A nanoparticle-based model delivery system to guide the rational design of gene delivery to the liver. 2. In vitro and in vivo uptake results

In Part 1 of our work (1), four nanoparticles were synthesized specifically for the purpose of identifying design constraints to guide next generation gene delivery to the liver. The four nanoparticles are Gal-50 and Gal-140 (galactosylated 50 and 140 nm nanoparticles) and MeO-50 and MeO-140 (methoxy-terminated 50 and 140 nm nanoparticles). All four particles have the same surface charge, and Gal-50 and Gal-140 have the same surface galactose density (ca. 25-30 pmol/cm2). Here, the hepatocyte uptake in vitro and hepatic distribution in vivo of these four nanoparticles is investigated. With freshly isolated hepatocytes, Gal-50 nanoparticles are taken up to a greater extent than are MeO-50, and both 50 nm beads are taken up to a much greater extent than either of the 140 nm nanoparticles. In mice, about 90% of the in vivo dose of Gal-140 nanoparticles is found within the liver 20 min after tail-vein injection. TEM and immunohistochemistry images confirm that Gal-140 nanoparticles are primarily internalized by Kupffer cells, though isolated examples of a few Gal-140 in hepatocytes are also observed. Gal-50 nanoparticles are overwhelmingly found in vesicles throughout the cytoplasm of hepatocytes, with only isolated examples of Kupffer cell uptake 20 min after tail vein injections in mice. Despite similar surface charge and ligand density, 50 nm nanoparticles are primarily found in hepatocytes while 140 nm nanoparticles are primarily observed in Kupffer cells. These results clearly show that slightly anionic, galactose-PEGylated nanoparticles with 25-30 pmol/cm2 galactose should be about 50 nm in diameter to preferentially target hepatocytes while they should be about 140 nm in diameter to selectively target Kupffer cells.     

Uptake and distribution of ceria nanoparticles in cucumber plants

The presence and release of nanoparticles (NPs) into the environment have important implications for human health and the environment. A critical aspect of the risk assessment of nanoparticles is to understand the interactions of manufactured nanoparticles with plants. In this study, the uptake and distribution characteristics of two types of ceria nanoparticles with sizes of ca. 7 nm and 25 nm in cucumber plants were investigated using a radiotracer method and other techniques. With increasing concentration of the nanoparticles, concentration dependent absorption by the plant roots was noticed, but the majority of the particles only loosely adhered to the root surface. The seedlings treated with 7 nm ceria particles showed significantly higher ceria contents in both roots and shoots than those exposed to 25 nm ceria particles at all test concentrations (2, 20, and 200 mg L(-1)). Only very limited amounts of ceria nanoparticles could be transferred from the roots to shoots because the entry of nanoparticles into the roots was difficult. However, the results of tissue distributions of ceria nanoparticles in the plants and two dimensional distributions of the particles in the leaves imply that once they have entered into the vascular cylinder, ceria nanoparticles could move smoothly to the end of the vascular bundle along with water flow. To the best of our knowledge, this is the first detailed study of uptake and distribution of metal oxide nanoparticles in plants.     

The role of reactive oxygen species in silicon dioxide nanoparticle-induced cytotoxicity and DNA damage in HaCaT cells

The increasing applications of silicon dioxide (SiO₂) nanomaterials have been widely concerned over their biological effects and potential hazard to human health. In this study, we explored the effects of SiO₂ nanoparticles (15, 30, and 100 nm) and their micro-sized counterpart on cultured human epidermal Keratinocyte (HaCaT) cells. Cell viability, cell morphology, reactive oxygen species (ROS), DNA damage (8-OHdG, γH2AX and comet assay) and apoptosis were assessed under control and SiO₂ nanoparticles exposed conditions. As observed in the Cell Counting Kit-8 (CCK-8) assay, exposure to 15, 30 or 100 nm SiO₂ nanoparticles at dosage levels between 0 and 100 μg/ml decreased cell viability in a concentration- and size dependent manner and the IC50 of 24 hour exposure was 19.4 ± 1.3, 27.7 ± 1.5 and 35.9 ± 1.6 μg/ml for 15, 30 and 100 nm SiO₂ nanoparticles, respectively. Morphological examination revealed cell shrinkage and cell wall missing after SiO₂ nanoparticle exposure. Increase in intracellular ROS level and DNA damage as well as apoptosis were also observed in SiO₂ nanoparticle-exposed HaCaT cells. Exposure to SiO₂ nanoparticles results in a concentration- and size-dependent cytotoxicity and DNA damage in cultural HaCaT cells which is closely correlated to increased oxidative stress.     

Multifunctional Nanoparticles for Prostate Cancer Therapy

The relapse of cancer after first line therapy with anticancer agents is a common occurrence. This recurrence is believed to be due to the presence of a subpopulation of cells called cancer stem cells in the tumor. Therefore, a combination therapy which is susceptible to both types of cells is desirable. Delivery of this combinatorial approach in a nanoparticulate system will provide even a better therapeutic outcome in tumor targeting. The objective of this study was to develop and characterize nanoparticulate system containing two anticancer agents (cyclopamine and paclitaxel) having different susceptibilities toward cancer cells. Both drugs were entrapped in glyceryl monooleate (GMO)-chitosan solid lipid as well as poly(glycolic-lactic) acid (PLGA) nanoparticles. The cytotoxicity studies were performed on DU145, DU145 TXR, and Wi26 A4 cells. The particle size of drug-loaded GMO-chitosan nanoparticles was 278.4 ± 16.4 nm with a positive zeta potential. However, the PLGA particles were 234.5 ± 6.8 nm in size with a negative zeta potential. Thermal analyses of both nanoparticles revealed that the drugs were present in noncrystalline state in the matrix. A sustained in vitro release was observed for both the drugs in these nanoparticles. PLGA blank particles showed no cytotoxicity in all the cell lines tested, whereas GMO-chitosan blank particles showed substantial cytotoxicity. The types of polymer used for the preparation of nanoparticles played a major role and affected the in vitro release, cytotoxicity, and uptake of nanoparticles in the all the cell lines tested.KEY WORDS: cancer stem cells, cyclopamine, glyceryl monooleate, nanoparticles, PLGA     

Investigation of siRNA-Loaded Polyethylenimine-Coated Human Serum Albumin Nanoparticle Complexes for the Treatment of Breast Cancer

Small interfering RNA (siRNA) molecules have great potential for developing into a future therapy for breast cancer. To overcome the issues related to rapid degradation and low transfection of naked siRNA, polyethylenimine (PEI)-coated human serum albumin (HSA) nanoparticles have been characterized and studied here for efficient siRNA delivery to the MCF-7 breast cancer cell line. The optimized nanoparticles were ~90 nm in size, carrying a surface charge of +26 mV and a polydispersity index (PDI) less than 0.25. The shape and morphology of the particles was studied using electron microscopy. A cytotoxicity assessment of the nanoparticles showed no correlation of cytotoxicity with HSA concentration, while using high molecular weight PEI (MW of 70 against 25 kDa) showed higher cytotoxicity. The optimal transfection achieved of fluorescin-tagged siRNA loaded into PEI-coated HSA nanoparticles was 61.66 ± 6.8%, prepared with 6.25 μg of PEI (25 kDa) added per mg of HSA and 20 mg/ml HSA, indicating that this nonviral vector may serve as a promising gene delivery system.     

Aqueous Synthesis and Concentration-Dependent Dermal Toxicity of TiO<Subscript>2</Subscript> Nanoparticles in Wistar Rats

A number of dermal toxicological studies using TiO₂ nanoparticles exist which are based on the study of various animal models like mice, rabbits etc. However, a well-defined study is lacking on the dermal toxic effects of TiO₂ nanoparticles on rats, which are the appropriate model for systemic absorption study of nanoparticles. Furthermore, toxicity of TiO₂ nanoparticles varies widely depending upon the size, concentration, crystallinity, synthesis method etc. This study was conducted to synthesize TiO₂ nanoparticles of different sizes (∼15 to ∼30 nm) by aqueous method, thereby evaluating the concentration-dependent toxicological effects of the ∼20-nm sized nanoparticles on Wistar rats. Characterization of the particles was done by transmission electron microscope, dynamic light scattering instrument, X-ray diffractrometer, and ultraviolet spectrophotometer. The toxicity study was conducted for 14 days (acute), and it is observed that TiO₂ nanoparticles (∼20 nm) at a concentration of 42 mg/kg, when applied topically showed toxicity on rat skin at the biochemical level. However, the histopathological studies did not show any observable effects at tissue level. Our data suggest that well-crystallized spherical-shaped ∼20 nm anatase TiO₂ nanoparticles synthesized in aqueous medium can induce concentration-dependent biochemical alteration in rat skin during short-term exposure.     

pH-responsive sulfonamide/PEI system for tumor specific gene delivery: an in vitro study

The feasibility of pH-sensitive polymeric nanoparticles that effectively target the acidic extracellular matrix of tumors is demonstrated. Plasmid DNA was complexed with polyethyleneimine (PEI) and further with a pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-block-PEG (PSD-b-PEG), to obtain naonparticles. The shielding/deshielding of nanoparticles was tested along with cell viability and transfection efficiency at physiological and tumor pH. The nanoparticles composed of DNA/PEI/PSD-b-PEG were 300 nm in size and showed low cytotoxicity and transfection at pH 7.4 due to shielding of PEI by PSD-b-PEG. The PSD-b-PEG bound to PEI/DNA complex decreased the interaction of PEI positive charges with cells and reduced the cytotoxicity by 60%. At pH 6.6, the nanoparticles demonstrated high cytotoxicity and transfection, indicating PSD-b-PEG detachment from the nanoparticles and permit PEI to interact with cells. PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues and hence has remarkable potential in drug targeting to tumor areas.     

Nano-sized and micro-sized polystyrene particles affect phagocyte function

Adverse effect of nanoparticles may include impairment of phagocyte function. To identify the effect of nanoparticle size on uptake, cytotoxicity, chemotaxis, cytokine secretion, phagocytosis, oxidative burst, nitric oxide production and myeloperoxidase release, leukocytes isolated from human peripheral blood, monocytes and macrophages were studied. Carboxyl polystyrene (CPS) particles in sizes between 20 and 1,000 nm served as model particles. Twenty nanometers CPS particles were taken up passively, while larger CPS particles entered cells actively and passively. Twenty nanometers CPS were cytotoxic to all phagocytes, ≥500 nm CPS particles only to macrophages. Twenty nanometers CPS particles stimulated IL-8 secretion in human monocytes and induced oxidative burst in monocytes. Five hundred nanometers and 1,000 nm CPS particles stimulated IL-6 and IL-8 secretion in monocytes and macrophages, chemotaxis towards a chemotactic stimulus of monocytes and phagocytosis of bacteria by macrophages and provoked an oxidative burst of granulocytes. At very high concentrations, CPS particles of 20 and 500 nm stimulated myeloperoxidase release of granulocytes and nitric oxide generation in macrophages. Cytotoxic effect could contribute to some of the observed effects. In the absence of cytotoxicity, 500 and 1,000 nm CPS particles appear to influence phagocyte function to a greater extent than particles in other sizes.     

Characterization of apoM in normal and genetically modified mice

A novel human apolipoprotein [apolipoprotein M (apoM)] was recently described and demonstrated to be a lipocalin. We have now examined apoM in wild-type mice and mice with genetically altered lipoprotein metabolism. Liver and kidney showed high mRNA expression, whereas spleen, heart, brain, and testis demonstrated low expression. ApoM gene expression was initiated on embryonic day 10. Western blot analysis of plasma suggested that mouse apoM, like its human counterpart, is secreted with a retained signal peptide, but unlike human apoM it is not glycosylated. Gel filtration of plasma showed apoM to be associated with HDL-sized particles in wild-type and apoA-I-deficient mice and with HDL- and LDL-sized particles in LDL receptor-deficient mice, whereas apoM was mainly found in VLDL-sized particles in high-fat, high-cholesterol-fed apoE-deficient mice. The plasma concentration of apoM was similar in wild-type, LDL receptor-deficient, and apoE-deficient mice but was reduced to 33% in apoA-I-deficient compared with wild-type mice (P = 0.007). These data suggest that apoM mainly associates with HDL in normal mice but also with the pathologically increased lipoprotein fraction in genetically modified mice. The substantially decreased apoM levels in apoA-I-deficient mice suggest a connection between apoM and apoA-I metabolism.     

Green synthesis of lead sulfide nanoparticles by the lead resistant marine yeast, Rhodosporidium diobovatum

Biosynthesis of nanoparticles using microorganisms has attracted a lot of attention in recent years as this route has the potential to lead to synthesis of monodisperse nanoparticles. Here, we report the intracellular synthesis of stable lead sulfide nanoparticles by a marine yeast, Rhodosporidium diobovatum. The PbS nanoparticles were characterized by UV-visible absorption spectroscopy, X-ray diffraction (XRD) and energy dispersive atomic spectroscopy (EDAX). UV-visible absorption scan revealed a peak at 320 nm, a characteristic of the nanosize range. XRD confirmed the presence of PbS nanoparticles of cubic structure. Crystallite size as determined from transmission electron microscopy was found to be in the range of 2-5 nm. Elemental analysis by EDAX revealed the presence of particles composed of lead and sulfur in a 1:2 ratio indicating that PbS nanoparticles were capped by a sulfur-rich peptide. A quantitative study of lead uptake through atomic absorption spectrometry revealed that 55% of lead in the medium was accumulated in the exponential phase, whereas a further 35% was accumulated in the stationary phase; thus, the overall recovery of PbS nanoparticles was 90%. The lead-exposed yeast displayed a marked increase (280% over the control) in nonprotein thiols in the stationary phase.     

Exposure to Silica Nanoparticles Causes Reversible Damage of the Spermatogenic Process in Mice

Environmental exposure to nanomaterials is inevitable, as nanomaterials have become part of our daily life now. In this study, we firstly investigated the effects of silica nanoparticles on the spermatogenic process according to their time course in male mice. 48 male mice were randomly divided into control group and silica nanoparticle group with 24 mice per group, with three evaluation time points (15, 35 and 60 days after the first dose) per group. Mice were exposed to the vehicle control and silica nanoparticles at a dosage of 20 mg/kg every 3 days, five times over a 13-day period, and were sacrificed at 15, 35 and 60 days after the first dose. The results showed that silica nanoparticles caused damage to the mitochondrial cristae and decreased the levels of ATP, resulting in oxidative stress in the testis by days 15 and 35; however, the damage was repaired by day 60. DNA damage and the decreases in the quantity and quality of epididymal sperm were found by days 15 and 35; but these changes were recovered by day 60. In contrast, the acrosome integrity and fertility in epididymal sperm, the numbers of spermatogonia and sperm in the testes, and the levels of three major sex hormones were not significantly affected throughout the 60-day period. The results suggest that nanoparticles can cause reversible damage to the sperms in the epididymis without affecting fertility, they are more sensitive than both spermatogonia and spermatocytes to silica nanoparticle toxicity. Considering the spermatogenesis time course, silica nanoparticles primarily influence the maturation process of sperm in the epididymis by causing oxidative stress and damage to the mitochondrial structure, resulting in energy metabolism dysfunction.     

B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.     

Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles

This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40-200 nm as determined using photon correlation spectroscopy (PCS) and 40-80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding beta-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect and Lipofectin were also used. In contrast to chitosan particles, the duration of expression for both SuperFect and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect and Lipofectin however, were observed to engender marked cytotoxicity. Poly-D,L-lactide (PLA) nanoparticles (40-80 nm) and poly-L-lactide (PLLA) lamellae (2-6 microm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.     

Toxic effects of titanium dioxide nanoparticles on microbial activity and metabolic flux

The purpose of this research is to estimate and quantify the toxicity of titanium dioxide (TiO₂) nanoparticles in microorganisms. Nano-sized particles of TiO₂ were more toxic compared to micro-sized particles. Three microorganismal species, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, were used to test TiO₂ antimicrobial effects. E. coli showed the lowest survival rate (36%), while S. cerevisiae showed the highest survival rate (71%). The antimicrobial effect of TiO₂ was also dependent on ultraviolet ray wavelength. The survival ratio of E. coli was 40% at a 254 nm wavelength and 80% at 365 nm. To observe the effect of TiO₂ on the intracellular metabolism, a metabolic flux analysis and the measurement of in vivo glucose-6-phosphate were performed. G6P concentration in cells exposed to TiO₂ increased, and glycolysis flux was also higher than the controls.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.     

Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on human cervix epithelioid carcinoma cell line (HeLa). Nickel oxide precursors were synthesized by an nickel sulphate-excess urea reaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm) were investigated by X-ray diffraction analysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in 50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy. The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for 2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiO nanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in culture on the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.     

Ligand density effect on biorecognition by PEGylated gold nanoparticles: regulated interaction of RCA120 lectin with lactose installed to the distal end of tethered PEG strands on gold surface

PEGylated gold nanoparticles (diameter: 20 nm) possessing various functionalities of lactose ligand on the distal end of tethered PEG ranging from 0 to 65% were prepared to explore the effect of ligand density of the nanoparticles on their lectin binding property. UV-visible spectra of the aqueous solution of the nanoparticles revealed that the strong steric stabilization property of the PEG layer lends the nanoparticles high dispersion stability even under the physiological salt concentration (ionic strength, I = 0.15 M). The number of PEG strands on a single particle was determined to be 520 from thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) observation under controlled acceleration voltage revealed the thickness of the PEG layer on the nanoparticle to be approximately 7 nm. The area occupied by a single lactose molecule on the surface of PEGylated gold nanoparticles was then calculated based on TGA and SEM results and was varied in the range of 10-34 nm2 depending on the lactose functionality (65 approximately 20%). PEGylated gold nanoparticles with 40% and 65% lactose functionality showed a selective and time-dependent aggregation in phosphate buffer with the addition of Ricinus communis agglutinin (RCA120) lectin, a bivalent galactose-specific protein. The aggregates can be completely redispersed by adding an excess amount of galactose. Time-lapse monitoring of UV-visible spectra at 600-750 nm revealed that the aggregation of PEGylated gold nanoparticles was accelerated with an increase in both RCA120 concentration in the solution and the lactose density of the nanoparticles. Furthermore, the sensitivity of lectin detection could be controlled by the regulation of lactose density on the particle surface. Interestingly, there was a critical lactose density (>20%) observed to induce detectable particle aggregation, indicating that the interaction between the particles is triggered by the multimolecular bridging via lectin molecules.     

NanoFerrite particle based radioimmunonanoparticles: binding affinity and in vivo pharmacokinetics

Dextran and PEG-coated iron oxide nanoparticles (NP), when suitably modified to enable conjugation with molecular targeting agents, provide opportunities to target cancer cells. Monoclonal antibodies, scFv, and peptides conjugated to 20 nm NP have been reported to target cancer for imaging and alternating magnetic field (AMF) therapy. The physical characteristics of NPs can affect their in vivo performance. Surface morphology, surface charge density, and particle size are considered important factors that determine pharmacokinetics, toxicity, and biodistribution. New NanoFerrite (NF) particles having improved specific AMF absorption rates and diameters of 30 and 100 nm were studied to evaluate the variation in their in vitro and in vivo characteristics in comparison to the previously studied 20 nm superparamagnetic iron oxide (SPIO) NP. SPIO NP 20 nm and NF NP 30 and 100 nm were conjugated to (111)In-DOTA-ChL6, a radioimmunoconjugate. Radioimmunoconjugates were conjugated to NPs using 25 microg of RIC/mg of NP by carbodiimide chemistry. The radioimmunonanoparticles (RINP) were purified and characterized by PAGE, cellulose acetate electrophoresis (CAE), live cell binding assays, and pharmacokinetics in athymic mice bearing human breast cancer (HBT 3477) xenografts. RINP (2.2 mg) were injected iv and whole body; blood and tissue data were collected at 4, 24, and 48 h. The preparations used for animal study were >90% monomeric by PAGE and CAE. The immunoreactivity of the RINP was 40-60% compared to (111)In-ChL6. Specific activities of the doses were 20-25 microCi/2.2 mg and 6-11 microg of mAb/2.2 mg of NP. Mean tumor uptakes (% ID/g +/- SD) of each SPIO 20 nm, NF 30 nm, and 100 nm RINP at 48 h were 9.00 +/- 0.8 (20 nm), 3.0 +/- 0.3 (30 nm), and 4.5 +/- 0.8 (100 nm), respectively; the ranges of tissue uptakes were liver (16-32 +/- 1-8), kidney (7.0-15 +/- 1), spleen (8-17 +/- 3-8), lymph nodes 5-6 +/- 1-2), and lung (2.0-4 +/- 0.1-2). In conclusion, this study demonstrated that 100 nm NF NP could be conjugated to (111)In-mAb so that the resulting RINP had characteristics suitable for AMF therapy. Although 100 nm RINP targeted tumor less than 20 nm SPIO RINP, their heating capacity is typically 6 times greater, suggesting the 100 nm NF RINP could still deliver better therapy with AMF.     

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.