Delivery Of Large Cargo Research Articles

Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

Metallic micro-ring device for highly efficient large cargo delivery in mammalian cells using infrared light pulses.

Uniform transfection of biomolecules into live cells with high delivery efficiency and cell viability is an immensely important area of biological research and has many biomedical applications. In the present study, we report highly efficient, uniform parallel intracellular delivery of small to very large biomolecules into diverse cell types using a titanium micro-ring (TMR) device activated by infrared (IR) light pulse. A TMR array device (2 cm × 2 cm) consists of a 10 μm outer diameter and 3 μm inner diameter for each micro-ring, and 10 μm interspacing between two micro-rings. Upon IR (1050 nm) pulse laser irradiation on the TMR device, photothermal cavitation bubbles are generated, disrupting the cell plasma membrane, and biomolecules are gently delivered into the cells by a simple diffusion process. This TMR device successfully delivered diverse types of small to very large biomolecules such as propidium iodide (PI; 668.4 Da) dye, dextran (3 kDa), small interfering RNA (13.3 kDa), enhanced green fluorescent protein expression plasmid DNA (6.2 kb), and β-galactosidase enzyme (465 kDa) into human cervical (SiHa), mouse fibroblast (L929), and mouse neural crest-derived (N2a) cancer cells. For smaller molecules (PI dye), delivery efficiency and cell viability were achieved at ∼96% and ∼97%, respectively, with a laser fluence of 21 mJ cm-2 for 250 pulses. In contrast, ∼85% transfection efficiency and ∼90% cell viability were achieved for plasmid DNA with 45 mJ cm-2 laser fluence for 250 pulses in SiHa cells. Moreover, the intracellular delivery of β-galactosidase enzyme was confirmed with confocal microscopy and flow cytometry analysis resulting in ∼83% co-staining of β-galactosidase enzyme and calcein AM. Based on these efficient deliveries of diverse types of biomolecules in different cell types, the device has the potential for cellular diagnostic and therapeutic applications.

High-Throughput and Dosage-Controlled Intracellular Delivery of Large Cargos by an Acoustic-Electric Micro-Vortices Platform.

A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min−1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.

Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells.

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus "piggy-backed" on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a "driver" to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.

A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells.

Development of safe and efficient nanoscale vehicles that can deliver large molecular cargos into human cells could transform future human therapies and personalized medicine. Here, we design a hybrid viral vector composed of a prokaryotic virus (bacteriophage T4) and a eukaryotic virus [adeno-associated virus (AAV)]. The small 25-nm AAV is attached to the large 120 nm × 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc and Hoc. AAV "piggy-backed" on T4 capsid, by virtue of its natural ability to enter human cells acted as an efficient "driver," delivering the largest payloads of foreign DNA (up to 170 kb) and protein (up to 1025 molecules) reported to date, and elicited robust immune responses in mice against flu and plague pathogens and conferred complete protection against lethal pneumonic plague challenge. The T4-AAV represents a unique platform for assembly of natural building blocks into potential therapeutics against genetic and infectious diseases.

Mesoporous Silica and Organosilica Nanoparticles: Physical Chemistry, Biosafety, Delivery Strategies, and Biomedical Applications.

Predetermining the physico-chemical properties, biosafety, and stimuli-responsiveness of nanomaterials in biological environments is essential for safe and effective biomedical applications. At the forefront of biomedical research, mesoporous silica nanoparticles and mesoporous organosilica nanoparticles are increasingly investigated to predict their biological outcome by materials design. In this review, it is first chronicled that how the nanomaterial design of pure silica, partially hybridized organosilica, and fully hybridized organosilica (periodic mesoporous organosilicas) governs not only the physico-chemical properties but also the biosafety of the nanoparticles. The impact of the hybridization on the biocompatibility, protein corona, biodistribution, biodegradability, and clearance of the silica-based particles is described. Then, the influence of the surface engineering, the framework hybridization, as well as the morphology of the particles, on the ability to load and controllably deliver drugs under internal biological stimuli (e.g., pH, redox, enzymes) and external noninvasive stimuli (e.g., light, magnetic, ultrasound) are presented. To conclude, trends in the biomedical applications of silica and organosilica nanovectors are delineated, such as unconventional bioimaging techniques, large cargo delivery, combination therapy, gaseous molecule delivery, antimicrobial protection, and Alzheimer's disease therapy.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery

The delivery of large cargos of diameter above 15nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~534kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

Mitochondrial Transfer by Photothermal Nanoblade Restores Respiration in Mammalian Cells with Dysfunctional Mitochondria

The reliable delivery of large cargo into mammalian cells could have profound impacts on cell engineering and medicine. Current techniques, such as electroporation, sonoporation, and microcapillary injection, can deliver DNAs, RNAs, and proteins into cells, but they are limited by cargo size, endosomal cargo degradation, and poor efficiency. To circumvent these problems, we invented a photothermal nanoblade. The nanoblade is a titanium‐coated glass pipet that is placed near a cell membrane and heated by a laser pulse. A resulting vapor bubble generates a transient membrane incision that allows the pressure driven delivery of cargo up to 3 μm in size into cells with high efficiency and cell viability. Here, we evaluated whether the nanoblade could transfer isolated mitochondria into cells to rescue mitochondrial dysfunction. The transfer of MDA‐MB‐453 mitochondria into 143BTK‐ρ0 cells, which lack mtDNA, do not respire, and require uridine supplementation to proliferate, resulted in the emergence of three rescued clones in uridine‐free medium 2 weeks after delivery. Interestingly, clones 1 and 3 had ATP levels, oxygen consumption rates, and ETC complex activities similar to the donor and parent cells, while clone 2 continued to have abnormal levels. qRT‐PCR of genes encoding anaplerotic and catapleurotic enzymes showed that rescue lines 1 and 3 were similar to the 143 BTK‐ parent, whereas clone 2 remained similar to the 143BTK‐ ρ0 cells. This distinction between the three clones was maintained in the metabolic profiling of TCA cycle and cytosolic metabolites by mass spectrometry. In conclusion, the ability of transferring functional mitochondria to cells by the photothermal nanoblade is not only important for basic research studies, but also has potential for therapies targeted at correcting mitochondrial diseases.Support or Funding InformationUCLA Tumor Immunology Training Grant (Ruth L. #Kirschstein Institutional National Research Service Award # T32 CA 009120)

Mitochondrial Transfer by Photothermal Nanoblade Restores Respiration in Mammalian Cells with Dysfunctional Mitochondria

The reliable delivery of large cargo into mammalian cells could have profound impacts on cell engineering and medicine. Current techniques, such as electroporation, sonoporation, and microcapillary injection, can deliver DNAs, RNAs, and proteins into cells, but they are limited by cargo size, endosomal cargo degradation, and poor efficiency. To circumvent these problems, we invented a photothermal nanoblade. The nanoblade is a titanium-coated glass pipet that is placed near a cell membrane and heated by a laser pulse. A resulting vapor bubble generates a transient membrane incision that allows the pressure driven delivery of cargo up to 3 µm in size into cells with high efficiency and cell viability. Here, we evaluated whether the nanoblade could transfer isolated mitochondria into cells to rescue mitochondrial dysfunction. The transfer of MDA-MB-453 mitochondria into 143BTK-ρ0 cells, which lack mtDNA, do not respire, and require uridine supplementation to proliferate, resulted in the emergence of three rescued clones in uridine-free medium 2 weeks after delivery. Interestingly, clones 1 and 3 had ATP levels, oxygen consumption rates, and ETC complex activities similar to the donor and parent cells, while clone 2 continued to have abnormal levels. qRT-PCR of genes encoding anaplerotic and catapleurotic enzymes showed that rescue lines 1 and 3 were similar to the 143 BTK- parent, whereas clone 2 remained similar to the 143BTK- ρ0 cells. This distinction between the three clones was maintained in the metabolic profiling of TCA cycle and cytosolic metabolites by mass spectrometry. In conclusion, the ability of transferring functional mitochondria to cells by the photothermal nanoblade is not only important for basic research studies, but also has potential for therapies targeted at correcting mitochondrial diseases.

Massively parallel delivery of large cargo into mammalian cells with light pulses.

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

3T3 세포에 의한 피브로인 마이크로스피어의 흡수

Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.

Mapping Photothermally Induced Gene Expression in Living Cells and Tissues by Nanorod-Locked Nucleic Acid Complexes

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40,000, and vesicles with a diameter of 1-2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.

In vitro cellular uptake of fibroin microspheres and its dependency on the cell cycle stage

Cellular uptake of a protein microsphere was investigated in vitro. Fibroin microspheres with an average diameter of approximately 1 µm were prepared with Fluorescein isothiocyanate–dextran for the fluorescent core. Tomography, performed using confocal laser scanning microscopy, confirmed microsphere uptake into the cytoplasmic area of 3T3 cells. Flow cytometry showed that cellular uptake was proportional to the co-incubation time and the microsphere concentration. It also revealed that fibroin microspheres were internalised by 3T3 cells at different rates depending on the cell cycle stage. The following cell stages had higher concentrations of internalised microspheres: G2/M > S > G0/G1, when using 0.1 mg of microspheres per 106 cells. The internalised microspheres per cell also increased in the same order of cell cycle stages when co-incubating cells with 1 mg of microspheres. These results provide information that can be used to develop fibroin microspheres for intracellular delivery of large cargos.

1311 DEVELOPMENT OF A NOVEL NANOENCAPSULATED CONTRAST AGENT DELIVERED INTRACELLULARLY TO ENHANCE MR IMAGING OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 20121311 DEVELOPMENT OF A NOVEL NANOENCAPSULATED CONTRAST AGENT DELIVERED INTRACELLULARLY TO ENHANCE MR IMAGING OF PROSTATE CANCER Joel Slaton, Bruce Hammer, Gregory Metzger, and Gretchen Unger Joel SlatonJoel Slaton Minneapolis, MN More articles by this author , Bruce HammerBruce Hammer Minneapolis, MN More articles by this author , Gregory MetzgerGregory Metzger Minneapolis, MN More articles by this author , and Gretchen UngerGretchen Unger Chaska, MN More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1658AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Systemic MR contrast agents, whether traversing primarily in the extracellular compartments or attaching onto the cell surface, can enhance imaging of tumor anatomy. However, these agents are nonspecific and cannot always differentiate between neoplastic and normal tissue. We have developed a novel contrast agent encapsulating dysprosium dextran (DyDex) into sub-50 nanometer capsule, a delivery technology capable of intracellular delivery of large cargos in a tissue- and cell-specific manner. We present our preliminary work demonstrating the ability of this agent to provide an intracellular positive signal on T1-weighted MR imaging of prostate cancer. METHODS DyDex (which provides a positive MR signal on T1 imaging) was incorporated into nanocapsules coated with tenfibgen which selectively targets integrins. It was discovered Dy retained red fluorescence within the hydrophobic inner environment of the s50 capsule. We performed in vitro and in vivo toxicity studies. We conducted pilot imaging studies in male mice bearing the lymph node-metastatic tumor model, PC3LN4 with DyDex encapsulated in s50 tenfibgen capsules. Mice were imaged using a 5 T scanner with a spin echo sequence. RESULTS PC3LN4 cells were treated with PBS suspensions of 1) naked DyDex 2) naked Gd-DTPA or 3) s50 Tbg capsules bearing DyDex and assessed for cell growth by thymidine incorporation. s50 encapsulated DyDextran showed no negative impact on growth vs. naked Dy3+ ion. For in vivo assessment, male mice received single i.v. injections of DyDex and controls. The results indicate that the s50 capsules at a 10x dose (5 umol/kg) did not significantly perturb the blood chemistry or hematology values (p < 0.05). Imaging demonstrated that good tumor contrast was still apparent at 10 nmol/kg equivalent to a dose of 50 ng of DyDex in prostatic PC3-LN4 flank tumor. This dosage level is 10,000-fold less than standard gadolinium dose of 0.1 mmol/kg which has been associated with significant kidney toxicity in some patients. Two mm lymph node masses were easily seem at 5T MR. Bright nuclear red signal that colocalized in confocal images with cytoplasmic capsule signal was observed in 10 nmol/kg-treated primary tumor and 100 nmol/kg-treated primary tumor and lymph node metastases. CONCLUSIONS We have demonstrated intracellular nontoxic uptake of a novel nanoencapsulated DyDex agent with corresponding positive T1-weighted signal on MRI in human prostate cancer xenografts. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e531 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joel Slaton Minneapolis, MN More articles by this author Bruce Hammer Minneapolis, MN More articles by this author Gregory Metzger Minneapolis, MN More articles by this author Gretchen Unger Chaska, MN More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Photothermal nanoblade for large cargo delivery into mammalian cells.

It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integration of the metallic nanostructure with a micropipet, the nanoblade generates a micrometer-sized membrane access port for delivering highly concentrated cargo (5 × 10(8) live bacteria/mL) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads, to 2 μm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult cargo into mammalian cells.

Viral membrane proteins

This special issue on ‘Viral membrane proteins’ incorporates a series of research articles that were presented at the ‘Conference on Viral Membrane Proteins’, held at the University of Heidelberg, Germany, on 12–14 December 2008. The aim of the symposium was to involve all areas of research in this emerging Weld focusing on biophysical and clinically relevant approaches as well as on the fundamental science. The interest in viral membrane proteins has diVerent foundations. The most obvious is that viral membrane proteins can serve as targets for a new generation of antiviral drugs. But in addition to this, viral membrane proteins reveal many unique structural and functional features that can provide a blueprint for developing molecular tools for bioand nanotechnology as well as biomedicine. Whilst the membrane proteins of the host comprise up to 50% of all drug targets, this frequency is not yet the case for viral membrane proteins. Today most antiviral drugs target globular proteins with only a few targeting membrane proteins. To highlight some of these membrane proteins: they include entry inhibitors for HIV-1 and inXuenza as well as channel blockers for the latter virus. A better understanding of the role and also of the structure of viral membrane proteins can be anticipated to be directly relevant for the assessment of these proteins as potential drug targets in the very near future. At the current state of knowledge, viral membrane proteins in particular are very likely to become very important drug targets, as an increasing number of companies are dealing with this type of protein. Apart from their role as drug targets, viral membrane proteins also have many other interesting features. They may, for example, serve as templates for unique techniques for proteinaceous systems which enable the delivery of large cargo across the lipid membrane. The “technology” used by the viruses includes fusion and transduction. Some viruses have coupled membrane protein-based sensor systems including proteins that ultimately perform the fusion event. One such coupled protein system is the neuraminidase (sensor)/hemaglutinin (fusion) system of inXuenza. Other viruses such as HIV-1 have non-covalently linked their fusion machinery, gp41, with a sensor protein, gp120, with the latter placed like a backpack on top of the former. A completely diVerent strategy relies on formation of channels and pores that facilitate the Xux of the viral genome into the cell (polio virus, tobacco mosaic virus). In the case of transduction, certain sequences of viral proteins are highly enriched with positively charged amino acids known to facilitate transduction. Used as a short sequence, these positively charged peptides may work as artiWcial vehicles for cargo delivery across the lipid membrane. Whatever one’s interest in viral membrane proteins is, whether it is as a drug target or a shuttle system, the Wrst, most essential step is to achieve a profound understanding of the structural and functional aspects of these proteins. To achieve the necessary level of understanding, a broad range of techniques has to be employed in the investigation of the proteins. Since all techniques cover speciWc time and size W. B. Fischer (&) Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang-Ming University, 155, Sec. 2, Li-Nong St., Taipei 112, Taiwan e-mail: wWscher@ym.edu.tw