Augmentation of C17.2 Neural Stem Cell Differentiation via Uptake of Low Concentrations of ssDNA-Wrapped Single-Walled Carbon Nanotubes.

Nanostructured biomaterials are extensively explored in clinical imaging and in gene/drug delivery applications. However, limited studies are performed that examine the influence that nanomaterials may have on cell behavior over long time scales at nonlethal concentrations. This study is designed to investigate whether carbon nanotubes are able to augment cell behavior at low concentrations. Single-walled carbon nanotubes are introduced to neural stem cells at different stages of differentiation at concentrations as low as 5 ng mL-1 . Results demonstrate that in this particular cell model, nanotube uptake is mediated by endocytosis. Differentiation is augmented, especially when nanotubes are introduced to cells in an actively dividing state. Significant increases in neuronal cell population are observed over the control specimens. While the mechanisms behind this observation are yet unknown, this study demonstrates that low concentrations of internalized nanomaterials can significantly alter the differentiation profile of a stem cell line.

Highly Stabilized Core-Satellite Gold Nanoassemblies in Vivo: DNA-Directed Self-Assembly, PEG Modification and Cell Imaging.

Au nanoparticles (NPs) have important applications in bioimaging, clinical diagnosis and even therapy due to its water-solubility, easy modification and drug-loaded capability, however, easy aggregation of Au NPs in normal saline and serum greatly limits its applications. In this work, highly stabilized core-satellite Au nanoassemblies (CSAuNAs) were constructed by a hierarchical DNA-directed self-assembly strategy, in which satellite Au NPs number could be effectively tuned through varying the ratios of core-AuNPs-ssDNA and satellite-AuNPs-ssDNAc. It was especially interesting that PEG-functionalized CSAuNAs (PEG-CSAuNAs) could not only bear saline solution but also resist the enzymatic degradation in fetal calf serum. Moreover, cell targeting and imaging indicated that the PEG-CSAuNAs had promising biotargeting and bioimaging capability. Finally, fluorescence imaging in vivo revealed that PEG-CSAuNAs modified with N-acetylation chitosan (CSNA) could be selectively accumulate in the kidneys with satisfactory renal retention capability. Therefore, the highly stabilized PEG-CSAuNAs open a new avenue for its applications in vivo.

The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates.

The sequence-dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR-A) and caveolae-mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR-A, and it has been proposed that interaction of poly G with SR-A is dependent on the formation of G-quadruplexes. Since G-rich oligonucleotides are known to interact strongly with SR-A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug-SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Steered molecular dynamics simulation study on dynamic self-assembly of single-stranded DNA with double-walled carbon nanotube and graphene.

In the present work, we explored the diameter selectivity of dynamic self-assembly for the single-strand DNA (ssDNA) encapsulation in double-walled nanotubes (DWNTs) via molecular dynamics simulation method. Moreover, the pulling out process was carried out by steered molecular dynamics simulations. Considering π-π stacking and solvent accessibility together, base-CNT binding should be strongest on a graphene sheet and weakest on the inner CNT surface. When pulling the ssDNA out of the single-walled carbon nanotube (SWNT), the force exhibits characteristic fluctuations around a plateau about 300 pN. Each fluctuation force pulse to pull ssDNA corresponds to the exit of one base. In addition, the solvents used for the system are also of significant interest. Water does play an important role in encapsulation process but doesn't in the pulling out process.

DNA nanotubes as combinatorial vehicles for cellular delivery.

This work explores using self-assembled DNA nanostructures as carriers for drug delivery. We have recently developed an organic nanotube system that is assembled from a single component: a 52-base-long DNA single strand. In this work, functional agents (folate as a cancer cell target agent and Cy3 as a fluorescence imaging agent) are conjugated with the DNA strands; the conjugates self-assemble into micrometers-long nanotubes (NTs). The conjugated DNA-NTs can be effectively taken by cancer cells as demonstrated by fluorescence imaging and fluorescence-activated cell sorting. No obvious toxicity has been observed under current experimental conditions.

Kinetic limitations of cooperativity-based drug delivery systems.

We study theoretically a novel drug delivery system that utilizes the overexpression of certain proteins in cancerous cells for cell-specific chemotherapy. The system consists of dendrimers conjugated with "keys" (ex: folic acid) which "key-lock" bind to particular cell-membrane proteins (ex: folate receptor). The increased concentration of "locks" on the surface leads to a longer residence time for the dendrimer and greater incorporation into the cell. Cooperative binding of the nanocomplexes leads to an enhancement of cell specificity. However, both our theory and detailed analysis of in vitro experiments indicate that the degree of cooperativity is kinetically limited. We demonstrate that cooperativity and hence the specificity to particular cell type can be increased by making the strength of individual bonds weaker, and suggest a particular implementation of this idea.

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase.

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.

Surfactant-DNA gel particles: formation and release characteristics.

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report on the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant cetyltrimetrylammonium bromide (CTAB). By using CTAB, the formation of DNA reservoir gel particles, without adding any kind of cross-linker or organic solvent, has been demonstrated. Particles have been characterized with respect to the degree of DNA entrapment, surface morphology, and secondary structure of DNA in the particles. The swelling/deswelling behavior and the DNA release have been investigated in response to salt additions. Analysis of the data has suggested a higher degree of interaction between ssDNA and the cationic surfactant, confirming the stronger amphiphilic character of the denatured DNA. Fluorescence microscopy studies have suggested that the formation of these particles is associated with a conservation of the secondary structure of DNA.

Scleral permeability of a small, single-stranded oligonucleotide.

Developing more effective ocular drug delivery systems is essential to improving the treatment of posterior segment eye disease. The large target area provided by the sclera and potentially less vision threatening complications are advantages of transscleral administration compared to more traditional modalities of drug delivery to the posterior segment. We aimed to determine the permeability coefficient for the in vitro diffusion of a small, single-stranded, oligonucleotide across human sclera. Transscleral permeability was measured by placing 100 microL of 2.96 x 10(-4) mol single-stranded, fluorescein-labeled oligonucleotide (MW = 7998.3) on the episcleral surface of sclera mounted in a perfusion chamber. Fractions of choroidal perfusate were collected hourly for 24 hours. The permeability constant or K(trans) for the transscleral diffusion of the naked, single-stranded, fluorescein-labeled oligonucleotide was 7.67 +/- 1.8 x 10(-7) cm/s (mean +/- SEM, N = 7). The permeability constant or K(trans) after intrascleral injection of the same fluorescein-labeled oligonucleotide was 1.32 +/- 0.42 x 10(-7) (mean +/- SEM, N = 4). This analysis demonstrates that diffusion of a naked, 24-base, single-stranded, fluorescein-labeled oligonucleotide can be accomplished by both of the described methods. The ability to deliver single-stranded oligonucleotides across the sclera may prove to be advantageous given the development of several novel therapeutic strategies that use similar molecules.

Cellular uptake and metabolism of DNA frayed wires.

DNA frayed wires are a novel, multistranded form of DNA that arises from interactions between single-stranded oligodeoxyribonucleotides with the general sequence d(N(x)G(y)) or d(G(y)N(x)), where y > 10 and x > 5. Frayed wires exhibit greater stability with respect to thermal and chemical denaturation than single- or double-stranded DNA molecules and, thus, may have potential usefulness for DNA drug delivery. However, the stability and uptake of frayed wires have not been investigated in biological systems. Our objective was to examine the cellular uptake and stability of frayed wires in cultured hepatic cells. In these studies, the parent oligonucleotide d(A(15)G(15)) was used to form DNA frayed wires (DNA(FW)) while a random 30-mer oligonucleotide was used as the control nonaggregated DNA (DNA(SS)). Uptake and metabolism studies of DNA(FW) were performed in cultured human hepatoma, HepG2 cells and compared to DNA(SS). Our results indicate that DNA(FW) are not cytotoxic and that their intracellular uptake in HepG2 cells is 2-3.5-fold greater than that of DNA(SS) within the first 2 h (p < 0.05). Similarly, nuclear localization of DNA(FW) is 10-13-fold higher than that of DNA(SS) (p < 0.05). As both internalized and extracellular DNA(FW) appear to be more stable in vitro than DNA(SS), the enhanced uptake may be due to either increased stability or enhanced intracellular transport. These studies also indicate that uptake of DNA(FW) likely occurs via active processes such as receptor-mediated endocytosis similar to mechanisms which have been proposed for DNA(SS). The internalization pathways of DNA(FW) may differ somewhat from that of DNA(SS) insofar as chloroquine does not appear to alter DNA(FW) uptake and degradation, as is the case with DNA(SS).

Toward third-generation aptamers: Spiegelmers and their therapeutic prospects.

Single-stranded mirror-image oligonucleotides, which are highly resistant to nuclease degradation and are capable of tightly and specifically binding to protein targets to inhibit their function, have been developed as potential therapeutic agents. The scientific discoveries that led to the development of the Spiegelmer technology are described in this review, along with recent preclinical developments of the first therapeutic Spiegelmers.

Cellular uptake, distribution, and stability of 10-23 deoxyribozymes.

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.

Solid-phase genetic engineering with DNA immobilized on a gold surface.

A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.

Anti-L-selectin aptamers: binding characteristics, pharmacokinetic parameters, and activity against an intravascular target in vivo.

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.

Pharmacokinetics of 99mTc-labeled oligonucleotides.

Oligonucleotides labeled with gamma-emitting radionuclides are likely to find eventual applications as radiopharmaceuticals. Accordingly, methods of radiolabeling single-stranded DNA by chelation with the gamma-emitting radionuclide 111In and, more importantly, with 99mTc, have been developed. As an emerging technology, the results of only two pharmacokinetic investigations with 99mTc-labeled DNA have been reported thus far, both from this laboratory. This review focuses on the pharmacokinetic properties in mice of 99mTc when radiolabeled by one method (SHNH) to a 22-base native phosphodiester and phosphorothioate DNA. The labeled phosphodiester DNA displayed an affinity for proteins through its 99mTc-SHNH chelate. The affinity for proteins of the labeled phosphorothioate DNA was even greater and was attributed in this case to both the chelate and to the modified, and lipophilic, DNA backbone. As a consequence of this binding, and the recognized increased stability of the phosphorothioate DNA towards nucleases, probably explained the long-term retention of label in organs such as liver, spleen and kidney. In conclusion, under the conditions of these investigations, the labeled phosphodiester and phosphorothioate DNAs studied were both judged to be unsuitable for most applications as radiopharmaceuticals.

Comparative properties of a technetium-99m-labeled single-stranded natural DNA and a phosphorothioate derivative in vitro and in mice.

Oligonucleotides, particularly single stranded, may ultimately be of considerable use as radiopharmaceuticals. We have compared a synthetic 22-base single-stranded phosphodiester DNA with its phosphorothioate analog after both were radiolabeled with 99mTc via the hydrazino nicotinamide chelator. Whole body clearance of the label in mice was much slower when introduced on the phosphorothioate (30% vs. 75% clearance at 6 hr) because of immediate and persistent accumulation in liver (47% vs. 2% injected dose/g at 4 hr). The label in both cases was present in urine primarily on low molecular weight catabolites. High-performance liquid chromatography analysis of 37 degrees C serum incubates showed serum protein binding of 99mTc in both cases (about 100% bound at 24 hr) but to different proteins. Different behavior with respect to protein binding was also observed in the analysis of liver and kidney homogenates: the phosphodiester label was almost quantitatively converted to lower molecular weight catabolites after only 15 min, whereas the phosphorothioate label was primarily on proteins. The rapid digestion of the phosphodiester by nucleases was not observed, probably because protein binding of the labeled oligonucleotides stabilized against degradation. Thus the phosphodiester DNA may be the preferred 99mTc-labeled oligonucleotide in certain circumstances to avoid the high and persistent liver uptake observed with the phosphorothioate DNA.

Hairpin, dumbbell, and single-stranded phosphodiester oligonucleotides exhibit identical uptake in T lymphocyte cell lines.

The cellular binding, uptake, and intracellular distribution of structured double-stranded phosphodiester oligonucleotides (decoys) have been examined in T lymphocytes using fluorescein-labeled molecules. Intracellular localization of hairpin and dumbbell decoys was similar to that of single-stranded oligonucleotides. At short incubation times, oligonucleotides were localized only in cytoplasmic vesicles, whereas at longer times, they were also found in the nucleus. Cellular uptake was dependent on temperature, time, and extracellular concentration. Oligonucleotide efflux was similar for all types of molecules and was very rapid (t1/2 = 10-15 minutes). These results imply that phosphodiester double-stranded oligonucleotides can enter cellular compartments of interest for their potential biologic function and, thus, provide a potential tool to control gene transcription.

Protein labelling via deoxyribonucleic acid hybridization.

A novel method of radiolabelling antibodies and other proteins is described. A small single-stranded DNA was covalently conjugated to an antibody and labelled by hybridization following the addition of the complementary single-stranded DNA labelled with technetium-99m (99Tc(m)) or indium-111 (111In). Antibody labelling efficiencies were 100% in about 1 h at room temperature with specific activities of up to 30 microCi micrograms-1 of IgG for 99Tc(m). Both diester and thioate DNAs were used. Both the diester- and thioate-labelled antibodies showed complete label stability in 37 degrees C saline. After 24 h in 37 degrees C serum, however, about 40% of the label in the case of the diester antibody was on low molecular weight species--probably labelled catabolites from nuclease degradation of the phosphodiester DNA. In contrast, the 99Tc(m) label on the thioate antibody was immediately and quantitatively bound to serum proteins--probably due to non-specific binding through the sulphur groups. Biodistribution studies in normal mice reflect these in vitro observations: 99Tc(m) on the diester antibody was rapidly cleared through the kidneys, probably as low molecular weight catabolite, while on the thioate antibody, the 99Tc(m) label was predominately deposited in the liver. In conclusion, by modifying with a single-stranded DNA, proteins may be readily labelled with a variety of radionuclides by DNA hybridization. The properties of the radiolabel are strongly influenced by the nature of the DNA.