Effect of crystal size and surface functionalization on the cytotoxicity of silicalite-1 nanoparticles.

In this report, we describe the synthesis and characterization of nanocrystalline silicalite (the purely siliceous form of the zeolite, ZSM-5) of defined crystal size and surface functionalization and determine the effect on the type and degree of cytotoxicity induced in two distinct model cell lines. The silicalite materials were characterized by powder X-ray diffraction, dynamic light scattering and zeta potential, solid state NMR, thermal gravimetric analysis, and nitrogen adsorption using the BET method to determine specific surface area. The silicalite samples were functionalized with amino, thiol, and carboxy groups and had crystal sizes of approximately 30, 150, and 500 nm. The cytotoxicities of the silicalite samples with different crystal sizes and different surface functional groups were investigated using human embryonic kidney 293 (HEK-293) cells and RAW264.7 macrophage cell lines. We used the lactic dehydrogenase release assay to measure damage to the cell membrane, the caspase 3/7 activity assay to measure key molecules involved in apoptosis, and the Annexin V-propidium iodide staining method to provide visual confirmation of the types of cell death induced. We have shown that the impact of size and surface functionalization of silicalite nanoparticles on cell toxicity and mechanism of cell death is cell type-dependent. Thirty nanometer silicalite nanoparticles were nontoxic in RAW264.7 cells relative to untreated controls but caused necrosis in HEK293 cells. Carboxy-functionalized 500 nm silicalite nanoparticles resulted in apoptosis and necrosis in RAW264.7 cells and predominantly activated apoptosis in HEK293 cells.

Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal caco-2 cells.

Oxidative stress has been linked to the development of various chronic diseases. Vegetables and fruits, which contain polyphenols, were shown to have protective effects. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol abundant in tea, has been shown to have antioxidant activities in cell-free conditions and this study focused on the effect of cellular EGCG. Using an intestinal cell model to examine the oxidative stress induced by hydroxyl radicals, we report here that physiological concentrations (0.1-1 microM) of EGCG have dose- and incubation duration-dependent cell-associated lipid antioxidant activity (measuring malondialdehyde production). Vitamin E and vitamin C at 10-40 microM also showed cell-associated lipid antioxidant activities under shorter incubation durations. When EGCG was included in the incubation with vitamin E or C, more antioxidant activities were consistently observed than when vitamins were added alone. Catechin (widely present in fruits and vegetables) at 1 microM also significantly increased the antioxidant activity of vitamins E and C. Previous studies examining cell-associated activity of EGCG mainly focused on the 10-100 microM concentration range. Our results suggest that although the physiological level (0.1-1 microM) of dietary catechins is much lower than that of vitamins, they further contribute to the total antioxidant capacity even in the presence of vitamins.

Evidence for decreased interaction and improved carotenoid bioavailability by sequential delivery of a supplement.

Despite the notable health benefits of carotenoids for human health, the majority of human diets worldwide are repeatedly shown to be inadequate in intake of carotenoid-rich fruits and vegetables, according to current health recommendations. To address this deficit, strategies designed to increase dietary intakes and subsequent plasma levels of carotenoids are warranted. When mixed carotenoids are delivered into the intestinal tract simultaneously, competition occurs for micelle formation and absorption, affecting carotenoid bioavailability. Previously, we tested the in vitro viability of a carotenoid mix designed to deliver individual carotenoids sequentially spaced from one another over the 6 hr transit time of the human upper gastrointestinal system. We hypothesized that temporally and spatially separating the individual carotenoids would reduce competition for micelle formation, improve uptake, and maximize efficacy. Here, we test this hypothesis in a double-blind, repeated-measure, cross-over human study with 12 subjects by comparing the change of plasma carotenoid levels for 8 hr after oral doses of a sequentially spaced carotenoid mix, to a matched mix without sequential spacing. We find the carotenoid change from baseline, measured as area under the curve, is increased following consumption of the sequentially spaced mix compared to concomitant carotenoids delivery. These results demonstrate reduced interaction and regulation between the sequentially spaced carotenoids, suggesting improved bioavailability from a novel sequentially spaced carotenoid mix.

Rational design, fabrication, characterization and in vitro testing of biodegradable microparticles that generate targeted and sustained transgene expression in HepG2 liver cells.

Poly(lactide-co-glycolide) (PLGA) microparticles have significant potential for sustained delivery of plasmid DNA (pDNA). However, unmodified PLGA microparticles have poor transfection efficiencies. In this study, we use several approaches to enhance the transfection efficiencies of PLGA microparticles in a HepG2 liver cell line. Polyethylenimine (PEI) is used to condense the pDNA prior to loading into the PLGA microparticles. This provides enhanced loading efficiencies and greater protection to the pDNA during the entrapment process. In addition, the pDNA used (ApoE) incorporates a hybrid liver-specific murine albumin enhancer/α1 antitrypsin promoter (AlbE/hAAT) to enhance transgene expression in human liver (HepG2) cells. The percentage of surfactant used in the preparation of the microparticles, the polymer composition of the PLGA, the ratio of the PEI to pDNA (N/P), the structure of the PEI and the potential utility of a galactose targeting ligand were then investigated to further optimize the efficacy of the cationic microparticle non-viral delivery system in transfecting HepG2 cells. For each PLGA PEI-pDNA microparticle formulation prepared, we evaluated particle size, ζ-potential, loading of pDNA, cytotoxicity, and transgene expression in HepG2 cells and control human embryonic kidney (HEK293) and monkey African green kidney fibroblast-like (COS7) cells. Loading PLGA particles with PEI-ApoE pDNA complexes resulted in a significant reduction in particle size when compared to PLGA microparticles loaded with ApoE pDNA alone. Scanning electron microscopy images showed that all the particle formulations were smooth and spherical in appearance. Incorporation of the cationic PEI in the PLGA particles changed the ζ-potential from negative to positive. Complexing PEI with ApoE pDNA increased the loading efficiency of the ApoE pDNA into the PLGA microparticles. The cytotoxicity of PLGA particles loaded with PEI-ApoE pDNA complexes was similar to PLGA particles loaded with ApoE pDNA alone. The transfection efficiency of all particle formulations prepared with ApoE pDNA was significantly higher in HepG2 cells when compared to HEK293 and COS7 cell lines. The release of PEI-pDNA complexes from particles prepared with different PLGA polymer compositions including PLGA 50-50, PLGA 75-25, and PLGA 85-15 was sustained in all cases but the release profile was dependent on the polymer composition. Transmission electron microscopy images showed that PEI-pDNA complexes remained structurally intact after release. The optimum formulation for PLGA particles loaded with PEI-ApoE pDNA complexes was prepared using 2% polyvinyl alcohol, 50-50 PLGA compositions and N/P ratios of 5-10. Strong sustained transgene expression in HepG2 cells was generated by PLGA PEI-ApoE pDNA particles up to the full 13 days tested.

Immunostimulatory sutures that treat local disease recurrence following primary tumor resection.

Neuroblastoma is a common childhood cancer that often results in progressive minimal residual disease after primary tumor resection. Cytosine-phosphorothioate-guanine oligonucleotides (CpG ODN) have been reported to induce potent anti-tumor immune responses. In this communication, we report on the development of a CpG ODN-loaded suture that can close up the wound following tumor excision and provide sustained localized delivery of CpG ODN to treat local disease recurrence. The suture was prepared by melt extruding a mixture of polylactic acid-co-glycolic acid (PLGA 75:25 0.47 dL g⁻¹) pellets and CpG ODN 1826. Scanning electron microscopy images showed that the sutures were free of defects and cracks. UV spectrophotometry measurements at 260 nm showed that sutures provide sustained release of CpG ODN over 35 days. Syngeneic female A/J mice were inoculated subcutaneously with 1 × 106 Neuro-2a murine neuroblastoma wild-type cells and tumors were grown between 5 to 10 mm before the tumors were excised. Wounds from the tumor resection were closed using CpG ODN-loaded sutures and/or polyglycolic acid Vicryl suture. Suppression of neuroblastoma recurrence and mouse survival were significantly higher in mice where wounds were closed using the CpG ODN-loaded sutures relative to all other groups.

Fabrication, characterization and in vitro evaluation of poly(D,L-lactide-co-glycolide) microparticles loaded with polyamidoamine-plasmid DNA dendriplexes for applications in nonviral gene delivery.

We report, for the first time, on the preparation, characterization and in vitro testing of poly(D,L-lactide-co-glycolide) (PLGA) microparticles loaded with polyamidoamine (PAMAM)-plasmid DNA (pDNA) dendriplexes. Loading of pDNA into the PLGA microparticles increased by 150% when pDNA was first complexed with PAMAM dendrimers relative to loading of pDNA alone. Scanning electron microscopy (SEM) showed that the presence of PAMAM dendrimers in the PLGA microparticles created porous features and indentations on the surface of the microparticles. Loading PLGA microparticles with PAMAM-pDNA dendriplexes lowered the average PLGA microparticle size and changed the surface charge of the microparticles from negative to positive when compared to PLGA microparticles loaded with pDNA alone. The zetapotential and buffering capacity of the microparticles increased as the generation of the PAMAM dendrimer loaded in the PLGA microparticles increased. Gel electrophoresis assays showed that all the PLGA microparticle formulations were able to entrap the pDNA within the PLGA matrix. There was no significant difference in the cytotoxicity of PLGA microparticles loaded with PAMAM-pDNA dendriplexes when compared to PLGA microparticles loaded with pDNA alone. Furthermore, and in contrast to PAMAM dendrimers alone, the generation of the PAMAM dendrimer loaded in the PLGA microparticles had no significant impact on cytotoxicity or transfection efficiencies in human embryonic kidney (HEK293) or Monkey African green kidney fibroblast-like (COS7) cells. The transfection efficiency of PLGA microparticles loaded with generation 3 (G3) PAMAM-pDNA dendriplexes was significantly higher than PLGA microparticles loaded with pDNA alone in HEK293 and COS7 cells. PLGA microparticles loaded with G3 PAMAM-pDNA dendriplexes generated equivalent transfection efficiencies as (G3 to G6) PAMAM-pDNA dendriplexes alone in COS7 cells when the transfection was carried out in serum containing media. The delivery system developed in this report has low toxicity, high pDNA loading efficiencies and high transfection efficiencies that are not reduced in the presence of serum. A delivery system with these characteristics is expected to have significant potential for translational applications.

Characterization of the transgene expression generated by branched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo.

Polyethylenimine (PEI) is a cationic polymer that has shown significant potential for delivering genes in vitro and in vivo. Mixing cationic PEI with negatively charged plasmid DNA (pDNA) results in the spontaneous electrostatic formation of stable nanoparticle complexes. The structure of PEI can be branched or linear. In this study, we show that branched PEI has a stronger electrostatic interaction with pDNA than linear PEI, which accounts for greater compaction, higher zeta potentials and smaller nanoparticle sizes at equivalent pDNA concentrations. For both linear and branched PEI, increasing the concentration of pDNA mixed in the same volume and at the same nitrogen to phosphate (N:P) ratio results in larger average particle sizes. Increasing the N:P ratio increases luciferase activity generated by branched PEI-pDNA nanoparticles and linear PEI-pDNA nanoparticles in HEK293, COS7 and HeLa cell lines. Increasing the N:P ratio at which branched PEI-pDNA nanoparticles are prepared also increases luciferase expression in HepG2 cells but does not increase luciferase expression generated by linear PEI-pDNA nanoparticles. In all of the cell lines, branched PEI-pDNA nanoparticles prepared at N:P ratios of 10 and above generated significantly higher luciferase activity than linear PEI-pDNA nanoparticles. Luciferase activity was highest in the HEK293 cells and luciferase expression in each of the cell lines followed the order of HEK293>COS7>HepG2>HeLa. Intraperitoneal (IP) injection of PEI-pDNA nanoparticles is attractive because it is simple, reproducible and often leads to a depot effect of nanoparticle complexes residing in the peritoneum. The IP route of administration avoids PEI-pDNA nanoparticle accumulation in the lung and the nanoparticles do not pass through the blood-brain barrier. In this study, using bioluminescent imaging (BLI), we show that changing the PEI structure and dose of the PEI-pDNA nanoparticles has a significant impact on the strength and duration of transgene expression after IP injection in vivo but increasing the N:P ratio does not. Increasing the dose and N:P ratio for all the PEI-pDNA nanoparticle formulations injected IP did not reduce mice survival and all mice remained in good health as determined by the Body Condition Scoring (BCS) technique.

Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods.

Controlled release of plasmid DNA (pDNA) from biodegradable poly lactic-co-glycolic acid (PLGA) microparticles has the potential to enhance transgene expression. However, barriers to this approach include limited encapsulation efficiency, pDNA damage during fabrication and confinement of the microparticles inside phagolysosomal compartments. Combining PLGA with poly ethyleneimine (PEI) can improve protection of pDNA during fabrication, increase encapsulation efficiencies and impart the PLGA microparticles with the capacity to escape the phagolysosomal compartments. This study compares three promising formulation methods for preparing PLGA PEI pDNA microparticles and evaluates for buffering capacity, cellular uptake, transfection efficiency and toxicity. In the first method, PLGA PEI pDNA microparticles are prepared by entrapping pDNA in blended PLGA/PEI using the double emulsion water-in-oil-in-water solvent evaporation technique (PA). In a second approach, PEI-pDNA polyplexes are prepared and then entrapped in PLGA microparticles using a double emulsion solvent evaporation method (PB). Microparticles prepared using formulation methods PA and PB are then compared against PLGA microparticles with PEI conjugated to the surface using carbodiimide chemistry (PC); 0.5% PVA is identified as the optimum concentration of surfactant for generating the strongest transfection efficiencies. N:P ratios of 5 and 10 are selected for preparation of each group. Gel electrophoresis demonstrates that all PLGA microparticle formulations have strong pDNA binding capacity. An MTT assay shows that in vitro cytotoxicity of PLGA PEI microparticles is significantly lower than PEI alone. PLGA PEI pDNA microparticles mediate higher cellular uptake efficiency and consequently higher transgene expression than unmodified PLGA microparticles in COS7 and HEK293 cells. Preparing PEI-pDNA polyplexes prior to entrapment in PLGA microparticles (PB) results in the highest pDNA loading. This is 2.5-fold higher than pDNA loading in unmodified PLGA microparticles. PLGA PEI pDNA microparticles prepared using method PB generates the strongest transfection efficiencies, which are 500-fold higher than unmodified PLGA pDNA microparticles in HEK293 cells and 1800-fold higher in COS-7 cells. The highest transfection efficiencies generated from microparticles prepared using method PB is achieved using an N:P ratio of 5.

Pulsatile release of biomolecules from polydimethylsiloxane (PDMS) chips with hydrolytically degradable seals.

We demonstrate, for the first time, a robust novel polydimethylsiloxane (PDMS) chip that can provide controlled pulsatile release of DNA based molecules, proteins and oligonucleotides without external stimuli or triggers. The PDMS chip with arrays of wells was constructed by replica molding. Poly(lactic acid-co-glycolic acid) (PLGA) polymer films of varying composition and thickness were used as seals to the wells. The composition, molecular weight and thickness of the PLGA films were all parameters used to control the degradation rate of the seals and therefore the release profiles. Degradation of the films followed the PLGA composition order of 50:50 PLGA>75:25 PLGA>85:15 PLGA at all time-points beyond week 1. Scanning electron microscopy images showed that films were initially smooth, became porous and ruptured as the osmotic pressure pushed the degrading PLGA film outwards. Pulsatile release of DNA was controlled by the composition and thickness of the PLGA used to seal the well. Transfection experiments in a model Human Embryonic Kidney 293 (HEK293) cell line showed that plasmid DNA loaded in the wells was functional after pulsatile release in comparison to control plasmid DNA at all time-points. Thicker films degraded faster than thinner films and could be used to fine-tune the release of DNA over day length periods. Finally the PDMS chip was shown to provide repeated sequential release of CpG oligonucleotides and a model antigen, Ovalbumin (OVA), indicating significant potential for this device for vaccinations or applications that require defined complex release patterns of a variety of chemicals, drugs and biomolecules.

Conjugation of polyamidoamine dendrimers on biodegradable microparticles for nonviral gene delivery.

We report on the preparation and characterization of poly(D, L-lactide-co-glycolide) (PLGA) microparticles with surface-conjugated polyamidoamine (PAMAM) dendrimers of varying generations. The buffering capacity and zeta-potential of the PLGA PAMAM microparticles increased with increasing generation level of the PAMAM dendrimer conjugated. Conjugation of the PAMAM dendrimer to the surface of the PLGA microparticle removed generation-dependent cytotoxicity in HEK293 and COS7 cell lines. PLGA PAMAM pDNA microparticles displayed similar cytotoxicity profiles to unmodified PLGA pDNA microparticles in COS7 cells. A generation three PAMAM dendrimer conjugated to PLGA microparticles significantly increased transfection efficiencies in comparison to unmodified PLGA microparticles.