Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery

Brushed polymers composed of a backbone of poly(hydroxyethyl methacrylate) (pHEMA) onto which poly(2-(dimethylamino)ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable linker were synthesized and evaluated as nonviral gene delivery vectors. Both pDMAEMA and pHEMA polymers with controlled molecular weights and narrow distributions were synthesized by controlled atom transfer radical polymerization (ATRP). The azide initiator was used to ensure complete and monoazide functionalization of the pDMAEMA polymer chains. Click reaction between pHEMA with alkyne side groups and the azide end group in the pDMAEMA resulted in a high-molecular-weight polymer composed of low-molecular-weight constituents via an easily degradable carbonate ester linker. The length of the pDMAEMA grafts as well as the number of grafts of the brushed pHEMA-pDMAEMA can be easily varied. At physiological conditions (pH 7.4 and 37 degrees C), the brushed polymer degraded by hydrolysis of the carbonate ester with a half-life of 96 h. The molecular weights of the formed degradation products was very close to that of the starting pDMAEMA, which is likely below the renal excretion limit (<30 kDa). It was shown that the degradable brushed pHEMA-pDMAEMAs were able to condense plasmid DNA into positively charged nanosized particles. The resulting polyplexes were able to transfect cells efficiently in the presence of the endosomal membrane disrupting INF-7 peptide, and all these degradable polymers showed lower cellular toxicity compared to a high-molecular-weight pDMAEMA reference. On the other hand, the low-molecular-weight pDMAEMA used for the grafting to pHEMA was neither able to condense the structure of DNA nor able to transfect cells. This study demonstrates that grafting a low-molecular-weight cationic polymer via a hydrolyzable linker to a neutral hydrophilic polymer is an effective approach to modulate the transfection activity and toxicity profile of gene delivery polymers.     

Epidermal growth factor (EGF) receptor targeted delivery of PEGylated adenovirus

A biotin-polyethylene glycol (PEG)-epidermal growth factor (EGF) conjugate was immobilized onto the surface of avidin-modified adenovirus (ADV-Avi) via biotin-avidin interaction to deliver ADV specifically to EGF receptor over-expressing cancer cells. ADV-Avi/biotin-PEG-EGF complexes showed greatly enhanced intracellular uptake of ADV particles for an EGF receptor positive cell line (A431 cells), compared to naked or PEG alone immobilized ADV. ADV coding an exogenous GFP gene was used to quantitatively evaluate the level of GFP expression. ADV-Avi/biotin-PEG-EGF complexes also exhibited significantly increased extent of GFP expression for A431 cells, but not for MCF-7 cells (an EGF receptor deficient cell line), suggesting that retargeting of ADV to specific cells occurred by tethering of a cell-specific targeting ligand to the distal end of a PEG chain anchored onto the surface of ADV. This study demonstrates that ADV-Avi/biotin-PEG-EGF construct systems can be applied for cell-specific delivery of ADV with simultaneously reducing innate immune responses.     

Lectin functionalized nanocarriers for gene delivery

Gene therapy has emerged as one of the most promising therapeutic methods to treat various diseases. However, inadequate gene transfection efficacy during gene therapy demands further development of more efficient gene delivery strategies. Targeting genetic material to specific sites of action endows numerous advantages over non-targeted delivery. An ample variety of non-viral gene delivery vectors have been developed in recent years owing to the safety issues raised by viral vectors. Non-viral gene delivery vectors containing specific targeting ligands on their surfaces have been reported to enhance the gene transfection efficiency via receptor-mediated endocytosis for gene delivery. Among various targeting moieties investigated, carbohydrates and lectins (carbohydrate-binding proteins) played an essential role in gene delivery via either direct or reverse lectin targeting strategies. Lectins have a specific carbohydrate binding domain that can bind specifically to the carbohydrates. This review sheds light on various gene delivery nanovectors conjugated with either lectins or carbohydrates for enhanced gene transfection.     

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.     

Cyclodextrin-polyethylenimine conjugates for targeted in vitro gene delivery

Many human gene therapies will require cell-specific targeting. Though recombinant viruses are much more efficient than nonviral vectors, the latter, especially polymers, have the advantage of being targetable via conjugation of cell-specific ligands, including sugars, peptides, and antibodies, which can be covalently attached to the polymer using a variety of chemistries. Cyclodextrin, which forms inclusion complexes with small hydrophobic molecules, has been incorporated into a gene-delivery polymer and may provide a facile and versatile attachment site for targeting ligands. Polyethylenimine (PEI) was derivatized with beta-cyclodextrin on approximately 10% of the polymer's amines (termed CD-PEI). Human insulin was also derivatized with a hydrophobic palmitate group (pal-HI), which could anchor the protein to CD-PEI/DNA polyplexes. CD-PEI was essentially nontoxic to HEK293 cells at concentrations optimal for gene delivery and mediated nearly 4-fold higher gene expression than unmodified PEI, which is relatively toxic to these cells. More importantly, addition of the pal-HI to CD-PEI enhanced gene expression by more than an order of magnitude compared to unmodified PEI, either with or without the pal-HI. Because of the relative ease with which CD-binding moieties may be attached to various types of ligands, CD-PEI may be a generally useful material for testing novel cell-specific targeting compounds.     

Synthesis of galactosyl compounds for targeted gene delivery

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.     

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.     

Preparation of diazenecarboxamide-carboplatin conjugates by click chemistry

An alternative, mild and highly efficient synthetic approach to platinum complexes with bioactive carrier ligands features a platinum-complex-tolerant copper(I)-catalyzed 1,3-dipolar cycloaddition. As demonstrated by the preparation of novel diazenecarboxamide-carboplatin conjugates, this approach is superior to other methodologies.     

Epidermal growth factor-conjugated poly(ethylene glycol)-block- poly(delta-valerolactone) copolymer micelles for targeted delivery of chemotherapeutics

Epidermal growth factor (EGF)-conjugated copolymer micelles were prepared from a mixture of diblock copolymers of methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) (MePEG-b-PVL) and EGF-PEG-b-PVL for targeted delivery to EGF receptor (EGFR)-overexpressing cancers. The block copolymers and functionalized block copolymers were synthesized using PEG as the macroinitiator and HCl-diethyl ether as the catalyst. The MePEG-b-PVL and the carboxyl-terminated PEG-b-PVL (HOOC-PEG-b-PVL) copolymers were found to have molecular weights of 5940 and 5900, respectively, as determined by gel permeation chromatography (GPC) analyses. The HOOC-PEG-b-PVL copolymers were then activated by N-hydroxysuccinimide and subsequently reacted with EGF to form the EGF-PEG-b-PVL copolymers. The efficiency for the conjugation of EGF to the copolymer was found to be 60.9%. A hydrophobic fluorescent probe, CM-DiI, was loaded into both the nontargeted MePEG-b-PVL micelles and the targeted EGF-conjugated PEG-b-PVL micelles. The effective mean diameters of the CMDiI-loaded nontargeted and the CMDiI-loaded targeted micelles were found to be 32 +/- 1 nm and 45 +/- 2 nm, respectively, as determined by dynamic light scattering (DLS). The zeta potentials for the nontargeted micelles (no CM-DiI-loaded), CM-DiI-loaded nontargeted micelles, and CM-DiI-loaded targeted micelles were found to be -6.5, -8.7, and - 13.5 mV, respectively. Evaluation of the in vitro release of CM-DiI from the MePEG-b-PVL micelles in phosphate buffer saline (0.01 M, pH = 7.4) containing 10% (v/v) fetal bovine serum at 37 degrees C revealed that approximately 20% of the probe was released within the first 2 h. Confocal laser scanning microscopy (CLSM) analysis revealed that the targeted micelles containing CM-DiI accumulated intracellularly in EGFR-overexpressing MDA-MB-468 breast cancer cells following a 2 h incubation period, while no detectable cell uptake was observed for the nontargeted micelles. Results obtained from the confocal images were confirmed in an independent study by measuring the intracellular CM-DiI fluorescence in cell lysate. In addition, the presence of free EGF was found to decrease the extent of uptake of the targeted micelles. Nuclear staining of the cells with Hoechst 33258 indicated that the targeted micelles mainly localized in the perinuclear region and some of the micelles were localized in the nucleus. These results demonstrate that the EGF-conjugated copolymer micelles developed in this study have potential as vehicles for targeting hydrophobic drugs to EGFR-overexpressing cancers.     

Self-assembled terplexes for targeted gene delivery with improved transfection

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.     

Search for inhibitors of AminoAcyl-tRNA synthases by virtual click chemistry

The increase of multidrug-resistant strains of bacteria to known classes of antibiotics present a severe challenge for modern medicine. The most promising strategy to combat pathogenic bacteria is to discover new drug targets. In this regard, aminoacyl-tRNA synthetases are particularly well suited to develop novel drugs that show no cross-resistance to other classical antibiotics. To date various chemical structures that inhibit AA-RS have been identified. In this report we present an interesting approach towards generating of Leu-RS inhibitors by virtual click chemistry. That is we identified key fragments for ligand binding within catalytic pocket of Leu-RS, generated the collection of similar fragments with the use of Ligand.Info, identified the fragments that are most strongly bound in different areas within the catalytic pocket, and finally with the use of virtual click chemistry we generated a set of molecules which are most likely to act as highly potent bacterial Leu-RS inhibitors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Design of functionalized cellulosic honeycomb films: site-specific biomolecule modification via "click chemistry"

Value-added materials from naturally abundant polymers such as cellulose are of significant importance. In particular, cellulosic open-framework structures with controlled chemical functionality of the internal surface have great potential in many biosensor applications. Although various cellulose derivatives can form porous honeycomb structured materials, solubility issues and problems with film formation exist. To address this, we have generated robust cellulosic open-framework structures that can be post-functionalized through site-specific modification. Regioselectively modified amphiphilic cellulose azides, 3-O-azidopropoxypoly(ethylene glycol)-2,6-di-O-thexyldimethylsilyl cellulosics, were synthesized, and honeycomb-patterned films were readily produced by the simple breath figures method. Changing the degree of polymerization (DP) of the pendent ethylene glycol (EG(DP)) groups from 22 to 4 increased the corresponding honeycomb film pore diameters from ~1.2 to ~2.6 μm, enabling the potential tuning of pore size. Moreover, these novel azido-functionalized honeycomb films were easily functionalized using Cu(I)-catalyzed alkyne-azide [2 + 3] cycloaddition reaction; biotin was "clicked" onto the azide functionalized cellulosic honeycomb films without any effect to the film structure. These results indicate this system may serve as a platform for the design and development of biosensors.     

Labeling live cells by copper-catalyzed alkyne--azide click chemistry

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells.     

Probing gorge dimensions of cholinesterases by freeze-frame click chemistry

Freeze-frame click chemistry is a proven approach for design in situ of high affinity ligands from bioorthogonal, reactive building blocks and macromolecular template targets. We recently described in situ design of femtomolar reversible inhibitors of fish and mammalian acetylcholinesterases (EC 3.1.1.7; AChEs) using several different libraries of acetylene and azide building blocks. Active center gorge geometries of those AChEs are rather similar and identical triazole inhibitors were detected in situ when incubating the same building block libraries in different AChEs. Drosophila melanogaster AChE crystal structure and other insect AChE homology models differ more in their overall 3D structure than other members of the cholinesterase family. The portion of the gorge proximal to the catalytic triad and choline binding site has a approximately 50% reduction in volume, and the gorge entrance at the peripheral anionic site (PAS) is more constricted than in the fish and mammalian AChEs. In this communication we describe rationale for using purified recombinant Drosophila AChE as a template for in situ reaction of tacrine and propidium based libraries of acetylene and azide building blocks. The structures of resulting triazole inhibitors synthesized in situ are expected to differ appreciably from the fish and mammalian AChEs. While the latter AChEs exclusively promote synthesis of syn-substituted triazoles, the best Drosophila AChE triazole inhibitors were always anti-substituted. The anti-regioisomer triazoles were by about one order of magnitude better inhibitors of Drosophila than mammalian and fish AChEs. Moreover, the preferred site of acetylene+azide reaction in insect AChE and the resulting triazole ring formation shifts from near the base of the gorge to closer to its rim due to substantial differences of the gorge geometry in Drosophila AChE. Thus, in addition to synthesizing high affinity, lead inhibitors in situ, freeze-frame, click chemistry has capacity to generate species-specific AChE ligands that conform to the determinants in the gorge.     

99mTc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a ^99mTc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with ^99mTc at an efficiency of >95% and was radiochemically stable. ^99mTc–HYNIC tetrazine reacted with the TCO–CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of ^99mTc–HYNIC–tetrazine for tumor imaging with pretargeted mAbs.     

Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)