[Clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency by neonatal screening].

Objective: To investigate the incidence, clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency (SCADD). Method: From January, 2009 to October, 2015, a retrospective analysis of the urine organic acids and acyl-coenzyme A dehydrogenase (ACADS) gene mutation characteristics of patients diagnosed as SCADD by newborn screening using tandem mass spectrometry in Department of Genetics and Metabolism (Newborn screening Center of Zhejiang Province), Children's Hospital, Zhejiang University School of Medicine. Dietary guidance, life management and supplementation of L-carnitine were conducted, and growth and intelligence development were observed during follow-up among the SCADD patients. Result: A total of 1 430 024 neonates, seventeen cases were diagnosed with SCADD with an incidence of 1/84 117. All patients had no clinical symptoms, and intelligence and physical development were normal. Blood butylacyl-carnitine (C4) levels and the ratios increased, C4 0.713.14 µmol/L(reference value 0.03-0.48 µmol/L), C4/C2 0.07-0.23(reference value 0.01-0.04), C4/C3 0.65-2.04(reference value 0.05-0.39). Thirteen with increased urinary ethyl malonic acid (9.30-90.99 mg/g creatinine (reference value 0-6.20 mg/g creatinine )), one patient was accompanied by increased methyl succinic acid (12.33 mg/g creatinine(reference value 0-6.40 mg/g creatinine)), one subject with increased acetylglycine (3.52 mg/g creatinine(reference value 0-0.70 mg/g creatinine)). A total of 13 known mutations were detected in the ACADS gene, 1 homozygous mutation (c.1031A>G), the others are compound heterozygous mutations. One frameshift mutation (c.508_509delGC) and 12 missense mutations were detected. Common mutation were c. 1031A>G(35.3%), c. 164C>T(20.6%) and c. 991G>A(11.8%). SCADD in newborn screening program had no clinical symptoms and normal growth development after 8-42 months follow-up. Conclusion: Cases with SCADD had no clinical symptoms with an incidence of 1/84117. The c. 164C>T and c. 1031A>G may be the common mutations.

Single-dose microparticle delivery of a malaria transmission-blocking vaccine elicits a long-lasting functional antibody response.

Malaria sexual stage and mosquito transmission-blocking vaccines (SSM-TBV) have recently gained prominence as a necessary tool for malaria eradication. SSM-TBVs are unique in that, with the exception of parasite gametocyte antigens, they primarily target parasite or mosquito midgut surface antigens expressed only inside the mosquito. As such, the primary perceived limitation of SSM-TBVs is that the absence of natural boosting following immunization will limit its efficacy, since the antigens are never presented to the human immune system. An ideal, safe SSM-TBV formulation must overcome this limitation. We provide a focused evaluation of relevant nano-/microparticle technologies that can be applied toward the development of leading SSM-TBV candidates, and data from a proof-of-concept study demonstrating that a single inoculation and controlled release of antigen in mice, can elicit long-lasting protective antibody titers. We conclude by identifying the remaining critical gaps in knowledge and opportunities for moving SSM-TBVs to the field.

Protection against titanium particle-induced osteoclastogenesis by cyclooxygenase-2 selective inhibitor.

Wear particle-induced osteoclastogenesis is the most common cause of aseptic loosening in total joint arthroplasty. Although cyclooxygenase (COX)-2, an inducible regulator of prostaglandin E2 (PGE2) synthesis, is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In this study, we investigated the role of COX-2 in the regulation of osteoclast differentiation in the osteoclast precursor cell line RAW264.7 stimulated with titanium (Ti) particles. The results showed COX-2 expression in the early stages of RAW264.7 differentiation when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL) and Ti particles. Blockade of COX-2 by celecoxib, a COX-2 selective inhibitor, effectively reduced the expression of PGE2 and inhibited differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells. Quantitative real-time polymerase chain reaction revealed that celecoxib inhibited mRNA expression of RANK, cathepsin K (CPK), TRAP, and the nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells stimulated by Ti particles and RANKL. Moreover, exogenous PGE2 reversed the inhibitory effects of celecoxib. These results provide direct evidence that COX-2 dependent PGE2 induced by RANKL and Ti particles is required for osteoclastogenesis and suggests that reduced production of PGE2 by inactivation of COX-2 would provide a promising therapeutic target for the treatment of osteoclastogenesis induced by wear particles.

A novel sheep vertebral bone defect model for injectable bioactive vertebral augmentation materials.

New injectable bone substitutes have been developed that are, unlike polymethylmethacrylate, biologically active and have an osteogenic effect leading to osteogenesis and bone remodeling for vertebroplasty or kyphoplasty. In this study, we developed a sheep vertebral bone defect model to evaluate the new bioactive materials and assessed the feasibility of the model in vivo. Bone voids were experimentally created on lumbar vertebrae L2-L5 with L1 and L6 left intact as a normal control in mature sheep. The defect vertebrae L2-L5 in each sheep were randomized to receive augmentation with calcium phosphate cement (CPC) or sham. Vertebrae (L1-L6) were collected after 2 and 24 weeks of the cement augmentation and their strength and stiffness, as well as osseointegration activity and biodegradability, were evaluated. Finally, CPC significantly improved the strength and stiffness of vertebrae but did not yet restore it to the normal level at 24 weeks. Osteogenesis occurred at a substantially high level after 24 weeks of CPC augmentation or sham. Therefore, the sheep vertebral model with one void, 6.0 mm in diameter and 15.0 mm in depth, is replicable and can be used for evaluating the new injectable bioactive materials in vertebral augmentation or reconstruction.

A comparison of the proximal femoral nail antirotation device and dynamic hip screw in the treatment of unstable pertrochanteric fracture.

This prospective randomized study compared the outcome of elderly patients with an unstable pertrochanteric fracture, treated with a proximal femoral nail antirotation device (PFNA; n = 51) or a dynamic hip screw (DHS; n = 55). All patients in the DHS group and nine in the PFNA group had open reductions. Incisions were significantly shorter for the PFNA than the DHS group. Blood loss and the number of patients requiring post-operative blood transfusions were significantly greater, but operation and fluoroscopy times were significantly shorter, for the DHS versus the PFNA group. Time to mobilization with a frame was significantly shorter in the PFNA group, and post-operative complications were more common in the DHS group. Poor fracture reduction led to three revisions. All fractures in both groups united during follow-up. The PFNA allowed earlier mobilization and faster recovery than the DHS. The PFNA is a highly acceptable, minimally invasive implant for unstable fractures.

Hybrid braided 3-D scaffold for bioartificial liver assist devices.

Three-dimensional ex vivo hepatocyte culture is a tissue-engineering approach to improve the treatment of liver disease. The extracorporeal bioartificial liver (BAL) assists devices that are used in patients until they either recover or receive a liver transplant. The 3-D scaffold plays a key role in the design of bioreactor that is the most important component of the BAL. Presently available 3-D scaffolds used in BAL have shown good performance. However, existing scaffolds are considered to be less than ideal in terms of high-density cultures of hepatocytes maintaining long-term metabolic functions. This study aims to develop a 3-D hybrid scaffold for a BAL support system that would facilitate high-density hepatocyte anchorage with long-term metabolic functions. The scaffolds were fabricated by interlacing polyethylene terephthalate (PET) fibers onto the polysulfone hollow fibers utilizing a modern microbraiding technique. Scaffolds with various pore sizes and porosities were developed by varying braiding angle which was controlled by the gear ratio of the microbraiding machine. The morphological characteristics (pore size and porosity) of the scaffolds were found to be regulated by the gear ratio. Smaller braiding angle yields larger pore and higher porosity. On the other hand, a larger braiding angle causes smaller pore and lower porosity. In hepatocyte culture it was investigated how the morphological characteristics (pore size and porosity) of scaffolds influenced the cell anchorage and metabolic functions. Scaffolds with larger pores and higher porosity resulted in more cell anchorage and higher cellular functions, like albumin and urea secretion, compared to that of smaller pores and lower porosity.

Polyphosphoramidate gene carriers: effect of charge group on gene transfer efficiency.

Cationic polymeric carriers have been widely used for gene delivery. However, the structure-function relationship, especially the effect of charge groups of cationic polymeric carriers on the transfection activity, is poorly understood. To examine this important parameter, a series of cationic polymers, polyphosphoramidates (PPAs) with an identical backbone, same side chain spacer, similar molecular weights but different charge groups containing primary to quaternary amino groups (PPA-EA, PPA-MEA, PPA-DMA and PPA-TMA, Figure 1) were synthesized. The DNA-binding affinity of these four PPAs increased in the order of PPA-EAPPA-MEA>PPA-DMA>PPA-TMA. Particle size and zeta potential of four different types of PPA/DNA nanoparticles did not show significant correlation with PPA structure. These PPAs did not show significant buffering capacity within pH 5-7, even though transfection mediated by PPA-EA was the only one that seemed to be limited by endolysomal escape. Endocytosis of DNA mediated by PPAs was also similar (17-22%) for all four PPAs. However, the transfection efficiency of these PPAs varied significantly. In vitro transfection efficiency of PPAs decreased in the order of PPA-EA>PPA-MEA>PPA-DMA approximately PPA-TMA. Nanoparticles with PPA-EA containing primary amino groups gave the highest transfection efficiency in cell lines at the charge ratios from 6/1 to 20/1 (+/-). Matching the trend of transfection efficiency observed in vitro, PPA-EA mediated the highest transgene expression, comparable to that of polyethylenimine, in the spinal cord following intrathecal injection of the nanoparticles. These results establish that PPA gene carriers with primary amino group side chains are more potent than those with secondary, tertiary or quaternary amino groups in vitro and in the intrathecal gene delivery model.

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes.

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.

Immobilization of galactose ligands on acrylic acid graft-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture.

Surface modification of argon-plasma-pretreated poly(ethylene terephthalate) (PET) films via UV-induced graft copolymerization with acrylic acid (AAc) was carried out. Galactosylated surfaces were then obtained by coupling a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the AAc graft chains with the aid of a water-soluble carbodiimide (WSC) and N-hydroxysulfosuccinimide (sulfo-NHS). The modified PET films were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact-angle measurements. The galactosylated PET films were used as substrates for hepatocyte culture. The effects of surface carboxyl group concentration on the extent of galactose ligand immobilization, the extent of hepatocyte attachment, and the surface morphology were investigated. The amount of the galactose ligands immobilized on the PET surface increased with the AAc polymer graft concentration. AFM images revealed that the surface roughness of the PET film increased after graft copolymerization with AAc, but did not change appreciably with the subsequent immobilization of the galactose ligands. At the surface carboxyl group concentration of about 0.56 micromol/cm(2) or galactose ligand concentration of about 0.51 micromol/cm(2), the hepatocyte culture on the galactosylated surface exhibited the optimum concentration and physiological functions and formed aggregates or spheroids after just 1 day of culture. The albumin and urea synthesis functions of these hepatocytes were comparable to or higher than those of the hepatocytes cultured on the collagen-modified PET substrates.

Enhanced gene expression in mouse muscle by sustained release of plasmid DNA using PPE-EA as a carrier.

Delivery of plasmid DNA by nanoparticles improves the DNA bioavailability, for instance in intramuscular administration, by localizing the DNA in the muscle tissue. Extracellular sustained release of the DNA may lead to more prolonged transgene expression. The present study describes a novel controlled gene delivery system based on a water soluble and biodegradable polyphosphoester, poly(2-aminoethyl propylene phosphate) (PPE-EA). The polymer degraded in PBS at 37 degrees C through the cleavage of the backbone phosphate bonds, and it was synthesized with a relative high molecular weight to ensure a suitable hydrolytic stability as a gene carrier. The tissue response and cytotoxicity study demonstrated a better tissue compatibility of PPE-EA in mouse muscle compared with commonly used polyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently and protected DNA from nuclease and serum degradation. Sustained release of plasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EA degradation. The DNA release profiles appear to be predominantly controlled by carrier degradation and the release rate of plasmid could be adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P (amino to phosphate groups) ratio of 1, a 46% burst was observed for the first day, followed by about 4% release per day (24 microg DNA/day/mg of complex) for 12 days. Higher charge ratios reduced both the DNA release rate and the burst effect. The released DNA retained its structural and functional integrity. Intramuscular injection of PPE-EA-p43-LacZ complexes at N/P ratios of 0.5 and 1 resulted in enhanced beta-galactosidase expression in anterior tibialis muscle in Balb/c mice, as compared with naked DNA injections. Similarly, PPE-EA/IFN(alpha)2b DNA complexes generated an increased systemic level of interferon-alpha2b in mouse serum following intramuscular injection, as compared with naked DNA injection.

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.

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.

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.

Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency.

Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 microg/ml, the size of particles was optimized to approximately 100--250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINE-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.

Interactions of phospholipid bilayer with chitosan: effect of molecular weight and pH.

Chitosan has demonstrated its potentials as a gene carrier and a membrane perturbant for subsequent drug delivery to cells. However, there is currently a lack of experimental correlation between the physiochemical properties of chitosan and the resulting degree of lipid bilayer destabilization. In this study, the effect of pH and chitosan molecular weight on the interaction between chitosan and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer was examined with cross-polarization microscopy, differential scanning calorimetry (DSC), and Fourier transform- (FT-) Raman spectroscopy. Cross-polarized images showed that the direct hydration of the DPPC/chitosan mixture led to the formation of larger DPPC multilamellar vesicles (MLV), and pure chitosan also induced fusions of individual MLV. Under the influence of chitosan, the calorimetric enthalpy of DPPC was reduced in a concentration-dependent manner, and a new phase appeared at 28 degrees C during sample cooling. Even the lowest chitosan mole fraction of 0.04% reduced the cooperative unit of the DPPC bilayer by more than 70%. In addition, the electrostatic effect between chitosan and DPPC tuned the degree of membrane bilayer perturbation. Reduction of pH increased the number of protonated amines on the chitosan backbone and caused further disruption on the membrane organization. Mixing DPPC with chitosan in an organic medium before hydration enhanced the hydrophobic interactions between the two molecules and greatly reduced the cooperative unit among individual lipids during the main phase transition. The increase of chitosan molecular weight also affected the cooperativity in the thermotropic transition of DPPC bilayer. FT-Raman spectroscopy provided additional evidence that chitosan directly perturbed the organizations of the hydrophobic inner core of the DPPC bilayer.

Stereoselective determination of fenfluramine enantiomers in rat liver microsomal incubates.

An enantioselective assay for l- and d-fenfluramine in rat liver microsomal incubates was developed. The method involves extraction of fenfluramine from the microsomal incubates, and formation of fenfluramine diastereomeric derivatives with the chiral reagent S-(-)-N-trifluoroacetyl prolyl chloride. Separation and quantitation of the diastereomeric fenfluramine derivatives are carried out by a capillary gas chromatographic system with flame ionization detection. The assay is linear from 1 to 50 microg/ml for each enantiomer. The analytical method affords average recoveries of 92.28 and 96.44% for l- and d-fenfluramine, respectively. The limits of detection and quantitation for the method are 0.1 and 1.0 microg/ml for the l- and d-fenfluramine isomers, respectively. The reproducibility of the assay was <10% (RSD). The method allowed study of the depletion of l- and d-fenfluramine in rat liver microsomal incubates. The stereoselectivity of fenfluramine phase I metabolism was observed.

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.

Gene transfer by DNA-gelatin nanospheres.

A DNA and gelatin nanoparticle coacervate containing chloroquine and calcium, and with the cell ligand transferrin covalently bound to the gelatin, has been developed as a gene delivery vehicle. In this study, the coacervation conditions which resulted in the formation of distinct nanoparticles are defined. Nanospheres formed within a narrow range of DNA concentrations and achieved incorporation of more than 98% of the DNA in the reaction. Crosslinking of gelatin to stabilize the particles does not effect the electrophoretic mobility of the DNA. DNA in the nanosphere is partially resistant to digestion with concentrations of DNase I that result in extensive degradation of free DNA but is completely degraded by high concentrations of DNase. Optimum cell transfection by nanosphere DNA required the presence of calcium and nanospheres containing transferrin. The biological integrity of the nanosphere DNA was demonstrated with a model system utilizing DNA encoding the cystic fibrosis transport regulator (CFTR). Transfection of cultured human tracheal epithelial cells (9HTEo) with nanospheres containing this plasmid resulted in CFTR expression in over 50% of the cells. Moreover, human bronchial epithelial cells (IB-3-1) defective in CFTR-mediated chloride transport were complemented with effective transport activity when transfected with nanospheres containing the CFTR transgene.

DNA-polycation nanospheres as non-viral gene delivery vehicles.

Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such as gelatin and chitosan were evaluated as gene delivery vehicles. DNA-nanospheres in the size range of 200-750 nm could transfect a variety of cell lines. Although the transfection efficiency of the nanospheres was typically lower than that of lipofectamine and calcium phosphate controls in cell culture, the beta-gal expression in muscle of BALB/c mice was higher and more sustained than that achieved by naked DNA and lipofectamine complexes. This gene delivery system has several attractive features: (1) ligands can be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; (2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; (3) other bioactive agents or multiple plasmids can be co-encapsulated; (4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; (5) the nanosphere can be lyophilized for storage without loss of bioactivity.