Translating Nanomedicine to Comparative Oncology-the Case for Combining Zinc Oxide Nanomaterials with Nucleic Acid Therapeutic and Protein Delivery for Treating Metastatic Cancer.

The unique anticancer, biochemical, and immunologic properties of nanomaterials are becoming a new tool in biomedical research. Their translation into the clinic promises a new wave of targeted therapies. One nanomaterial of particular interest are zinc oxide (ZnO) nanoparticles (NPs), which has distinct mechanisms of anticancer activity including unique surface, induction of reactive oxygen species, lipid oxidation, pH, and also ionic gradients within cancer cells and the tumor microenvironment. It is recognized that ZnO NPs can serve as a direct enzyme inhibitor. Significantly, ZnO NPs inhibit extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) associated with melanoma progression, drug resistance, and metastasis. Indeed, direct intratumoral injection of ZnO NPs or a complex of ZnO with RNA significantly suppresses ERK and AKT phosphorylation. These data suggest ZnO NPs and their complexes or conjugates with nucleic acid therapeutic or anticancer protein may represent a potential new strategy for the treatment of metastatic melanoma, and potentially other cancers. This review focuses on the anticancer mechanisms of ZnO NPs and what is currently known about its biochemical effects on melanoma, biologic activity, and pharmacokinetics in rodents and its potential for translation into large animal, spontaneously developing models of melanoma and other cancers, which represent models of comparative oncology.

Effects of nanomaterials on luciferase with significant protection and increased enzyme activity observed for zinc oxide nanomaterials.

This principle goal of this research was to examine the effects of various nanomaterials on the activity and behavior of the firefly enzyme luciferase. Nanomaterials have been found to stabilize, and in some instances, shown to increase the activity of enzymes. In this study gold, manganese oxide (MnO), and zinc oxide (ZnO) nanomaterials were utilized in order to test their effects on enzyme activity. Luciferase was used because its activity is easy to analyze, as it typically produces a large amount of bioluminescence easily detected by a Microtiter plate reader. Following incubation with the various nanomaterials, luciferase was subjected to degradation by several protein denaturing agents, such as heat, SDS, urea, ethanol, protease, hydrogen peroxide, and pH changes. Results indicated that luciferase activity is indeed affected when combined with nanomaterials, accompanied by both increases and decreases in enzyme activity depending on the type of nanomaterial and denaturing agent used. In most of the experiments, when incubated with ZnO nanomaterials, luciferase depicted significant increases in activity and bioluminescence. Additional experiments, in which human A375 cells were treated with luciferase-nanomaterial mixtures, also depicted increased enzyme activity and bioluminescence for luciferase incubated with ZnO nanomaterials. Ultimately, our findings indicated that when luciferase was subjected to multiple types of denaturation, zinc oxide nanomaterials dramatically preserved and increased enzyme activity and bioluminescence.

Interaction of MnO and ZnO nanomaterials with biomedically important proteins and cells.

Zinc and manganese nanomaterials may have potential for biomedical nanotechnology. Here first generation Zn and Mn oxide nanomaterials were prepared as determined by XRD. Transmission electron microscopy confirmed their nanoscale in two dimensions and revealed a rod or belt-like morphology for MnO or ZnO respectively. Association of MnO and ZnO to three model biomedically important proteins (albumin, protamine and thrombin) has been characterized by ultra-violet and dynamic laser light spectroscopy, UVS and DLLS respectively. UVS demonstrated a concentration-dependent loss of protein from the supernatant upon sedimentation of MnO or ZnO. Shifts in the surface charge of the MnO or ZnO by DLLS confirmed the protein's adsorption to the surface. MnO and ZnO were incubated with live human cells in culture (HeLa, A375 or 1321N1). A marked difference was observed for the two nanomaterials behavior in cell culture where the MnO could be discerned associating at the cell surface whereas the ZnO caused the cells to exhibit a rounded up morphology. Trypan blue dye exclusion studies demonstrated cytotoxicity of the ZnO at high concentrations 62.5-31.5 microg/mL whereas surprisingly the MnO demonstrated no cytotoxicity at any of the concentrations tested.

Challenges and potential for RNA nanoparticles (RNPs).

The current research of our group focuses on delivery of oligonucleotides and gene vaccines targeted against infectious disease and cancer. Recently we reported a strategy for associating DNA to gold via protamine delivery enhancing material which here we suggest might be applicable to RNA and to other nanoparticles. An important new modality for such RNA based nanoparticles (RNPs) is the myriad of genes now known to undergo alternative splicing. Here we will review some important issues for the binding, stabilization and delivery of RNA, particularly splice-site switching oligomers (SSOs) via these RNPs in order to achieve selective molecular therapeutic effects and unlock their potential as chemotherapeutic agents.

R4 peptide-pDNA nanoparticle coated HepB vaccine microparticles: sedimentation, partitioning, and spray freeze dry bioprocesses.

Broad therapeutic application of nucleic acid micro- and nanoparticles will require bioprocesses capable of achieving high loads of structurally intact and functionality active DNA. Here we report condensation of pDNA into nanoparticles by sedimentation through R4 peptide and partitioning at a hydrophobic interface. > or = 90% coating efficiency onto microparticles is achieved via this combined bioprocess with the pDNA retaining 85-90% intact supercoil after bioprocessing. SEM analyses of the microparticles produced therefrom reveals bound pDNA and R4 peptide nanoparticles. HPLC and chemical analyses afford quantification of the particle-associated pDNA and R4 peptide along with lactose, raffinose, or trehalose carbohydrate stabilizer, surface coatings uniformly applied by spray freeze-drying. Administration of these particles by gene gun demonstrates delivery to the nucleus of expressive nanoparticles and into rodents and pigs pronounced immunogenicity even after bioprocessing and accelerated degradation. These data support the discovery of a robust bioprocess platform for preparing macromolecule bound bioparticles with potential relevance beyond simple preparation of bioactive DNA vaccine.

pDNA bioparticles: comparative heterogeneity, surface, binding, and activity analyses.

New applications for nucleic acid-bound micro/nanoparticles are emerging in drug delivery, biocatalysis, diagnostics, and toxicology. Bioactivity of viral or liposomal based technologies is limited by heterogeneity, partitioning, aggregation, and protein binding in physiological fluids, underlying immunotoxicity, and poor in vitro and cell-culture corollaries. Here we have systematically investigated novel pDNA bioparticles formed through complexation to model non-viral/non-lipid materials, peptides, aminoglycans, and small molecules (polybrene, chitosan, butirosin, protamine, Lys10, RGDS, bupivacaine, and chlorpromazine). On the basis of characterization by heterogeneity, kinetics, partitioning in physiological fluid and serum protein-binding, surface, size and electrophoretic behavior, transfection, and immunotoxicity, notably protamine, and chitosan DNA particles gave a long lifetime (12-18h), low protein-binding (<10microg/ml), good transfection activity (10(2)-10(4)RLU/mg cell protein), and low immunotoxicity. Our results support further evaluation of these materials as potential alternatives to viral or liposomal approaches, in combination with pDNA as binding, expression or therapeutic agents.

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.

Comparative pharmacokinetics, tissue distribution, and tumor accumulation of phosphorothioate, phosphorodithioate, and methylphosphonate oligonucleotides in nude mice.

The goals of this study were to systematically compare the pharmacokinetics and tissue distribution of phosphorothioate (PS), methylphosphonate (MP), and phosphorodithioate (PS2) oligonucleotide analogs; 15-mers of sequence d-TAC GCC AAC AGC TCC (5'-3') complementary to the AUG region of K-ras were radiolabeled with carbon-14. Oligomers were administered as a single dose in the tail vein of nude mice harboring a K-ras-dependent human pancreatic tumor (CFPAC1). The kinetics of PS, PS2, and MP oligomer availability in the bloodstream was followed. Concentration versus time profiles for all oligomers were biphasic, indicative of a two-compartment model. A rapid distribution phase with t1/2 alpha values of 1 minute or less and an elimination phase with average t1/2 beta values of 24-35 minutes were observed. Volumes of distribution (Vd) were 3.2, 4.8, and 6.3 ml for PS2, MP, and PS, respectively, in comparison to 3.6 ml for sucrose, a fluid-phase marker. Relative tissue drug levels obtained at 1 and 24 hours after administration were kidney > liver > spleen > tumor > muscle. Total kidney and liver oligonucleotide accumulation was approximately 7%-15% of the initial dose, with tumor accumulating 2%-3%. Intact compound was recovered from all tissues, including tumor, as assessed by high-pressure reversed-phase HPLC coupled to radiometric detection. Integrity of the oligonucleotides ranged from 73% in blood to 43%-46% in kidney and liver. Kidney and liver appear to be the primary sites of metabolism. These results demonstrate widespread tissue availability of these compounds and suggest their development as potential antitumor agents.

Hepatic distribution and clearance of antisense oligonucleotides in the isolated perfused rat liver.

PURPOSE: This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. METHODS: The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5'-TAC-GCC-AAC-AGC-TCC-3'), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr: perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. RESULTS: MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [CI = 1.05 +/- 0.21 mL/min; extraction ratio = 0.06 +/- 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 +/- 2.2%) than for the MP oligonucleotide (53.0 +/- 4.7%). CONCLUSIONS: Backbone modifications significantly-influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.

Interactions between single-stranded DNA binding protein and oligonucleotide analogs with different backbone chemistries.

Chemical modification of backbone structures has been an important strategy in designing oligonucleotides capable of improved antisense effects. However, altered backbone chemistry may also affect the binding of oligonucleotides to key cellular proteins, and thus may impact on the overall biological action of antisense agents. In this study we have examined the binding of oligonucleotides having four different backbone chemistries to single-strand binding protein (SSB), a protein having a key role in DNA repair and replication. The oligomers tested had the same sequence, while the internucleoside linkages were phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), or methylphosphonate (MP). We found that both PS and PS2 oligomers bound to SSB with higher affinity than PO oligonucleotides, while MP oligonucleotides did not bind appreciably at the concentrations tested. Oligonucleotide length was also an important factor in binding to SSB, but sequence was less critical. These observations indicate that backbone chemistry is an important factor in interactions between oligonucleotides and critical cellular proteins, and thus may be a key determinant of the biological effects of antisense oligonucleotides.

Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.

Inhibition of human collagenase activity by antisense oligonucleoside methylphosphonates.

Oligodeoxyribonucleoside methylphosphonates (d-OMP) were synthesized whose sequences are complementary to sequences found in the mRNA coding for the 72-kDa (MMP-2) or 92-kDA (MMP-9) forms of human collagenase i.v., matrix metalloproteinases (MMP) whose excessive secretion correlates with the metastatic potential of tumor cells. The effects of these oligomers on MMP-2 and MMP-9 activities secreted by HT1080 cells, a human fibrosarcoma cell line, were studied using a gelatin zymography assay. A d-OMP, M2.3, complementary to nucleotides 14 to 28 of the initiation codon region of MMP-2 mRNA selectively inhibited MMP-2 activity, whereas a d-OMP, M9.1, which was targeted to nucleotides -19 to -5 of the 5'-untranslated region of MMP-9 mRNA selectively inhibited MMP-9 activity over the concentration range 5-50 microM. At 100 microM concentration, both M2.3 and M9.1 inhibited the activities of both MMP-2 and MMP-9. These oligomers were completely stable under cell culture conditions and did not appear to adversely affect cell growth after 48 hours at concentrations up to 100 microM, although 100 microM M9.1 did reduce cell growth 30% after prolonged, 120-hours exposure. Other d-OMP tested either had no effect on collagenase activity or inhibited both MMP-2 and MMP-9 activities. The latter oligomer was complementary to MMP-2 mRNA and partially complementary to MMP-9 mRNA. Oligomer M2.3 was also tested for its effects on the morphology of malignant human lung cells, BZR-T33, growing on the surface of reconstituted base membrane, Matrigel, in culture. In absence of oligomer, the BZR-T33 cells formed extensive networks indicative of the ability of the cells to invade the Matrigel substrate. In the presence of 100 microM M2.3, BZR-T33 formed colonies of rounded cells, a morphology typical of noninvasive cells. Other non-complementary d-OMP had no effect on the morphology of BZR-T33 under these conditions. These results suggest that antisense d-OMP may be useful for inhibiting expression of collagenase in human tumor cells and for studying the role of collagenase expression in tumor cell metastasis.