Delivery of shRNA using gold nanoparticle-DNA oligonucleotide conjugates as a universal carrier

The efficient delivery of nucleic acids into mammalian cells is a central aspect of research involving cell biology and medical applications, including the clinical treatment of genetic disorders. We report an efficient small hairpin RNA (shRNA) delivery system that utilizes a single species of gold nanoparticle-DNA oligonucleotide conjugate (AuNP-DNA oligo) as a universal carrier. In vitro synthesized shRNA that is specific to the p53 gene was efficiently delivered into HEK293 and HeLa human cell lines using an AuNP-DNA oligo. The delivery resulted in an 80-90% knockdown of p53 expression. The same AuNP-DNA oligo was also efficient for the delivery of another shRNA, which is specific to the Mcl-1 gene, as well as the repression of MCL-1 expression. The knockdown efficiency of shRNA that was delivered using an AuNP-DNA oligo was comparable with that of a liposome-based shRNA delivery method. Our results offer an alternate delivery system for shRNA that can be used on any gene of interest.     

Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure.

The staining efficiency of peroxidase labeled immunoglobulin conjugate, used either as antigen or as antibody, has been compared with that of peroxidase-anti-peroxidase complex (PAP) on ultrathin sections of araldite embedded material. The conjugate gave positive results in a two layer method as well as in a three layer method when used as antibody. No staining was observed when it was used as antigen. The conjugation seemed to impair the antigenic reactivity of immunoglobulin. The conjugate when used as antibody in the three layer method gave approximately the same staining efficiency as PAP.     

Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene

To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.     

Selective recognition of glutathiolated aldehydes by aldose reductase

In this study, the selectivity and specificity of aldose reductase (AR) for glutathionyl aldehydes was examined. Relative to free aldehydes, AR was a more efficient catalyst for the reduction of glutathiolated aldehydes. Reduction of glutathionyl propanal [gammaGlu-Cys(propanal)-Gly] was more efficient than that of Gly-Cys(propanal)-Gly and gamma-aminobutyric acid-Cys(propanal)-Gly suggesting a possible interaction between alpha-carboxyl of the conjugate and AR. Two active site residues, Trp20 or Ser302, were identified by molecular modeling as potential sites of this interaction. Mutations containing tryptophan-to-phenylalanine (W20F) and serine-to-alanine (S302A) substitutions did not significantly affect reduction of free aldehydes but decreased the catalytic efficiency of AR for glutathiolated aldehydes. Combined mutations indicate that both Trp20 and Ser302 are required for efficient catalysis of the conjugates. The decrease in efficiency due to W20F mutation with glutathionyl propanal was not observed with gamma-aminobutyric-Cys(propanal)-Gly or Gly-Cys-(propanal)-Gly, indicating that Trp20 is involved in binding the alpha-carboxyl of the conjugate. The effect of the S302A mutation was less severe when gammaGlu-Cys(propanal)-Glu rather than glutathionyl propanal was used as the substrate, consistent with an interaction between Ser302 and Gly-3 of the conjugate. These observations suggest that glutathiolation facilitates aldehyde reduction by AR and enhances the range of aldehydes available to the enzyme. Because the N-terminal carboxylate is unique to glutathione, binding of the conjugate with the alpha-carboxyl facing the bottom of the alpha/beta-barrel may assist in the exclusion of unrelated peptides and proteins.     

Bile acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated perfused rat liver.

In studies of cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate comprised 15-27% of the biliary bile acids in these experiments, was less polar than cholylglycine on thin-layer chromatography using butanol, acetic acid, and water, and had an apparent molecular weight greater than that of cholyltaurine on gas-liquid chromatography. Amino acid analysis of the hydrolyzed conjugate demonstrated the presence of arginine. Perfusion studies with radioactive arginine, and mass spectrometric analysis proved that the conjugate was cholylarginine. Secretion of this conjugate does not represent a deficiency of available glycine and taurine.     

The antiviral action of a modified bacterial ribonuclease]

The antiviral activity of a bacterial ribonuclease conjugate with chitosane of Kamchatka crab (in a form of water soluble chito-oligosaccharides) has been studied. The conjugate inhibitory activity for A and B viruses as well as to Sindbis arbovirus in tissue cultures is shown. The preparation efficiency at intramuscular and intranasal administration was observed at experimental influenza infection of white mice.     

Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.     

Ubiquitin in Chlamydomonas reinhardii. Distribution in the cell and effect of heat shock and photoinhibition on its conjugate pattern

Ubiquitin, a highly conserved 76-amino-acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high-molecular-mass conjugates (apparent molecular mass greater than 45 kDa) were observed. Exposure of cells to a 41.5 degrees C heat shock in both the dark and light caused the disappearance of the 29-kDa conjugate and an increase in the high-molecular-mass conjugates. After step down to 25 degrees C the 29-kDa conjugate reappeared while the levels of high-molecular-mass conjugates decreased. In light, the recovery of the 29-kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.     

Cellular entry and nuclear targeting by a highly anionic molecular umbrella

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.     

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg? > Arg? ? Arg?. The conjugate with Arg? exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg?-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg?-1 and 17-desacetylvinblastineTrp-Arg?-2 are epimers at the tryptophan α carbon atom.     

The requirement for glutathione S-transferase in the conjugation of activated aflatoxin B1 during aflatoxin hepatocarcinogenesis in the rat

The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.     

Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-peptide analogue of insulin-like growth factor 1

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.     

Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.     

Synthesis and biological evaluation of Doxorubicin-containing conjugate targeting PSMA

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), has recently emerged as a prominent biomarker of prostate cancer (PC) and as an attractive protein trap for drug targeting. At the present time, several drugs and molecular diagnostic tools conjugated with selective PSMA ligands are actively evaluated in different preclinical and clinical trials. In the current work, we discuss design, synthesis and a preliminary biological evaluation of PSMA-specific small-molecule carrier equipped by Doxorubicin (Dox). We have introduced an unstable azo-linker between Dox and the carrier hence the designed compound does release the active substance inside cancer cells thereby providing a relatively high Dox concentration in nuclei and a relevant cytotoxic effect. In contrast, we have also synthesized a similar conjugate with a stable amide linker and it did not release the drug at all. This compound was predominantly accumulated in cytoplasm and did not cause cell death. Preliminary in vivo evaluation has showed good efficiency for the degradable conjugate against PC3-PIP(PSMA⁺)-containing xenograft mine. Thus, we have demonstrated that the conjugate can be used as a template to design novel analogues with improved targeting, anticancer activity and lower rate of potential side effects. 3D molecular docking study has also been performed to elucidate the underlying mechanism of binding and to further optimization of the linker area for improving the target affinity.     

Poly(l-glutamic acid) Gd(III)-DOTA conjugate with a degradable spacer for magnetic resonance imaging

The clinical application of macromolecular Gd(III) complexes as MRI contrast agents is impeded by their slow excretion and potential toxicity due to the release of Gd(III) ions caused by the metabolism of the agents. A polymer Gd(III) chelate conjugate with a cleavable spacer has been designed to solve this problem. Poly(l-glutamic acid)-cystamine-[Gd(III)-DOTA] was prepared by the conjugation of DOTA to PGA (MW = 50,000) via cystamine, a cleavable disulfide spacer, followed by the complexation with GdCl(3). A Gd(III) DOTA chelate derivative was readily released from the polymer conjugate in the incubation with cysteine, an endogenous plasma thiol. The conjugate produced significant MRI blood pool contrast enhancement in nude mice bearing OVCAR-3 human ovarian carcinoma xenographs. Less significant contrast enhancement was observed for a small molecular contrast agent, Gd(DTPA-BMA). The pharmacokinetic MRI study showed that the Gd(III) chelate from the conjugate accumulated in the urinary bladder in a similar kinetic pattern to Gd(DTPA-BMA), suggesting that the chelate was released by the endogenous thiols and excreted through renal filtration. The preliminary results suggest that this novel design has a great potential to solve the safety problem of macromolecular MRI contrast agents.     

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.     

Evaluation on the use of reactive dye-modified polylysine as the biomarker in immunochromatographic test application

An improved dye immunochromatographic test (DICT) using polylysine (PL) as conjugate spacer loading dye molecules to enhance chromophor color intensity with the potential of a simultaneous multicolored assay has been developed. To construct this new effective chromophor, a dyeing process coupling a reactive dye, Procion Blue MX-7RX (PB7RX), with PL of different molecular weights was performed. The optimal conjugate condition between PB7RX and PL was studied. It showed that under the optimized dyeing conditions, a PL molecular weight of 189.4 kDa and a molar ratio (mol dye/mol amine group in PL) of 1.5 were obtained. The resulting dyed PL chromophor, used as both a spacer and a color intensifier, was further labeled to a model antibody, anti-human serum albumin (anti-HSA), to build a PB7RX–PL–anti-HSA (PPA) conjugate. The PPA obtained in this way generated the highest color intensity of 19,455 assayed by immunochromatographic test strip under densitometer scanning. A competitive DICT for determination of HSA was carried out. A linear range between 0 and 18.77 μg/ml with a detection limit of 0.49 μg/ml was observed. A test for using dyed PL chromophors as biomarkers was also performed to demonstrate the feasibility of a multianalyte immunoassay.     

RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping

Cyclic Arg-Gly-Asp-D-Phe-Lys [c(RGDfK)] targeted poly(L-glutamic acid) (PGA)-(Gd-DO3A) conjugate with a biodegradable cystamine spacer was prepared and evaluated for in vivo detection of an angiogenesis biomarker, alpha(v)beta3 integrin, in neoplastic tissues with T1 mapping, a quantitative magnetic resonance imaging (MRI) technique. The binding activity of the c(RGDfK) containing conjugate was investigated using in vitro vitronectin assay with human prostate carcinoma DU145 cell line and Kaposi's sarcoma SLK cell line. The peptide c(RGDfK) and PGA-cystamine-(Gd-DO3A) conjugate were used as controls. The binding affinity of polymer bound c(RGDfK) was slightly lower than free c(RGDfK) peptide. The RGD targeted conjugate had higher binding affinity to the DU145 cells than the SLK cells, which was consistent to free c(RGDfK). The imaging of alpha(v)beta3 integrin with targeted PGA-cystamine-(Gd-DO3A) was evaluated in nude mice bearing DU145 and SLK xenografts at a dose of 5 micromol-Gd/kg. The targeted conjugate demonstrated higher in vivo binding affinity to the DU145 xenografts than the SLK xenografts, resulting in a significant decrease of T1 values of water protons in the periphery of the DU145 tumors as shown in the MR T1 maps. No significant decrease of T1 values was observed in the SLK tumor with the targeted conjugate and in both tumors with the non-targeted conjugate. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging with quantitative MR T1 mapping.