Oral gene delivery with chitosan--DNA nanoparticles generates immunologic protection in a murine model of peanut allergy.

Food allergy is a common and often fatal disease with no effective treatment. We describe here a new immunoprophylactic strategy using oral allergen-gene immunization to modulate peanut antigen-induced murine anaphylactic responses. Oral administration of DNA nanoparticles synthesized by complexing plasmid DNA with chitosan, a natural biocompatible polysaccharide, resulted in transduced gene expression in the intestinal epithelium. Mice receiving nanoparticles containing a dominant peanut allergen gene (pCMVArah2) produced secretory IgA and serum IgG2a. Compared with non-immunized mice or mice treated with 'naked' DNA, mice immunized with nanoparticles showed a substantial reduction in allergen-induced anaphylaxis associated with reduced levels of IgE, plasma histamine and vascular leakage. These results demonstrate that oral allergen-gene immunization with chitosan-DNA nanoparticles is effective in modulating murine anaphylactic responses, and indicate its prophylactic utility in treating food allergy.

Scaffolding in tissue engineering: general approaches and tissue-specific considerations.

Scaffolds represent important components for tissue engineering. However, researchers often encounter an enormous variety of choices when selecting scaffolds for tissue engineering. This paper aims to review the functions of scaffolds and the major scaffolding approaches as important guidelines for selecting scaffolds and discuss the tissue-specific considerations for scaffolding, using intervertebral disc as an example.

Real-time observation of leukocyte-endothelium interactions in tissue-engineered blood vessel.

Human cell-based 3D tissue constructs play an increasing role in disease modeling and drug screening. Inflammation, atherosclerosis, and many autoimmune disorders involve the interactions between immune cells and blood vessels. However, it has been difficult to image and model these interactions under realistic conditions. In this study, we fabricated a perfusion and imaging chamber to allow the real-time visualization of leukocyte perfusion, adhesion, and migration inside a tissue-engineered blood vessel (TEBV). We monitored the elevated monocyte adhesion to the TEBV wall and transendothelial migration (TEM) as the TEBV endothelium was activated by the inflammatory cytokine TNF-α. We demonstrated that treatment with anti-TNF-α or an NF-kB signaling pathway inhibitor would attenuate the endothelium activation and reduce the number of leukocyte adhesion (>74%) and TEM events (>87%) close to the control. As the first demonstration of real-time imaging of dynamic cellular events within a TEBV, this work paves the way for drug screening and disease modeling in TEBV-associated microphysiological systems.

Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors.

Patients with malignant gliomas have a very poor prognosis. To explore a novel and more effective approach for the treatment of malignant gliomas, a strategy that combined tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and radiation treatment (RT) was designed in this study. Plasmid pE4-GFP was constructed by including the radioinducible early growth response gene 1 (Egr-1) promoter, and it yielded the best response with fractionated RT. Plasmid pE4-TRAIL was constructed by including the Egr-1 promoter and evaluated using U251 and U87 glioma cells. In the assay of apoptosis and killing activities, pE4-TRAIL exhibited radioresponse. pE4-TRAIL combined with RT is capable of inducing cell death synergistically. The expression of TRAIL death receptors was evaluated; which may be influenced by RT. Glioma cells with wild-type p53 showed upregulated expression of death receptors, and more synergistic effects on killing activities are expected. pE4-TRAIL was transfected into the subcutaneous U251 glioma cells in nude mice by the in vivo electroporation method. In the mice treated with pE4-TRAIL and RT, apoptotic cells were detected in pathological sections, and a significant difference of tumor volumes was observed when compared with the other groups (P<0.001). Our results indicate that radioresponsive gene therapy may have great potential as a novel therapy because this therapeutic system can be spatially or temporally controlled by exogenous RT and provides specificity and safety.

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets.

The rapid advances in synthetic biology and biotechnology are increasingly demanding high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescence signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing an error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl ß-d-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ∼100 bacteria per droplet generates detectable fluorescence signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.

Effects of nanoimprinted patterns in tissue-culture polystyrene on cell behavior.

Tissue engineering seeks to develop functional tissues in a biomimetic environment in vitro. As the extracellular environment in vivo is composed of numerous nanostructures, fabrication of nanostructured substrates will be valuable for tissue engineering applications. In this article, we report a simple nanoimprint lithography (NIL) process to pattern nanostructures directly on tissue-culture polystyrene plates. By repeating this NIL process, three-dimensional scaffolds consisting of multiple-layer nanostructures were also fabricated. Bovine pulmonary artery smooth muscle cells were cultured on imprinted gratings ranging from 350 nm to 10 µm. The smooth muscle cells attached and proliferated well on these imprinted substrates without additional surface treatment. Cell elongation and alignment were observed on the micro- and nanopatterns, with the effect significantly more pronounced on the nanostructures.

Controlled gene delivery by DNA-gelatin nanospheres.

A novel system for gene delivery, based on the use of DNA-gelatin nanoparticles (nanospheres) formed by salt-induced complex coacervation of gelatin and plasmid DNA, has been developed. These particles were spherical, with a size range of 200-700 nm, contained 25-30% (w/w) DNA, and were stabilized by cross-linking of gelatin. As a consequence of being controlled by the cross-linking density of the gelatin matrix, the average release rate of DNA from nanospheres synthesized under standard conditions was 2.2%/day in serum. Nanosphere DNA incubated in bovine serum was more resistant to nuclease digestion than was naked DNA. Various bioactive agents could be encapsulated in the nanospheres by ionic interaction with the matrix components, physical entrapment, or covalent conjugation. Transfection of cultured cells with a luciferase plasmid was enhanced by conjugating human transferrin onto the nanosphere and coencapsulating the endolysolytic agent chloroquine. Under our experimental conditions, gene expression in mice subsequent to intramuscular injection of nanospheres containing 1 microg of a beta-galactosidase plasmid was greater and more prolonged than was observed after injection of an equal amount of naked DNA or DNA complexed with Lipofectamine.

Antigen-specific induction of peripheral T cell tolerance in vivo by codelivery of DNA vectors encoding antigen and Fas ligand.

Fas ligand (FasL, CD95L) induces apoptosis in activated T cells with upregulated Fas (CD95) expression through the process termed activation-induced cell death (AICD). We postulated that coexpression of antigen and FasL within individual antigen-presenting cells would lead to antigen-specific activation of T cells and to their consequent deletion by FasL-mediated AICD. A DNA-gelatin coacervate containing transferrin cell ligand, calcium, and the lysosomatropic agent chloroquine, a formulation previously shown to achieve high-level transfection of immune and muscle cells in vivo, was used to codeliver plasmids encoding FasL and antigen. Mice developed a strong cytolytic T cell response to beta-Gal when injected with DNA encoding beta-galactosidase (LacZ) model antigen, either as naked DNA or DNA nanoparticles, but failed to respond when there was concomitant injection of nanoparticles containing both the LacZ and murine FasL DNA vectors. This loss of T cell response was systemic, specific for beta-Gal, complete when nanoparticles were administered before antigen challenge, and decreased the T cell response from prior immunization with LacZ DNA. In effect, this "tolerization" injection induced antigen-specific peripheral tolerance in study mice, and represents a possible approach to the treatment of autoimmune diseases and transplantation rejection.

Coacervate microspheres as carriers of recombinant adenoviruses.

The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

In vitro and in vivo studies of subcutaneous hydromorphone implants designed for the treatment of cancer pain.

Unrelieved cancer pain remains a significant problem worldwide. Patients receive inadequate analgesia for a variety of complex and multifactorial reasons. Limited availability of opioids secondary to concerns about potential diversion of these medications for illicit use and poor compliance with oral regimens are significant factors in many countries. This study was designed to develop and test an implantable opioid delivery device capable of releasing a potent opioid subcutaneously at a continuous rate for 4 weeks. A low temperature solvent casting technique was used to formulate ethylene vinyl acetate (EVA) copolymer disks containing 50% hydromorphone by weight. The release characteristics of disks of different height and diameter, coated and uncoated, and with and without a central uncoated channel were studied. The effect of temperature and pH were also evaluated. In vitro assessments were conducted in phosphate buffer using UV spectrophotometry. In vivo studies employed New Zealand White Rabbits and a radioimmunoassay. Plasma levels following hydromorphone delivery by polymer, osmotic pump, and intravenous administration were compared. In vitro, uncoated EVA polymer disks measuring 1.05 cm in diameter and 0.27 cm in height released an initial large burst of hydromorphone. Coating the disks with 100-200 microM of poly(methyl-methacrylate) prevented drug egress from the polymer. A central uncoated channel measuring 1.25 mm in diameter in an otherwise coated polymer virtually eliminated the initial burst of drug release and provided near zero-order hydromorphone release at an average rate of 164 micrograms per hour for 4 weeks. Doubling the height of the polymer approximately doubled the release rate while doubling the diameter of the polymer extended the duration of drug release to over 8 weeks. In rabbits, stable plasma hydromorphone concentrations (23-37 ng/ml) were sustained for 4 weeks following implantation of 2 polymers with an uncoated central channel. No initial burst of hydromorphone release was noted. Increasing the number of polymers produced sustained and predictable increases in plasma hydromorphone concentrations. Plasma levels were similar with subcutaneous hydromorphone delivered by polymer and osmotic pump and much less variable than with intravenous bolus hydromorphone. A uniquely configured implantable drug delivery device has been developed using materials which are approved for human use. It safely and reproducibly releases hydromorphone for weeks in vitro and in vivo without an initial burst of drug release. Varying the thickness, diameter, and number of implants provides flexibility in the release rate and duration of release. This implantable opioid delivery device could provide a sustained subcutaneous infusion of hydromorphone to patient with cancer pain in developed and developing nations without pumps, catheters, or extensive outpatient support services. In addition, it should improve compliance and reduce concern regarding illicit diversion of opioids.

The use of bioerodible polymers and 5-fluorouracil in glaucoma filtration surgery.

A study was performed to examine the effect of a localized and sustained delivery of 5-fluorouracil (5-FU) on the success of glaucoma filtration surgery in 18 rabbits in a prospective, randomized, double-masked and placebo-controlled fashion. A bioerodible polyanhydride composed of bis (p-carboxyphenoxy) propane and sebacic acid was used as the drug carrier. The polymer and 5-FU (20% by weight) were compressed into 3 mm diameter discs, 1 mm thick. The polymer with the 5-FU was randomized to one eye and the fellow eye received the blank polymer. The results showed that intraocular pressures (IOP) were lower in the experimental eyes during the 5th through 17th postoperative days, but eventually both experimental and control eyes returned to preoperative levels. Filtration blebs lasted longer in experimental eyes when compared to control eyes. Implant disappearance occurred after IOP elevations and bleb failure. Eventually, the filtration surgery failed in both the experimental and control rabbit eyes.

Glaucoma filtration surgery in nonhuman primates using taxol and etoposide in polyanhydride carriers.

PURPOSE: To determine the effect of taxol and etoposide, hydrophobic drugs with antifibrosis activity, on the outcome of filtration surgery in glaucomatous monkeys. METHODS: Elevated intraocular pressure was produced bilaterally in eight cynomolgus monkeys by laser treatment of the trabecular meshwork. Four animals subconjunctivally received a polyanhydride disk containing 1 mg etoposide at the time of posterior lip sclerectomy in one eye; the other eye received an identical disk without drug. Similarly, four animals received a disk containing 50 micrograms of taxol in one eye and a blank disk in the other. RESULTS: Eyes treated with taxol had lower intraocular pressures than control eyes from 20 days after surgery until death. Eyes with satisfactory filtration bleb appearance and patent fistulae on histologic examination had lower intraocular pressures. The intraocular pressure was lower and the duration of success longer in the etoposide-treated eyes (mean, 16 days) compared to that of the fellow eyes (mean, 10 days), but the difference was not statistically significant. CONCLUSIONS: Use of polyanhydride disks containing taxol, but not etoposide, had a marked beneficial effect on intraocular pressure and bleb appearance after experimental filtration surgery in monkeys. The difference between the two agents may result from the greater antiproliferative potency of taxol and its greater duration of release from the polymer.

Polyanhydrides for controlled release of bioactive agents.

This report is a review of the development of a drug delivery system based on biorodible polyanhydrides. With the water labile anhydride linkage, a wide range of matrix degradation and drug release rates can be obtained from these drug-carriers. In addition to monolithic formulations, the feasibility of an injectable system by microencapsulation is demonstrated. The possibility of enhancing the release externally by an ultrasonic source has also been explored. The polymers tested showed good tissue biocompatibility and their breakdown products showed no adverse toxicological effects. Preliminary in vivo results confirmed the efficacy of these devices.

Fabrication of poly(phosphoester) nerve guides by immersion precipitation and the control of porosity.

Immersion precipitation was employed as a method for the fabrication of polymeric conduits from P(BHET-EOP/TC), a poly(phosphoester) with an ethylene terephthalate backbone, to be applied as guidance channels for nerve regeneration. Coatings of various porosities could be obtained by immersing mandrels coated with a solution of the polymer in chloroform into non-solvent immersion baths, followed by freeze or vacuum-drying. The porosity of the coatings decreased with an increase in polymer molecular weight, drying time before precipitation and concentration of polymer solution. The effects of these parameters can be rationalized by employing ternary phase diagrams, where porosity is directly related to the degree of phase separation available to the system before gelation occurs. To afford improved porosity control, a new system was developed which employed the contrasting phase-separation behavior of P(BHET-EOP/TC)/chloroform solution in methanol and water. As water is essentially a non-solvent for the polymer, the demixing boundary of the P(BHET-EOP/TC)-CHCl3-H2O system is located close to the polymer-solvent edge of the phase diagram, while that of the P(BHET-EOP/TC)-CHCl3-MeOH system is located further away. A mixture of methanol and water allows the demixing boundary to be shifted to intermediate coordinates. By immersing P(BHET-EOP/TC) coatings in immersion baths containing different ratios of water and methanol, then gradually titrating the bath with methanol to a concentration of 70% (v/v) methanol, surface porosities ranging from 2 to 58% could be achieved.

Development of bioabsorbable glass fibres.

Calcium-iron phosphate glasses with an iron oxide content ranging from 5 wt.% to 22 wt.% were prepared to investigate the effect of iron oxide on the properties of the glass. It was found that the dissolution rate, the fibre strength and the glass transition temperature were strongly affected by iron oxide. The glass dissolution rate exhibited a 50-fold reduction while the fibre strength doubled when the iron oxide content was increased from 5 wt.% to 22 wt.%. The phosphate glass containing 22 wt.% of iron oxide had a dissolution rate of about 5 micrograms/(cm2 day). The fibres drawn from this glass also exhibited the highest tensile strength over 1000 MPa. A cortical bone plug method was used to assess the biocompatibility of the glasses with the hard and soft tissues. The tissues surrounding the samples showed no inflammation at 9 wk.

Engineering of a sugar-derivatized porous network for hepatocyte culture.

Many tissue engineering applications require a scaffold or template conducive to cell attachment and maintenance of functions. It may also be advantageous in some cases for these scaffolds to have a controlled porous architecture to facilitate cellular or tissue ingrowth. In this study, we have engineered a porous carbohydrate-derivatized substrate for hepatocyte culture. Polystyrene foams, with pore sizes up to 100 microns, fabricated by phase separation from a homogeneous naphthalene solution, were derivatized with lactose and heparin, both of which are known to promote rat hepatocyte attachment and maintenance of its differentiated functions. Rat hepatocytes cultured on these derivatized foams exhibited a rounded cellular morphology with many microvilli evident on the surface of the cells. The hepatocytes showed an increase in albumin secretion for the first 3 days of culture in a defined, serum-free medium, and dropped back to initial levels by the end of 7 days. The production of cytochrome P450-dependent hydroxytestosterone metabolites were also measured. Two testosterone metabolites were maintained and five others were present but decreased over a culture period of 1 week. These carbohydrate-derivatized porous substrates may be useful for large-scale culture of hepatocytes, toxicology screening and for use in a liver assist device.

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

A new nerve guide conduit material composed of a biodegradable poly(phosphoester).

There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of fibroblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10 mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw 14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.

Fabrication of controlled release biodegradable foams by phase separation.

Highly porous biodegradable foams with controlled release function were fabricated by a phase separation technique. This technique involved inducing phase changes in a homogeneous solution of polymers with naphthalene or phenol used as solvents. A variety of foams with pore sizes ranging from 20 to 500 microm were made of poly(L-lactic acid) (PLLA), poly(bisphenol A-phenylphosphonate (BPA/PP), and its copolymer with poly[bis(2-ethoxy)- hydrophosphonic terephthalate] (PP/PPET). Controlled delivery capability was demonstrated by studying the release of sulforhodamine B and alkaline phosphatase (AP) from these highly porous structures. After an initial burst, AP was released from BPA/PP and PLLA foams at a near steady rate of 0.32 +/- 0.04 and 0.49 +/- 0.13 mg/day/g foam, respectively. These foams were intended for use as cell transplantation devices and tissue grafts such as synthetic bone grafts. Hydroxyapatite (HA) was added into the foams in an attempt to enhance interaction of these foams with bone. This composite was analyzed by energy dispersive spectroscopy, differential scanning calorimetry, and thermomechanical analysis. Since phosphates are known to have good affinity to calcium, poly(phosphoester) foams were treated with 1M calcium chloride solution in an attempt to study the possible interaction of the degrading poly(phosphoester) with calcium. After three weeks in 1 M calcium chloride solution, the complex modulus of the poly(phosphoester) foams changed from 40 to 1948 kPa, with a concurrent decrease in loss tangent from 0.349 to 0.170.

Hepatocyte encapsulation for enhanced cellular functions.

An efficient bioartificial liver-assisted device can sustain the lives of patients with acute liver failure. Among different configurations of the bioreactor design, hepatocyte encapsulation has important features that satisfy most requirements of the device. We have encapsulated rat hepatocytes in a two-layer polymeric membrane by complex coacervation using a simple setup and demonstrated enhanced cellular functions up to three times higher than those of the monolayer control. These microcapsules of the functioning hepatocytes have a 2- to 3-microm outer layer of synthetic polymer with 25% 2-hydroxyethyl methacrylate, 25% methacrylic acid, and 50% methyl methacrylate and an inner layer of positively charged modified collagen as a suitable substrate for the enhanced cellular functions. Permeable only to small molecules up to albumin, the microcapsules should allow unimpeded exchange of nutrients, oxygen, growth factors, and metabolites but prevent attack by immunoglobulins of the immune system, and no "skin effect" of the collagen has been observed. Mechanical properties of the microcapsules measured with a nano-indentation method suggest that the microcapsules should be suitable for use in a bioartificial liver-assisted device.