbreviations; FK506; tacrolimus; FKBP; FK506-binding protein; HSA; human serum albumin; LPS; bacterial lipopolysaccharide; NF-κB; nuclear factor-kappa B; MRP1; multidrug resistance associated protein; Pgp; P-glycoprotein; PMA; phorbol-12-myristate-13-acetate; SP-A; surfactant protein A; SP-R210; surfactant protein A receptor 210; TNF-α; tumor necrosis factor-α
Nanoparticles attenuate P-glycoprotein/MDR1 function in A549 human alveolar epithelial cells
by Johanna J. Salomon; Carsten Ehrhardt (pp. 392-397).
The effect of nanoparticles on the release of the P-gp substrate, rhodamine 123 from A549 human alveolar epithelial cell layers was studied. Cell layers were loaded with rhodamine 123 for 30min ( t0), and fluorescence was measured after further 30min of release ( t30). Our findings suggest that nanomaterials can attenuate membrane transporter function depending on size and surface properties and hence might influence the disposition of xenobiotics as well as endogenous substrates.P-glycoprotein/MDR1 (P-gp) is a well-characterised membrane transporter relevant in drug disposition and multi-drug resistance. In this study, we aimed to investigate how far nanoparticulates impair the function of the P-gp transport system and which particle properties govern these interactions.Expression and function of P-gp was confirmed in A549 cell monolayers. Rhodamine 123 (Rh123) release studies were carried out in the presence of known inhibitors of P-gp function (i.e., cyclosporine A and verapamil), under ATP depletion (NaN3/DOG) and after acute exposure to nanoparticles (NPs) with different surface modifications, ζ-potentials and sizes (plain, carboxylated, and amine- and sulphate-modified). The cytotoxic potential of NPs on A549 monolayers was evaluated by MTT assay. The effects on P-gp protein level, after incubation with NPs, were investigated by Western blot analysis of A549 cell lysate and supernatant.Cellular retention of Rh123 was significantly ( P<0.05) increased in the presence of carboxylated (100nm), amine- and sulphate-modified NPs. A slight, but not significant, decrease in Rh123 release was also observed for plain latex and carboxylated (500nm) NPs. The MTT assay demonstrated that most NPs caused only marginal levels of cytotoxicity (78–88% cell viability); the positively charged amine-NPs, however, were considerably more cytotoxic. Western blot showed that NPs did not cause any cell membrane disruption.Our findings suggest that nanomaterials can attenuate membrane transporter function depending on their size and surface properties and hence might influence the disposition of xenobiotics as well as endogenous substrates.
Keywords: A549 cells; Rhodamine 123; Drug transporter; Release study
An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier
by Andrea D. Lehmann; Nicole Daum; Michael Bur; Claus-Michael Lehr; Peter Gehr; Barbara M. Rothen-Rutishauser (pp. 398-406).
Laser scanning microscopy image of primary human alveolar epithelial cells. All the cells were stained for F-actin (green) and additionally for CD14, a surface marker for monocyte-derived macrophages (turquoise).A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o– epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o– containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o– were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods.It could be shown that the epithelial integrity of hAEpC (mean±SD, 1180±188Ωcm2) was higher than in A549 (172±59Ωcm2) but similar to 16HBE14o– cells (1469±156Ωcm2). The triple cell co-culture model with hAEpC (1113±30Ωcm2) showed the highest integrity compared to the ones with A549 (93±14Ωcm2) and 16HBE14o– (558±267Ωcm2). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o– were more regularly expressed but not in A549.The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.
Keywords: Abbreviations; AJ; Adherens junctions; EC; Epithelial cells; hAEpC; Human alveolar epithelial cells in primary culture; LSM; Confocal laser scanning microscopy; MDM; Monocyte-derived macrophages; MDDC; Monocyte-derived dendritic cells; NP; Nanoparticle(s); TEM; Transmission electron microscopy; TEER; Transepithelial electrical resistance; TJ; Tight junctions; ZO-1; Zonula occludens-1Lung cell culture models; Alveolar epithelial models; Primary alveolar type I cells; Macrophages; Dendritic cells
Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration
by Stephanie Hirn; Manuela Semmler-Behnke; Carsten Schleh; Alexander Wenk; Jens Lipka; Martin Schäffler; Shinji Takenaka; Winfried Möller; Günter Schmid; Ulrich Simon; Wolfgang G. Kreyling (pp. 407-416).
Twenty-four hours after intravenous injection, the biodistribution of five different sizes (1.4, 5, 18, 80, and 200nm) and 2.8nm gold nanoparticles (GNP) with opposite surface charges was quantitatively measured by gamma-spectrometry. The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4nm GNP to >99% of 200nm GNP. Hepato-biliary clearance of the GNP showed an inverse linear relationship to the GNP diameter over the entire size range of 2.8–200nm.Gold nanoparticles (GNP) provide many opportunities in imaging, diagnostics, and therapies of nanomedicine. Hence, their biokinetics in the body are prerequisites for specific tailoring of nanomedicinal applications and for a comprehensive risk assessment.We administered198Au-radio-labelled monodisperse, negatively chargedGNP of five different sizes (1.4, 5, 18, 80, and 200nm) and 2.8nm GNP with opposite surface charges by intravenous injection into rats. After 24h, the biodistribution of the GNP was quantitatively measuredby gamma-spectrometry.The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4nm GNP to >99% of 200nm GNP. In contrast, there was little size-dependent accumulation of 18–200nm GNP in most other organs. However, for GNP between 1.4nm and 5nm, the accumulation increased sharply with decreasing size; i.e. a linear increase with the volumetric specific surface area. The differently charged 2.8nm GNP led to significantly different accumulations in several organs.We conclude that the alterations of accumulation in the various organs and tissues, depending on GNP size and surface charge, are mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and dose estimates used in risk assessment.
Keywords: Gold nanoparticles; Intravenous injection; Female Wistar-Kyoto rat; In vivo biodistribution; Nanoparticle size and surface charge; Hepato-biliary and renal clearance
Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages
by Arnida; M.M. Janát-Amsbury; A. Ray; C.M. Peterson; H. Ghandehari (pp. 417-423).
Compared to PEGylated gold nanospheres with slightly negative charge, near neutral PEGylated gold nanorods had increased tumor accumulation, longer circulation time in blood, less uptake by the liver and reduced interaction with serum proteins.Spherical and rod-shaped gold nanoparticles with surface poly(ethylene glycol) (PEG) chains were characterized for size, shape, charge, poly dispersity and surface plasmon resonance. The nanoparticles were injected intravenously to 6–8-week-old female nu/nu mice bearing orthotopic ovarian tumors, and their biodistribution in vital organs was compared. Gold nanorods were taken up to a lesser extent by the liver, had longer circulation time in the blood, and higher accumulation in the tumors, compared with their spherical counterparts. The cellular uptake of PEGylated gold nanoparticles by a murine macrophage-like cell line as a function of geometry was examined. Compared to nanospheres, PEGylated gold nanorods were taken up to a lesser extent by macrophages. These studies point to the importance of gold nanoparticle geometry and surface properties on transport across biological barriers.
Keywords: Gold nanoparticles; Nanorods; Nanomedicine; Biodistribution; Ovarian tumor; Macrophages
Ultrasound active nanoscaled lipid formulations for thrombus lysis
by Andreas Becker; Elena Marxer; Jana Brüßler; Anne Sophia Hoormann; Daniela Kuhnt; Udo Bakowsky; Christopher Nimsky (pp. 424-429).
Ultrasound active nanocaled lipid formulation on a red blood cell.In the present study, we investigated the sonothrombolytic effect of new nanoscaled lipid formulations in human blood clots, using diagnostic ultrasound. Human blood clots of 1ml were incubated with 1μl of the different lipid dispersions DPPC/CH, DPPC/PEG40S, DSPC/PEG40S and the commercially available ultrasound contrast agent SonoVue®. Clots were stored for 3days at 5°C to obtain maximal clot retraction and lytic resistance. Each clot weight was determined before and after continuous insonation for 1h of insonation at 1.4MHz. The pressure in the insonation chamber was 80mm Hg to mimic middle arterial blood pressure. There were no significant differences in thrombus weight before insonation. All nanoscaled formulations and SonoVue® were able to reduce thrombus weight compared to the weight loss of clots that were not insonated but kept under pressure for one hour ( p<0.001). We found a highly significant weight reduction with DSPC/PEG40S compared to SonoVue® ( p=0.007).Nanoscaled DSPC/PEG40S dispersion could be a promising formulation in ultrasound enhanced thrombolysis even without thrombolytic drugs. Stable cavitation is a crucial parameter in fragmentation of thrombus architecture. Further studies of physicochemical properties of DSPC/PEG40S are necessary to corroborate our hypothesis.
Keywords: Ultrasonography; Nanomedicine; Thrombolysis; Contrast agent; Microbubbles
Development and characterization of new nanoscaled ultrasound active lipid dispersions as contrast agents
by Elena Eva Julianne Marxer; Jana Brüßler; Andreas Becker; Joachim Schümmelfeder; Rolf Schubert; Christopher Nimsky; Udo Bakowsky (pp. 430-437).
High resolution AFM (A) and cryo-TEM (B) images of ultrasound active discoid micelles.Ultrasound contrast agents are widely used in clinical diagnosis. In recent years, the use of ultrasound contrast agents as therapeutic agents has gained a lot of attention. Of special interest are ultrasound-enhanced gene delivery in various tissues (e.g. cardiac, vascular, skeletal muscle and tumor tissue), ultrasound-enhanced protein delivery (e.g. insulin delivery) and ultrasound-enhanced delivery of small chemicals (e.g. doxorubicin, vancomycin). Commercially available ultrasound contrast agents such as SonoVue® or Optison® are ranged in a size of 2–8μm. These micronscaled agents show a good ultrasound contrast enhancement and thus they are used for diagnostic imaging. But they are not suitable for targeted drug delivery to tumor tissues or blood clots because for these applications particles smaller than 700nm are needed. In the present study, we developed new nanoscaled ultrasound contrast agents with a size between 70 and 300nm. The lipid formulations show excellent contrast intensities using diagnostic ultrasound of about 1.4MHz. The negatively charged colloidal dispersions are long-time stable under physiological conditions without loss of ultrasound reflectivity. The adjustable supramolecular organization of the carriers depends on the composition and varies from micellar to liposomal structures. The small size and the circulation stability of these systems make them promising for novel diagnostics and controlled drug release applications.
Keywords: Ultrasound; Liposomes; Lipid formulations; Diagnostics; Controlled drug release; Nanoscaled ultrasound contrast agents
Polyethylenimines for RNAi-mediated gene targeting in vivo and siRNA delivery to the lung
by Melanie Günther; Jens Lipka; Anastasia Malek; Daniela Gutsch; Wolfgang Kreyling; Achim Aigner (pp. 438-449).
Based on electrostatic interactions, linear or branched polyethylenimines (PEI) form complexes with negatively charged short interfering RNA (siRNA) molecules. The administration of low molecular weight PEI/siRNA nanoplexes through instillation is an efficient strategy for pulmonary siRNA delivery with very little systemic availability. Biodistribution studies with 32P-labeled siRNAs also demonstrate that the protection, delivery and cellular internalization of intact siRNA molecules relies on their complexation with PEI. Several pre-clinical in vivo studies on the PEI-mediated delivery of therapeutic siRNAs have already been performed in various animal models and are summarized in this paper.RNA interference (RNAi) is a promising strategy to inhibit the expression of pathologically relevant genes, which show aberrant (over-)expression, e.g. in tumors or other pathologies. The induction of RNAi relies on small interfering RNAs (siRNAs), which trigger the specific mRNA degradation. Their instability and poor delivery into target tissues including the lung, however, so far severely limits the therapeutic use of siRNAs and requires the development of nanoscale delivery systems. Polyethylenimines (PEIs) are synthetic polymers, which are able to form non-covalent complexes with siRNAs. These nanoscale complexes (’nanoplexes’) allow the protection of siRNAs from nucleolytic degradation, their efficient cellular uptake through endocytosis and intracellular release through the ’proton sponge effect’. Chemical modifications of PEIs as well as the coupling of cell/tissue-specific ligands are promising approaches to increase the biocompatibility, specificity and efficacy of PEI-based nanoparticles.This review article gives a comprehensive overview of pre-clinical in vivo studies on the PEI-mediated delivery of therapeutic siRNAs in various animal models. It discusses the chemical properties of PEIs and PEI modifications, and their influences on siRNA knockdown efficacy, on adverse effects of the polymer or the nanoplex and on siRNA biodistribution in vivo. Beyond systemic application, PEI-based complexation allows the local siRNA application to the lung. Biodistribution studies demonstrate cellular uptake of PEI-complexed, but not of naked siRNAs in the lung with little systemic availability of the siRNAs, indicating the usefulness of this approach for the targeting of genes, which are pathologically relevant in lung tumors or lung metastases.Taken together, (i) PEI and PEI derivatives may represent an efficient delivery platform for siRNAs, (ii) siRNA-mediated induction of RNAi is a promising approach for the knockdown of pathologically relevant genes, and (iii) when sufficiently addressing biocompatibility issues, the locoregional delivery of PEI/siRNA complexes may become an attractive therapeutic strategy for the treatment of lung diseases with little systemic side effects.
Keywords: Abbreviations; PEI; Polyethylenimine; siRNA; Small interfering RNA; RNAi; RNA interferencePoly(ethylene imine); PEI; Gene targeting; RNAi; siRNA; Lung
Gene silencing activity of siRNA polyplexes based on biodegradable polymers
by Amir K. Varkouhi; Twan Lammers; Raymond M. Schiffelers; Mies J. van Steenbergen; Wim E. Hennink; Gert Storm (pp. 450-457).
Endocytosis of the siRNA polyplexes and PCI mediated endosomal escape.Cationic polymers are used as non-viral vectors for nucleic acid delivery. In this study, two biodegradable cationic polymers were evaluated for the purpose of siRNA delivery: pHPMA-MPPM (poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol)) and TMC (O-methyl-free N,N,N-trimethylated chitosan). The silencing activity and the cellular cytotoxicity of polyplexes based on these biodegradable polymers were compared with those based on non-biodegradable pDMAEMA (poly(2-dimethylamino)ethyl methacrylate) and PEI (polyethylenimine) and with the regularly used lipidic transfection agent Lipofectamine. To promote endosomal escape, either the endosomolytic peptide diINF-7 was added to the formulations or photochemical internalization (PCI) was applied. Incubation of H1299 human lung cancer cells expressing firefly luciferase with polyplexes based on pHPMA-MPPM and TMC showed 30–40% silencing efficiency. This silencing activity was equal to or better than that obtained with the standard transfectants. Under all experimental conditions tested, the cytotoxicity of the biodegradable polymers was low. The application of PCI, as well as the addition of the diINF-7 peptide to the formulations increased their silencing activity up to 70–80%. This demonstrates that pHPMA-MPPM- and TMC-based polyplexes benefit substantially from endosomal escape enhancement. Importantly, the polyplexes retained their silencing activity in the presence of serum, and they showed low cytotoxicity. These biodegradable vectors are therefore attractive systems for further in vivo evaluations.
Keywords: siRNA; Polymeric vectors; Photochemical internalization; Fusogenic peptides; Gene silencing; Cytotoxicity
A novel approach to identify non-palpable breast lesions combining fluorescent liposomes and magnetic resonance-guided high intensity focused ultrasound-triggered release
by Chris Oerlemans; Frank Nijsen; Miranda van Amersfoort; Louis van Bloois; Edwin Heijman; Peter Luijten; Willem Mali; Gert Storm (pp. 458-464).
Treatment planning of an agarose phantom containing fluorescein-loaded liposomes positioned on a MR image during MR-HIFU treatment. The region of interest (ROI) was heated to 42°C for 30s with MR-HIFU.The combination of fluorescein-containing liposomes (FCL) and magnetic resonance-guided high intensity focused ultrasound (MR-HIFU)-triggered release is a promising approach for lesion demarcation and more efficient removal of non-palpable breast lesions. Exposure of FCL to ablation temperatures (60°C) using MR-HIFU would result in palpable, stained tumors, which are more easy to identify during surgical resection. In this study, proof-of-concept concerning fluorescent FCL for MR-HIFU-triggered release and tumor demarcation of non-palpable breast lesions is presented. Ex vivo experiments in human blood and porcine muscle tissue showed increased label release from the liposomes, clear fluorescence enhancement and diffusion of the released compound after heating to 60°C. Next, fluorescein release of FCL was observed after MR-HIFU-mediated mild hyperthermia (42°C) and ablation temperature (60°C) for a short period (30s), which is in line with the clinically relevant MR-HIFU treatment parameters. These results indicate the potential of the FCL as a tool to improve tumor demarcation in patients by MR-HIFU-triggered release. Therefore, this method may offer a new tool for efficient surgical resection of non-palpable breast tumor lesions by enabling proper discrimination between tumor tissue and adjacent healthy tissue.
Keywords: Liposomes; Focused ultrasound; Tumor ablation; Triggered release; Non-palpable breast lesions; Tumor demarcation
Penetration and storage of particles in human skin: Perspectives and safety aspects
by J. Lademann; H. Richter; S. Schanzer; F. Knorr; M. Meinke; W. Sterry; A. Patzelt (pp. 465-468).
Follicular targeting. Four different target structures of interest within the hair follicle.The application of particles in dermatology and cosmetology represents an emerging field and is closely connected with the question of risk assessment as the potential for, and consequences of, penetration of such particles into the living tissue has not been determined conclusively. In the medical sector, extensive research activities are in progress to develop particles, which can be used as efficient carriers for drug delivery through the skin barrier. In contrast, in cosmetic products, particles are mostly required to remain on the skin surface to fulfill their beneficial effect. Whereas the intercellular penetration of particles seems to be unlikely, the hair follicle has been shown to be a relevant penetration pathway for particles as well as an important long-term reservoir. It has been demonstrated that the penetration depth of the particles can be influenced by their size resulting in the possibility of a differentiated targeting of specific follicular structures. In the present review, the follicular penetration mechanisms and storage properties of particles are discussed.
Keywords: Hair follicles; Drug delivery; Particle; Skin barrier; Follicular targeting
Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy
by Michael S. Roberts; Yuri Dancik; Tarl W. Prow; Camilla A. Thorling; Lynlee L. Lin; Jeffrey E. Grice; Thomas A. Robertson; Karsten König; Wolfgang Becker (pp. 469-488).
An example of multichannel FLIM data from endogenous fluorophores at the stratum granulosum level in human epidermis, recorded in three wavelength channels (350–450 nm, 450–515 nm, 515–620 nm) with excitation at 740 nm. The lower panel shows decay curves from a selected pixel fitted with a bi-exponential model. The three lifetime images in the upper panel show, from left to right, mainly NAD(P)H, both NAD(P)H and FAD and FAD only. The weak FAD signal and lack of cellular definition seen in the right FAD image indicates that 740 nm is not the optimal excitation wavelength for FAD.New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.
Keywords: Dermatology; Fluorescence lifetime; Penetration; Metabolism
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