http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_1 This is an overview of drug delivery systems (DDS), starting with various routes of drug administration. Various drug formulations, as well as devices used for drug delivery and targeted drug delivery, are then described. Delivery of proteins and peptides presents special challenges. Nanoparticles are considered to be important in refining drug delivery; they can be pharmaceuticals as well as diagnostics. Refinements in drug delivery will facilitate the development of personalized medicine, which includes pharmacogenomics, pharmacogenetics, and pharmacoproteomics. The ideal DDS, commercial aspects, current achievements, challenges, and future prospects are also discussed. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_10 Solid tumors have an acidic extracellular environment and an altered pH gradient across their cell compartments. Nanoparticles responsive to the pH gradients are promising for cancer drug delivery. Such pH-responsive nanoparticles consist of a corona and a core, one or both of which respond to the external pH to change their soluble/insoluble or charge states. Nanoparticles whose coronas become positively charged or become soluble to make their targeting groups available for binding at the tumor extracellular pH have been developed for promoting cellular targeting and internalization. Nanoparticles whose cores become soluble or change their structures to release the carried drugs at the tumor extracellular pH or lysosomal pH have been developed for fast drug release into the extracellular fluid or cytosol. Such pH-responsive nanoparticles have therapeutic advantages over the conventional pH-insensitive counterparts. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_11 Oral drug delivery is the largest and the oldest segment of the total drug delivery market. It is the fastest growing and most preferred route for drug administration. Use of hydrophilic matrices for oral extended release of drugs is a common practice in the pharmaceutical industry. This chapter presents different polymer choices for fabrication of monolithic hydrophilic matrices and discusses formulation and manufacturing variables affecting the design and performance of the extended-release product by using selected practical examples. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_2 Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_3 The gene silencing capability of RNA interference (RNAi) is being used to study individual gene's biological function and role in biochemical pathways. However, the efficacy of RNAi depends upon efficient delivery of the intermediates of RNAi, small interfering RNA (siRNA) oligonucleotides. The delivery challenge is even greater when the aim is to inhibit the expression of target genes in disease tissues. In vivo delivery of siRNA is complicated and challenging, and recent works on various animal disease models and early successes in human clinical trials are enlightening the tremendous potential of RNAi therapeutics. In this chapter, the latest developments of in vivo delivery of siRNA and the critical issues related to this effort are addressed. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_4 Methods to infuse drugs into the parenchyma of the central nervous system (CNS) have been reported as inconsistent or unpredictable. The source of variability appears to be a compromised seal between the tissue and the outer surface of the cannula. Failure of the tissue to seal to the cannula creates a path of least resistance. Rather than penetrate the target area, the drug backflows along the path of the cannula. This artifact can be difficult to detect because drugs enter the systemic circulation and provide some fraction of the intended therapy. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_5 Human skin serves a protective function by imposing physicochemical limitations to the type of permeant that can traverse the barrier. For a drug to be delivered passively via the skin it needs to have a suitable lipophilicity and a molecular weight < 500 Da. The number of commercially available products based on transdermal or dermal delivery has been limited by these requirements. In recent years various passive and active strategies have emerged to optimize delivery. The passive approach entails the optimization of formulation or drug carrying vehicle to increase skin permeability. However, passive methods do not greatly improve the permeation of drugs with molecular weights >500 Da. In contrast, active methods, normally involving physical or mechanical methods of enhancing delivery, have been shown to be generally superior. The delivery of drugs of differing lipophilicity and molecular weight, including proteins, peptides and oligonucletides, has been shown to be improved by active methods such as iontophoresis, electroporation, mechanical perturbation and other energy-related techniques such as ultrasound and needleless injection. This chapter details one practical example of an active skin abrasion device to demonstrate the success of such active methods. The in vitro permeation of acyclovir through human epidermal membrane using a rotating brush abrasion device was compared with acyclovir delivery using iontophoresis. It was found that application of brush treatment for 10 s at a pressure of 300 N m−2 was comparable to 10 min of iontophoresis. The observed enhancement of permeability observed using the rotating brush was a result of disruption of the cells of the stratum corneum, causing a reduction of the barrier function of the skin. However, for these novel delivery methods to succeed and compete with those already on the market, the prime issues that require consideration include device design and safety, efficacy, ease of handling, and cost-effectiveness. This chapter provides a detailed review of the next generation of active delivery technologies. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_6 The inhalation route is seen as the most promising non-invasive alternative for the delivery of proteins; however, the short duration of activity of drugs delivered via this route brought about by the activities of alveolar macrophages and mucociliary clearance means there is a need to develop controlled release system to prolong the activities of proteins delivered to the lung. Polymeric materials such as (d,l)-poly(lactic glycolic acid) (PLGA), chitosan and poly(ethylene glycol) (PEGs) have been used for controlled release of proteins. Other systems such as liposomes and microcrystallization have also proved effective. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_7 Pulmonary delivery of proteins requires particles for delivery to be in the aerodynamic size range 1–5 μm for deep lung deposition. However, the traditional particle size reduction technique of jet-milling normally used for inhalation is not suitable for processing these protein particles because of their lability brought about by the weak physical interactions making up their higher order structures. Advanced techniques such as spray drying, spray freeze drying and the use of supercritical fluid technology have been developed to produce particles in the suitable size range and morphology for deep long deposition without altering the native conformation of these biomolecules. Judicious use of excipients and operating conditions are some of the factors needed for a successful particle design. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_8 Drug delivery across the blood–brain barrier (BBB) is a major obstacle for the development of effective treatments of many central nervous system disorders. Sophisticated cell culture models of the BBB have helped us to identify, characterize, and validate a novel targeted drug delivery technology, designated 2B-Trans™, for the receptor-mediated uptake and transport of drugs across the BBB. This paper describes in great detail how such a BBB cell culture model should be prepared and handled, and applied for the use of targeted drug delivery across the BBB. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-210-6_9 There are various types of liposomes used for cancer therapy, but these can all be placed into three distinct categories based on the surface charge of vesicles: neutral, anionic and cationic. This chapter describes the more rigorous and easy methods used for liposome manufacture, with references, to aid the reader in preparing these formulations in-house. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_1 Twenty-five years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulse delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a crossroad in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here as an introduction to the large range of applications described in this book. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. The feasibility of electric pulse for gene transfer in humans is discussed taking into account that electric pulse delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments have made DNA electrotransfer more efficient and safer, this nonviral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and a respect for safe procedures will be key elements for the successful future transition of DNA electrotransfer to the clinics. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_10 Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_11 Natural killer (NK) cells are highly resistant to transfection by conventional methods such as electroporation and lipofection. Recently, we reported the employment of a novel electroporation-based method, called nucleofection, which for the first time enabled efficient nonviral gene transfer into NK cells. In this study, we aimed at developing optimized conditions for the transfection of different NK cell lines as well as primary NK cells. Using EGFP (enhanced green fluorescent protein) or luciferase as reporter genes, suitable buffer conditions as well as instrument settings were defined. The new transfection methodology represents a useful tool for the immunotherapeutic use of NK cells, with the potential to enhance cytotoxicity as well as retarget the specificity of cytotoxic lymphocytes in clinical therapy of cancer and viral infection. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_12 Cultured adherent cells can be electroporated in situ, as they grow on a glass slide coated with electrically conductive, optically transparent indium-tin oxide (ITO). Although the introduction of DNA is a common use, the technique of electroporation in situ is valuable for studying many aspects of signal transduction. This is because, under the appropriate conditions, in situ electroporation can be remarkably nontraumatic, while a large variety of molecules, such as peptides, oligonucleotides, or drugs, are introduced instantly and into essentially 100% of the cells, making this technique especially suitable for kinetic studies of effector activation. Following the introduction of the material, the cells can be either extracted or biochemically analyzed, or their morphology and gene expression can be examined by immunocytochemistry. In this chapter, we describe the introduction of a peptide blocking the Src-homology 2 domain of the adaptor Grb2 to inhibit the activation of the downstream effector Erk1/2 by EGF. The setup includes nonelectroporated, control cells growing side by side with the electroporated ones on the same type of ITO-coated surface. In a modified version, this assembly can be used very effectively for studying intercellular, junctional communication: cells are grown on a glass slide half of which is ITO-coated. An electric pulse is applied in the presence of the fluorescent dye lucifer yellow, causing its penetration into the cells growing on the conductive part of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_13 Electroporation has been adapted for the transfer of macromolecules into various cells of tissues in vivo. Although mature adipocytes constitute less than 20% of cells residing in adipose tissue, we have found that fat cells are susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. The procedures detailed here permit electrotransfer of plasmid DNA into mature fat cells with greater than 99% selectivity over other cells in the tissue. This “adiporation” technique can be used to image the subcellular targeting of fluorescent bioreporter molecules and to manipulate the activity of specific pathways within adipocytes in situ. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_14 An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as the study of gene function and gene therapy applications. Among the different nonviral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple methods. This technique is a physical method of gene delivery consisting of a local application of electric pulses after injection of DNA. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_15 Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimal conditions for individual species and muscle group are not well defined. Using a muscle-specific plasmid driving the expression of a secreted embryonic alkaline phosphatase (SEAP) reporter gene, we have optimized the electroporation conditions in a large mammal model, i.e. pig. The parameters optimized include electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Constant current pulses, between 0.4 and 0.6 A, applied 80 s after the injection of 0.5 mg SEAP-expressing plasmid in a total formulation volume of 2 mL produced the highest expression in semimembranosus muscle in pigs. These results could be extrapolated for a different muscle group in pigs, the biceps femoris, and may be an evaluation starting point for large muscle in veterinary species or humans (see Note 1 ). 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_16 Delivery of DNA into skin is an attractive method, because skin is the most accessible somatic tissue for gene transfer and can be monitored conveniently. Skin is especially suitable for immunization using plasmid-DNA-based vaccines; however, a low level of transfection is the major limitation to the use of DNA-based therapeutics. Several chemical and physical methods are being investigated to improve the transfection of target cells with plasmid DNA. Electroporation is a physical method of gene transfer by applying electric pulses to the target cells. Most of the electroporation studies involve insertion of electrode needles into the tissues. In this chapter, we discuss that the DNA delivery into skin can be greatly enhanced by topical electroporation of the DNA injection site in rabbits using a tweezer electrode. Furthermore, the immune responses following a DNA vaccine delivery by using electroporation have been explored. Electroporation shows great potential for enhancing the DNA delivery into the skin. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_17 Electroporation is a safe, efficient, and inexpensive method to transfer naked plasmid DNA into various tissues. For electroporation-mediated gene transfer to the mouse lung, a plasmid solution is delivered to the lungs via the trachea. Immediately after plasmid delivery, eight square wave pulses are delivered by two pregelled electrodes placed on each side of the chest. The optimal field strength is 200 V/cm, with a pulse duration of 10 ms each and a 1 s interval between pulses. High-level gene expression can be achieved within 24 h in all cell types in the lung, with very little inflammation and no apparent trauma. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_18 The possibility of in vivo gene transfer into the rat bladder by electroporation (EP) was evaluated. The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of marker genes, green fluorescent protein (GFP) and luciferase, and the neuronal nitric oxide synthase (nNOS) gene were then injected into the subserosal space of the bladder and EP was applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder, and immunohistochemical staining was used to detect nNOS. NOx released from isolated bladder strips was also assessed using microdialysis procedure. From the luciferase assay, 45 V, 1 Hz, 50 ms, and 8 pulses were selected as the optimum conditions for EP. Bladder specimens with GFP genes injected by EP showed numerous bright sites of GFP expression in the smooth-muscle layer. In rats with the nNOS gene injected by EP, there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than that in the control groups. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_19 The Retina is a good model system for studies of neural development and disease because of its simplicity and accessibility. To analyze gene function rapidly and conveniently, we developed an electroporation technique in mice and rats for use in vivo and in vitro. The efficiency of electroporation into the neonatal retina is quite good, and transgene expression persists for more than a month. With this technique, various types of DNA constructs, including RNA interference (RNAi) vectors, are readily introduced into the retina without DNA size limitation. In addition, more than two different DNA constructs can be introduced into the retina at once, with very high cotransfection efficiency. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_2 Cell membranes can be transiently permeabilized under application of electric pulses that allow hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs through simple diffusion while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven association of the DNA molecule with the destabilized membrane and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cell, migrates toward the nuclei. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_20 Short interfering RNAs (siRNAs) represent new potential therapeutic tools owing to their capacity to induce strong, sequence-specific gene silencing in cells. However, this development requires new, safe, and efficient in vivo siRNA delivery methods. In this study, we reported that gene silencing was efficiently obtained in vivo in an adult mammal (mouse) with chemically synthesized siRNA after its electrical delivery in muscles. The associated gene silencing was followed on the same animal and lasted more than 11 days. Gene silencing was obtained in muscles not only on young adult mice but also on much older animals. No tissue damage was detected under our electrical conditions. Therefore, this method should provide an efficient approach for gene function analysis by a localized delivery of siRNAs. 2007-12-21T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-194-9_21 We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3–6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 μg/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1α, and Xenopus EF-1α) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism. 2007-12-21T05:00:00Z