Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context.     

Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation.

Most molecules that are not actively imported by living cells are impermeable to cell membranes, including practically all macromolecules and even many small molecules whose physicochemical properties prevent passive membrane diffusion. The use of peptide vectors capable of transporting such molecules into cells in the form of covalent conjugates has become an increasingly attractive solution to this problem. Not only has this technology permitted the study of modulating intracellular target proteins, but it has also gained importance as an alternative to conventional cellular transfection with oligonucleotides. Peptide vectors derived from viral, bacterial, insect, and mammalian proteins endowed with membrane translocation properties have now been proposed as delivery vectors. These are discussed comprehensively and critically in terms of relative utility, applications to compound classes and specific molecules, and relevant conjugation chemistry. Although in most cases the mechanisms of membrane translocation are still unclear, physicochemical studies have been carried out with a number of peptide delivery vectors. Unifying and distinguishing mechanistic features of the various vectors are discussed. Until a few years ago speculations that it might be possible to deliver peptides, proteins, oligonucleotides, and impermeable small molecules with the aid of cellular delivery peptides not only to target cells in vitro, but in vivo, was received with scepticism. However, the first studies showing pharmacological applications of conjugates between macromolecules and peptide delivery vectors are now being reported, and therapies based on such conjugates are beginning to appear feasible.     

A protein assay based on colloidal gold conjugates with trypsin

The standard sol particle immunoassay (SPIA) is based on a biospecific aggregation of gold nanoparticle conjugates, followed by conventional spectrophotometry. Here we propose a novel SPIA format that uses microtitration immunological plates and an enzyme-linked immunosorbent assay reader. The novel and standard assays are exemplified by determination of immunoglobulin G by using 15-nm colloidal gold-protein A conjugates. We also describe a novel sol particle-trypsin assay using conjugates of gold nanoparticles with trypsin. The method is based on measuring spectral extinction changes caused by the addition of protein to a conjugate solution. The changes in the extinction spectra are presumed to be related to aggregation of gold nanoparticles caused by polyvalent binding of protein molecules to the trypsin molecules of the conjugates.     

Application of 2-amino-6-vinylpurine as an efficient agent for conjugation of oligonucleotides

Attempts have been made to conjugate a variety of molecules with oligonucleotides to achieve useful functions. In this study, we have established a new efficient method for post-synthetic conjugation of oligonucleotides with the use of the 2-amino-6-vinylpurine nucleoside. Amino nucleophiles form the corresponding conjugates under acidic conditions, whereas thiol nucleophiles reacted efficiently under alkaline conditions. Thus, glutathione and HS-Cys-(Arg)8 without protecting groups were efficiently conjugated to the 2-amino-6-vinylpurine-bearing ODN under alkaline conditions. The use of 2-amino-6-vinylpurine as an agent for conjugation is advantageous in that it is stable during the reaction and may be applied to conjugation of ODNs with multiple functional molecules.     

Use of a new violet-excitable AmCyan variant as a label in cell analysis

Here we report a new variant of AmCyan fluorescent protein that has been specifically designed for multicolor cell analysis. AmCyan is one of the existing violet fluorochromes for use in flow cytometers equipped with a violet (405 nm) laser. It is also widely used as a label in fluorescent spectroscopy. Limitations on its use are due to the significant AmCyan fluorescence spillover into the FITC detector, due to excitation of AmCyan by the blue (488 nm) laser. In order to resolve this problem, we modified the excitation profile of AmCyan. The new fluorescent protein that we developed, AmCyan100, has an emission profile similar to AmCyan with an emission maximum at 500 nm, but its excitation maximum is shifted to 395 nm, which coincides more closely with the violet laser line and decreases the excitation with the blue laser, thus reducing the spillover observed with the original AmCyan. Moreover, this new protein has a Stokes shift of more than 100 nm compared to the Stokes shift of 31 nm in its precursor. Our data also suggests that AmCyan100-mAb conjugates have brightness similar to AmCyan-mAb conjugates. In summary, AmCyan100 conjugates have minimum spillover into the FITC detector, and can potentially replace existing AmCyan conjugates in multicolor flow cytometry without any changes in instrumental setup and existing reagent panel design.     

Nuclease conjugates with ligand-free serum albumin]

In order to obtain nuclease and human serum albumin (HSA) conjugates with a high enzyme content it is proposed to use a ligand-free HSA. The ligands are removed with the help of a strong anion exchanger. A two-stage procedure of conjugate preparation is proposed. It consists in the complexation of ligand-free HSA and enzyme and subsequent co-condensation of protein molecules of the poly-complex with the aid of glutaric aldehyde. When the conjugates are administered to rabbits intravenously, the RNAase activity is manifested in blood for 3-5 days. Moreover, in the case of conjugates with a molecular weight of 80 kDa, the prolongation time is greater than for conjugate with a higher molecular weight.     

Specific killing of lymphocytes that cause experimental autoimmune myasthenia gravis by ricin toxin-acetylcholine receptor conjugates

Experimental autoimmune myasthenia gravis (EAMG) is an autoimmune disease in which antibodies to acetylcholine receptor (AChR) cause loss of AChR from muscle, thereby impairing neuromuscular transmission. Here we report the use of a hybrid molecule that contains ricin toxin, irreversibly coupled to AChR to specifically suppress the immune response to AChR in vitro. Lymph node cell cultures from rats with EAMG pretreated with ricin toxin-AChR conjugates exhibited suppressed T helper cell proliferation and B cell antibody synthesis in response to the subsequent addition of AChR. Nonspecific toxicity of the conjugates was measured by suppression of the T cell proliferative response to the mitogen concanavalin A and the antigen keyhole limpet hemocyanin (KLH), and B cell antibody production to KLH. We have evaluated different pretreatment conditions and ricin toxin covalently coupled to AChR in different molar ratios to optimize specific immunosuppression. By varying the number of ricin molecules covalently bound to AChR in the immunotoxin, we were able to minimize the nonspecific toxicity while still maintaining specific killing of AChR-reactive lymphocytes. Furthermore, B cells were more susceptible to specific killing than were the T cells. The specific immunosuppression was potentiated by performing the pretreatment with immunotoxin in the presence of chloroquine. Chloroquine raises lysosomal pH and probably delays the degradation of immunotoxin in the cell. It should be noted that ricin toxin was covalently coupled to AChR by using a novel, non-reducible reaction. These in vitro results suggest that it may be feasible to use immunotoxin molecules to specifically suppress this autoimmune response in vivo.     

Synthesis of 5'-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes

A hydrazone-based method for conjugating synthetic nucleic acids and reporter molecules for use as nonradioactive hybridization probes is presented. Oligonucleotides complementary to the hepatitis B virus were derivatized at their 5' ends with hydrazine or homobifunctional acyl hydrazides. These derivatives reacted facilely with aldehydes to give hydrazones, which were characterized by uv spectroscopy and HPLC. Coupling of aldehyde-modified alkaline phosphatase with carbohydrazide-oligonucleotide derivatives provided a mixture of two enzyme-nucleic acid conjugates in 80-85% yield. The conjugates had a 1:1 and a 2:1 oligonucleotide/enzyme ratio, respectively, and were separated by ion-exchange chromatography. Both conjugates were able to detect 7 amol of target DNA in 1 h, using a colorimetric assay. In contrast, oligonucleotide-horseradish peroxidase conjugates were 40-fold lower in sensitivity of detection.     

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.     

Highly parallel translation of DNA sequences into small molecules

A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 10(10) to 10(15) distinct molecules for the discovery of nanomolar-affinity ligands to proteins. Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands. Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons. Creating a collection of 10(10) to 10(15) small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments.     

Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.     

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.     

Inhibiting prolyl isomerase activity by hybrid organic–inorganic molecules containing rhodium(II) fragments

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site.     

Triple helix-forming oligonucleotides conjugated to new inhibitors of topoisomerase II: synthesis and binding properties

Triplex-forming oligonucleotides (TFOs) are among the most specific DNA ligands and represent an important tool for specific regulation of gene expression. TFOs have also been used to target DNA-modifying molecules to obtain irreversible modifications on a specific site of the genome. A number of molecules have been recognized to target topoisomerase II and stabilize double-stranded cleavage mediated by this enzyme thus determining permanent DNA damage. Among these poisons, etoposide (VP16), a 4'-demethylepipodophyllotoxin derivative, is widely used in cancer chemotherapy. In the aim to design DNA site-specific molecules, three analogues of VP16 (1, 2, and 3), recently described (Duca et al. J. Med. Chem. 2005, 48, 596-603), were attached to TFOs, together with a fourth one, of which the synthesis is reported here. Two different oligonucleotides, differing by the length (a 16-mer and a 20-mer), and two different linker arms between the oligonucleotide and the drug were used. The coupling reaction between the drug and the TFO was further improved. For the first time, we also report the synthesis of TFO conjugates bearing two molecules of inhibitor linked to the same oligonucleotide end. In total, 16 new conjugates were synthesized and evaluated for their ability to form triple helices. The loss in triplex stability due to the conjugation of the TFO to compounds that do not interact with DNA is compensated by the presence of the ethylene glycol linker arm. This stabilization effect is more pronounced at the 3' end than at the 5' end. All conjugates form a stable triplex selectively on the DNA target at 37 degrees C and pH 7.2.     

Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.     

Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies

We identify two tissue-specific molecules in the sea urchin embryo by an immunofluorescence technique capable of co-localizing monoclonal antibodies on the same tissue section. The technique uses monovalent Fab-fluorochrome conjugates as secondary reagents to avoid cross-talk of subsequent antibody probes. Using this technique, we show that two cell surface molecules are expressed by different cell populations in the embryo. The technique is generally applicable for antibodies regardless of species or subtype specificity, and uses commercially available reagents. The technique provides sufficient amplification and resolution for analytical work, yet is rapid enough for screening procedures. As a fluorescent counterstain, use of the dye 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) in the protocol provides a distinct fluorescent background staining of the tissue without interference with the specific antibody staining.     

Veratridine-Stimulated Release of Amine Conjugates from Centrifugal Fibers in the Limulus Peripheral Visual System

Centrifugal fibers that originate in the brain and project to the Limulus peripheral visual system synthesize and store octopamine and conjugates of octopamine and tyramine. In a previous study we showed that depolarization, induced by elevating extracellular K+, stimulated a preferential release of octopamine from these fibers. Here we show that veratridine-induced depolarization stimulates a rapid, transient release of octopamine and a delayed, sustained release of amine conjugates. Veratridine-stimulated release of both octopamine and amine conjugates depends on the influx of extracellular Ca2+ and is blocked by tetrodotoxin or the absence of extracellular Na+. The depolarization-stimulated release of amine conjugates raises the possibility that these molecules serve as intercellular messengers in the Limulus peripheral visual system.     

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.     

Lysine-spermine conjugates: hydrophobic polyamine amides as potent lipopolysaccharide sequestrants

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a focused library of lysine-spermine conjugates with lipopolysaccharide (LPS) have been characterized. Lysine-spermine conjugates with the epsilon-amino terminus of the lysinyl moiety derivatized with long-chain aliphatic hydrophobic substituents in acyl or alkyl linkage bind and neutralize bacterial lipopolysaccharides, and may be of use in the prevention or treatment of endotoxic shock states.     

Rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling

Increasing interest in the use of radiolabeled antibodies for cancer imaging and therapy drives the need for more efficient production of the antibody conjugates. Here, we illustrate a method for rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling. We apply this technique to the production of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated antibodies at the milligram and gram production scale and achieve radiolabeling efficiencies >95% using In-111. Using vacuum diafiltration, antibody-chelate conjugation and purification can be accomplished within the same vessel, and the entire process can be completed in <24 h. Vacuum diafiltration also offers safer and gentler processing conditions by eliminating the need to keep the retentate vessel under positive pressure through applied gas pressure or shear-inducing restriction points in the retentate flow path. Experimental data and mathematical model calculations suggest there exists a weak binding affinity (approximately 10(4)M(-1)) between the charged chelate molecules (e.g., DOTA) and the antibodies that slows the removal of excess chelate during purification. By analyzing the radiolabeling efficiency as a function of the number of diavolumes, we demonstrate the importance of balancing the removal of free chelate with the introduction of metal contaminants from the diafiltration buffer and also illustrate how to optimize radiolabeling of antibody conjugates under a variety of operating conditions. This methodology is applicable to the production of antibody conjugates in general.