Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.

Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood–brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood–brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by 31P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before 31P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Caracterização de nanopartículas pDNA-protamina e pDNA-protamina-lipossoma e avaliação da eficiência de entrega gênica a células de mamífero.

Em estudos de terapia gênica e vacinação por DNA, a eficiência e a segurança dos vetores que transportam o material genético terapêutico possuem papel fundamental. Vetores não virais são considerados mais seguros, mas menos eficientes em relação aos vetores virais. Em parte, isso se deve à falta de estudos sistemáticos e comparativos no que diz respeito às características físico-químicas desses vetores quando em soluções biológicas e o efeito delas sobre a eficiência de entrega gênica. O objetivo deste trabalho é avaliar o efeito do pH, da força iônica e do tipo tampão de complexação sobre as características físico-químicas de nanopartículas pDNA-protamina e pDNA-protamina-lipofectamina, visando à entrega gênica para diferentes linhagens celulares. Para isso, nanopartículas formadas em diferentes condições foram caracterizadas através de ensaios de espalhamento dinâmico de luz (DLS) e potencial zeta. Os estudos indicaram que o pH, a força iônica, o tipo de tampão e a presença de meio de cultura e soro no ambiente de complexação alteram significativamente o tamanho, a polidispersidade e o potencial zeta das partículas formadas. Finalmente, buscou-se avaliar o efeito dessas características sobre a eficiência de transfecção in vitro de células de macrófagos IC21 e células HeLa. Os estudos de transfecção em células Hela indicam que tanto a composição como as condições de formação das partículas influenciam significativamente a eficiência de transfecção.

Lipid, sugar and liposaccharide based delivery systems 2

The vast majority of biologically active compounds will never be considered as potential drugs due to inherently poor bioavailability. This review discusses the progress in the development of chemical systems to improve the metabolic stability, absorption and physicochemical properties of potential drugs. Delivery systems that involve the conjugation of lipid and/or sugar moieties are highlighted, as well as novel methods of conjugation of these groups to drugs. The use of sugar molecules to target drugs to particular organs or cells is also discussed, as is the use of lipids in the growing area of gene delivery. This is an update of a previous review [1].

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Specific modulation of airway epithelial tight junctions by apical application of an occludin peptide

Tight junctions are directly involved in regulating the passage of ions and macromolecules (gate functions) in epithelial and endothelial cells. The modulation of these gate functions to transiently regulate the paracellular permeability of large solutes and ions could increase the delivery of pharmacological agents or gene transfer vectors. To reduce the inflammatory responses caused by tight junction-regulating agents, alternative strategies directly targeting specific tight junction proteins could prove to be less toxic to airway epithelia. The apical delivery of peptides corresponding to the first extracellular loop of occludin to transiently modulate apical paracellular flux has been demonstrated in intestinal epithelia. We hypothesized that apical application of these occludin peptides could similarly modulate tight junction permeability in airway epithelia. Thus, we investigated the effects of apically applied occludin peptide on the paracellular permeability of molecular tracers and viral vectors in well differentiated human airway epithelial cells. The effects of occludin peptide on cellular toxicity, tight junction protein expression and localization, and membrane integrity were also assessed. Our data showed that apically applied occludin peptide significantly reduced transepithelial resistance in airway epithelia and altered tight junction permeability in a concentration-dependent manner. These alterations enhanced the paracellular flux of dextrans as well as gene transfer vectors. The occludin peptide redistributed occludin but did not alter the expression or distribution of ZO-1, claudin-1, or claudin-4. These data suggest that specific targeting of occludin could be a better-suited alternative strategy for tight junction modulation in airway epithelial cells compared with current agents that modulate tight junctions.

Inorganic nanoparticles as carriers for efficient cellular delivery

Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical proper-ties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted. (c) 2005 Elsevier Ltd. All rights reserved.

Structure, dynamics, and energetics of siRNA-cationic vector complexation:a molecular dynamics study

The design and synthesis of safe and efficient nonviral vectors for gene delivery has attracted significant attention in recent years. Previous experiments have revealed that the charge density of a polycation (the carrier) plays a crucial role in complexation and the release of the gene from the complex in the cytosol. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with six cationic carrier systems of varying charge and surface topology. The simulations reveal detailed molecular-level pictures of the structures and dynamics of the RNA-polycation complexes. Estimates for the binding free energy indicate that electrostatic contributions are dominant followed by van der Waals interactions. The binding free energy between the 8(+)polymers and the RNA is found to be larger than that of the 4(+)polymers, in general agreement with previously published data. Because reliable binding free energies provide an effective index of the ability of the polycationic carrier to bind the nucleic acid and also carry implications for the process of gene release within the cytosol, these novel simulations have the potential to provide us with a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance the rational design of nonviral gene delivery systems.

Novel bioadhesive formulations for mucosal and parenteral delivery of vaccines

In this work we have established the efficient mucosal delivery of vaccines using absorption enhancers and chitosan. In addition, the use of chitosan was shown to enhance the action of other known adjuvants, such as CTB or Quil-A. Collectively, the results presented herein indicate that chitosan has excellent potential as a mucosal adjuvant. We have evaluated a number of absorption enhancers for their adjuvant activity in vivo. Polyornithine was shown to engender high scrum immune reasons to nasally delivered antigens, with higher molecular weight polyornithine facilitating the best results. We have demonstrated for the first time that vitamin E TPGS can act as mucosal adjuvant. Deoxycholic acid, cyclodextrins and acylcarnitines were also identified as effective mucosal adjuvants and showed enhanced immune responses to nasally delivered TT, DT and Yersinia pestis V and F1 antigens. Previously, none of these agents, common in their action as absorption enhancing agents, have been shown to have immunopotentiating activity for mucosal immunisation. We have successfully developed novel surface modified microspheres using chitosan as an emulsion stabiliser during the preparation of PLA microspheres. It was found that immune responses could be substantially increased, effectively exploiting the immunopenetrating characteristics of both chitosan and PLA microspheres in the same delivery vehicle. In the same study, comparison of intranasal and intramuscular routes of administration showed that with these formulations, the nasal route could be as effective as intramuscular delivery, highlighting the potential of mucosal administration for these particulate delivery systems. Chitosan was co-administered with polymer microspheres. It was demonstrated that this strategy facilitates markedly enhanced immune responses in both magnitude and duration following intramuscular administration. We conclude that this combination shows potential for single dose administration of vaccines. In another study, we have shown that the addition of chitosan to alum adsorbed TT was able to enhance immune responses. PLA micro/nanospheres were prepared and characterised with discreet particle size ranges. A smaller particle size was shown to facilitate higher scrum IgG responses following nasal administration. A lower antigen loading was additionally identified as being preferential for the induction of immune responses in combination with the smaller particle size. This may be due to the fact that the number of particles will be increased when antigen loading is low, which may in turn facilitate a more widespread uptake of particles. PLA lamellar particles were prepared and characterised. Adsorbed TT was evaluated for the potential to engender immune responses in vivo. These formulations were shown to generate effective immune responses following intramuscular administration. Positively charged polyethylcyanoacrylate and PLA nanoparticies were designed and characterised and their potential as delivery vehicles for DNA vaccines was investigated. Successful preparation of particles with narrow size distribution and positive surface charge (imparted by the inclusion of chitosan) was achieved. In the evaluation of antibody responses to DNA encoded antigen in the presence of alum administered intranasally, discrimination between the groups was only seen following intramuscular boosting with the corresponding protein. Our study showed that DNA vaccines in the presence of either alum or Quil-A may advantageously influence priming of the immune system by a mucosal route. The potential for the combination of adjuvants, Quil-A and chitosan, to enhance antibody responses to plasmid encoded antigen co-administered with the corresponding protein antigen was shown and this is worthy of further investigation. The findings here have identified novel adjuvants and approaches to vaccine delivery. In particular, chitosan or vitamin E TPGS are shown here to have considerable promise as non-toxic, safe mucosal adjuvants. In addition, biodegradable mucoadhesive delivery systems, surface modified with chitosan in a single step process, may have application for other uses such as drug and gene delivery.

Gene therapy strategies for the treatment of spinal cord injury

Spinal cord injury is a complex pathology often resulting in functional impairment and paralysis. Gene therapy has emerged as a possible solution to the problems of limited neural tissue regeneration through the administration of factors promoting axonal growth, while also offering long-term local delivery of therapeutic molecules at the injury site. Of note, gene therapy is our response to the requirements of neural and glial cells following spinal cord injury, providing, in a time-dependent manner, growth substances for axonal regeneration and eliminating axonal growth inhibitors. Herein, we explore different gene therapy strategies, including targeting gene expression to modulate the presence of neurotrophic growth or survival factors and increase neural tissue plasticity. Special attention is given to describing advances in viral and non-viral gene delivery systems, as well as the available routes of gene delivery. Finally, we discuss the future of combinatorial gene therapies and give consideration to the implementation of gene therapy in humans. © 2014 Future Science Ltd.