Bioconjugate Chemistry

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.

Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3 µg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 µg/mL concentration (XTT assay). In the concentration range of 6-12 µg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.

A potent, long-lasting form of interferon R-2a mono-pegylated with a 40 kilodalton branched poly-(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon R-2a was comprised of four major positional isomers involving Lys 31 , Lys 121 , Lys 131 , and Lys 134 of interferon. The in vitro anti-viral activity of pegylated interferon R-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon R-2a. Pegylated interferon R-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon R-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon R-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon R-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon R-2a compared to unmodified interferon R-2a.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 ( 5% of the -amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A 1 , indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.

New folate-conjugated superparamagnetic maghemite nanoparticles have been synthesized for the intracellular hyperthermia treatment of solid tumors. These ultradispersed nanosystems have been characterized for their physicochemical properties and tumor cell targeting ability, facilitated by surface modification with folic acid. Preliminary experiments of nanoparticles heating under the influence of an alternating magnetic field at 108 kHz have been also performed. The nanoparticle size, surface charge, and colloidal stability have been assessed in various conditions of ionic strength and pH. The ability of these folate "decorated" maghemite nanoparticles to recognize the folate receptor has been investigated both by surface plasmon resonance and in folate receptor expressing cell lines, using radiolabeled folic acid in competitive binding experiments. The specificity of nanoparticle cellular uptake has been further investigated by transmission electron microscopy after incubation of these nanoparticles in the presence of three cell lines with differing folate receptor expression levels. Qualitative and quantitative determinations of both folate nanoparticles and nontargeted control nanoparticles demonstrated a specific cell internalization of the folate superparamagnetic nanoparticles.

Attachment of drugs to high molecular weight polymers can significantly improve both tumor targeting and therapeutic efficacy due to the enhanced permeability and retention effect observed in tumor tissue. However, the commercial availability of well-defined water-soluble polymeric systems with narrow polydispersities that are biocompatible, nontoxic, and nonimmunogenic is rather limited. To address this need, we have investigated dendritic polymers as promising scaffolds for the preparation of new soluble polymeric drug carriers due to their well-defined molecular architecture and their multiplicity of surface sites. Herein we show the design and synthesis of dendritic polyester systems based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid as a possible versatile drug carrier. The potent anticancer drug doxorubicin was attached via a pH-sensitive linkage to one of the carriers presented, demonstrating the feasibility of using these polyester dendritic structures to prepare a viable polymer-drug conjugate.

The semi-IPN hydrogels consisting of poly(methacrylic acid) and guar gum (GG) are prepared at room temperature using water as solvent. 5-aminosalicylic acid (5-ASA) is entrapped in the hydrogel in the synthesis of hydrogel and all entrapment efficiencies are found above 85%. The hydrogel shows excellent pH-sensitivity. It exhibited minimum swelling in an acidic pH medium through the formation of a complex hydrogen-bonded structure and maximal swelling due to the electrostatic repulsion due to the ionization of the carboxylic groups in pH 7.4 medium. The degradation in vitro shows that the degree of degradation (R%) depended on the concentration of cross-linking agent and content of GG. The hydrogel shows a minimum release of 5-ASA due to the complex hydrogen bonded structure of the hydrogels in the medium of pH 2.2. The enzymatic degradation of hydrogels by cecal bacteria can accelerate the release of 5-ASA entrapped in the hydrogel in pH 7.4 medium.

Water-soluble gadolinium (Gd) endohedral metallofullerenes have been synthesized as polyhydroxyl forms (Gd@C(82)(OH)(n)(), Gd-fullerenols) and their paramagnetic properties were evaluated by in vivo as well as in vitro for the novel magnetic resonance imaging (MRI) contrast agents for next generation. The in vitro water proton relaxivity, R(1) (the effect on 1/T(1)), of Gd-fullerenols is significantly higher (20-folds) than that of the commercial MRI contrast agent, Magnevist (gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) at 1.0 T close to the common field of clinical MRI. This unusually high proton relaxivity of Gd-fullerenols leads to the highest signal enhancement at extremely lower Gd concentration in MRI studies. The strong signal was confirmed in vivo MRI at lung, liver, spleen, and kidney of CDF1 mice after i.v. administration of Gd-fullerenols at a dose of 5 micromol Gd/kg, which was 1/20 of the typical clinical dose (100 micromol Gd/kg) of Gd-DTPA.

define which of the radionuclides are of interest, the following discussion is structured according to application. The presentation is not a review of the area, but rather an overview with basic information to acquaint the reader with some of the important aspects of the area. There will be many potential applications not mentioned herein, and many more that have not yet been envisioned. A. Halogen Radionuclides for in Vitro Applications. Iodine-125 has been the primary radionuclide for in vitro applications due to its 60-d half-life and low-energy photon emissions. The long half-life permits the preparation and storage of labeled protein for extended periods prior to usage. The lack of particulate emissions and lowenergy photon emission makes it particularly attractive for storage, as minimal radiation damage to the labeled protein is expected. Specific activities that are very near the theoretical value (17.2 Ci/mg, 2175 Ci/mmol) are obtained with iodine-125. Its cost and availability are also very reasonable. In review other halogen radionuclides, it appears that there are no other radionuclides that can readily be used in the place of iodine-125 for in vitro studies. B. Halogen Radionuclides for in Vitro Imaging. Any of the radionuclides of halogens which have X-ray or y-ray (photon) emissions might be considered for in vivo imaging applications, but the extent of usefulness of the radionuclide to following the distribution and pharmacokinetics of a radiolabeled protein in vivo will be dependent on a number of factors. Most important are the energy and abundance of the photon emission(s) and the biological system studied. The application of radionuclides for measurement of receptor-ligand binding in vivo (37) has become less restricted through the advent of new imaging equipment and related computer image enhancement employed in single photon emission tomography (SPECT) and positron emission tomography (PET). Advancements are rapidly changing the detection limitations of specific radionuclides as outlined in the scientific highlights of the annual meeting of The Society of Nuclear Medicine presented by Dr. Wagner (38-40). Evaluations of labeled proteins in vivo are very important applications of radiolabeled proteins. While analysis of materials in excrement (e.g. urine or fecal material) permits gaining information on the clearance rate of labeled protein and of some of the pathways in the metabolism of the labeled protein, it provides little knowledge of the in vivo distribution of protein. In vivo data can be obtained by sacrificing animals, excising tissue samples, and counting the radioactivity in an appropriate counter. Tritiumand carbon-14-labeled proteins can be used to obtain in vivo data, but due to the relative ease of counting tissue samples, the photo-emitting radionuclides iodine-125 and iodine-131 are routinely used. From experiments using iodine-125 and iodine-131, biodistribution data, pharmacokinetic data, and data on the metabolic fate of the radiolabel can readily be obtained. However, iodine-125 and iodine-131 are not optimal to assess the parameters in patients. Iodine-125, while it can be imaged by a thincrystal y-camera (marginally) when evaluating small animals such as mice, it is of little or no value in imaging patients due to the attenuation of the photons through tissues. Iodine-131 is often used for imaging with conventional y-cameras, but its high-energy y-emissions are not optimally counted, and its @-particle emission results in more radiation dose to the tissues than is desirable. An alternate radionuclide of iodine, iodine-123, has a reasonable half-life of 13 h and a nearly ideal y-emission of 159 keV, in high abundance (100%) for imaging with the current y-cameras. Unfortunately, production of this Table I. Selected Halogen Nuclides nuclide half-life major route of decap ref

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene-distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electronwithdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT) 10 CG)] 2 DNA were performed to rationalize the nature of binding of these conjugates.

The therapeutic application of siRNA shows promise as an alternative approach to small molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the non-viral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line, showed that siRNA conjugated to cholesterol, TAT(48-60) and penetratin but not siRNA alone achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localisation within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6hrs. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown whilst penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression whilst the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA mediated p38 CPP and cholesterol-siRNA conjugates in mouse lung Supporting Information Available: , dose-dependent percentile p38 MAP kinase mRNA levels in vitro following lipofection with three mouse p38 MAP kinase siRNA or two mismatch controls; , analytical HPLC and electrospray mass spectrograms of peptide-and cholesteryl-RNA constructs; , analytical gels of CPP and cholesterol conjugate annealing products; , cell viability following incubation with siRNA, CPP or siRNA conjugates; , p38 MAP kinase expression across a mouse tissue panel. This information is available free of charge via the Internet at MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.

Small interfering RNA (siRNA) has been chemically conjugated to a variety of bioactive molecules, lipids, polymers, peptides, and inorganic nanostructured materials to enhance their pharmacokinetic behavior, cellular uptake, target specificity, and safety. To efficiently deliver siRNAs to the target cells and tissues, many different siRNA bioconjugates were synthesized and characterized, and their gene silencing efficiencies were tested in vitro and in vivo. In this review, recent developments of siRNA bioconjugates are summarized.

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly(L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 (L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLLmodified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLLmodified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in Vitro and in ViVo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to nonconjugated QDs. This peptide-driven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is colocalized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay, and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.

Large macromolecular MRI contrast agents with albumin or dendrimer cores are useful for imaging blood vessels. However, their prolonged retention is a major limitation for clinical use. Although smaller dendrimer-based MRI contrast agents are more quickly excreted by the kidneys, they are also able to visualize vascular structures better than Gd-DTPA due to less extravasation. Additionally, unlike Gd-DTPA, they transiently accumulate in renal tubules and thus also can be used to visualize renal structural and functional damage. However, these dendrimer agents are retained in the body for a prolonged time. The purpose of this study was to obtain information from which a macromolecular dendrimer-based MRI contrast agents feasible for use in further clinical studies could be chosen. Six small dendrimer-based MRI contrast agents were synthesized, and their pharmacokinetics, wholebody retention, and dynamic MRI were evaluated in mice to determine an optimal agent in comparison to Gd-[DTPA]-dimeglumine. Diaminobutane (DAB) dendrimer-based agents cleared more rapidly from the body than polyamidoamine (PAMAM) dendrimer-based agents with the same numbers of branches. Smaller dendrimer conjugates were more rapidly excreted from the body than the larger dendrimer conjugates. Since PAMAM-G2, DAB-G3, and DAB-G2 dendrimer-based contrast agents showed relatively rapid excretion, these three conjugates might be acceptable for use in further clinical applications.

Cell adhesion molecules R v 3 and R v 5 play a pivotal role in tumor angiogenesis and metastasis. Antiangiogenic therapy by using small peptide antagonists of R v -integrins slows tumor growth and prevents tumor spread. The ability to visualize and quantify integrin expression will enable selection of appropriate patients for clinical trials, following determination of treatment efficacy and development of new potent drugs. We have previously labeled cyclic RGD peptide c(RGDyK) with 125 I and 18 F and applied the radiotracers to both subcutaneous and orthotopic brain tumor models. Here we conjugated c(RGDyK) with 1,4,7,10-tetraaza-1,4,7,10-tetradodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) and labeled the DOTA-RGD conjugate with 64 Cu (t 1/2 ) 12.8 h, 19% + ) in high radiochemical purity and specific activity. The tumor targeting ability and in vivo kinetics of 64 Cu-DOTA-RGD was compared with [ 18 F]FB-RGD and 125 I-RGD in orthotopic MDA-MB-435 breast cancer model. All three radiotracers revealed fast blood clearance and high tumor-to-blood and tumor-to-muscle ratios. 125 I-RGD had higher tumor uptake than the corresponding 18 F and 64 Cu analogues. [ 18 F]FB-RGD indicated a fast tumor washout rate and an unfavorable hepatobiliary excretion pathway, resulting in significant activity accumulation in gallbladder and intestines. 64 Cu-DOTA-RGD had prolonged tumor retention (1.44 ( 0.09 %ID/g at 4 h postinjection) and persistent uptake in the liver. All three tracers revealed receptor specific tumor accumulation which were illustrated by effective blocking via coinjection with a blocking dose of c(RGDyK). Static microPET imaging and whole-body autoradiography showed strong contrast from the contralateral background. In conclusion, overall molecular charge and characteristics of radiolabels have profound effects on tumor accumulation and in vivo kinetics of radiolabeled RGD peptide. Further modification of the RGD peptide and optimization of the tracer for prolonged tumor uptake and improved in vivo kinetics are being explored.

We report the synthesis and in vivo characterization of an 18 F modified trimodal nanoparticle (18 F-CLIO). This particle consists of cross-linked dextran held together in core-shell formation by a superparamagnetic iron oxide core and functionalized with the radionuclide 18 F in high yield via "click" chemistry. The particle can be detected with positron emission tomography, fluorescence molecular tomography, and magnetic resonance imaging. The presence of 18 F dramatically lowers the detection threshold of the nanoparticles, while the facile conjugation chemistry provides a simple platform for rapid and efficient nanoparticle labeling.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 e n e 4). The results showed an astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high ligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at 600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive quenching occurs in antibodies labeled with Cy5 and Cy7.

Targeted therapeutics using antibodies are an attractive option over conventional cancer chemotherapeutics due to their potential to deliver a therapeutic specifically to cancer tissue without damaging normal tissue. However, there are known problems with immunoconjugates such as decreased immunoreactivity and poor solubility. Using dendrimers as carriers for these agents has the potential to resolve these issues. We synthesized J591 anti-PSMA (prostate specific membrane antigen) antibody dendrimer conjugates containing fluorophores on the dendrimer. The in vitro studies of these conjugates show that they specifically bind to cells expressing PSMA. Confocal microscopy experiments document the binding and internalization of these conjugates. This research encourages the further study of antibody-dendrimer-drug conjugates for use in targeted therapeutics.

The biotechnology and pharmaceutical industries have developed a wide variety of potential therapeutics based on the molecules of biology: DNA, RNA, and proteins. While these therapeutics have tremendous potential, effectively formulating and delivering them have also been a widely recognized challenge. A variety of viruses and toxins have evolved multi-functional biomolecules to solve this problem by directing cellular uptake and enhancing biomolecular transport to the cytoplasm from the low pH endosomal compartment. In the study reported here, we have designed and synthesized bio-inspired, pH-responsive polymeric carriers, which we call "encrypted polymers", that mimic the multi-functional design of biology. These encrypted polymers target and direct cellular uptake, as well as enhance cytosolic delivery by disrupting endosomal membranes in a pH-dependent fashion. We show that the encrypted polymeric carriers significantly enhance the delivery of oligonucleotides and peptides to the cytoplasm of cultured macrophages, demonstrating the potential of this approach for delivery of biotherapeutics and vaccines.

To develop E-selectin-specific MRI robes for detection of the early stage of tumor angiogenesis, anti-kuman E-selectin antibody fra ment F(ab') has been con'ugated to cross-linked iron oxide ($10) nanopahcles. The d-selectin F(ab'),-CLIO conjugate retains its high specificity for E-selectin. E-selectin ex ression can be detected by MR imaging in human endothelial celfculture after IL-lbeta treatment. This work provides a preliminary evidence of the feasibility of E-selectin imaging and justify further development of MR-targeted agents for monitoring proinflammator markers associated with tumor vascular endothelial prolizration, angiogenesis and atherosclerosis.

The generator-produced positron-emitting 68 Ga (T 1/2 ) 68 min) is of potential interest for clinical PET. 68 Ga as a metallic cation is suitable for complexation reactions with chelators, naked or conjugated, with peptides or other macromolecules. Large 68 Ga generator eluate volumes, metal traces from the generator column material, or reaction reagents, however, disturb a fast, reliable, and quantitative labeling procedure. In this paper we describe a simple technique, based on anion exchange, aiming first, to increase the 68 Ga concentration, second to purify it from competing impurities, and third to obtain a fast and quantitative 68 Ga-labeled peptide conjugate that can be applied in humans without further purification. Within 5 min one can obtain from the original 6 mL generator eluate a 200 µL 68 Ga preparation (volume reduction by a factor 30) that is suitable for direct and quantitative labeling of peptide conjugates. DOTATOC (DOTA-D-Phe 1 -Tyr 3 -octreotide, DOTA ) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was used as a test tracer for comparing the labeling properties of the different 68 Ga preparations. In combination with microwave heating, peptide conjugates of 0.5-1 nmol quantities could be labeled within 10 min with the full 68 Ga activity of a generator. Further purification of the 68 Ga-labeled peptide conjugate was no longer required since the nuclide incorporation was quantitative. The specific radioactivity (with respect to the peptide) was improved by a factor ∼100 compared to the previously applied techniques using the original generator eluate. The commercial 68 Ge/ 68 Ga generator from Obninsk in combination with this system for purification and concentration with an integrated microwave-supported labeling technology resulted in a kitlike technology for 68 Ga-tracer production. The first automated prototype using this technology is being tested.

The progress achieved over the last three decades in the field of bioconjugation has enabled the preparation of sophisticated nanomaterial−biomolecule conjugates, referred to herein as bionanoconstructs, for a multitude of applications including biosensing, diagnostics, and therapeutics. However, the development of bionanoconstructs for the active targeting of cells and cellular compartments, both in vitro and in vivo, is challenged by the lack of understanding of the mechanisms governing nanoscale recognition. In this review, we highlight fundamental obstacles in designing a successful bionanoconstruct, considering findings in the field of bionanointeractions. We argue that the biological recognition of bionanoconstructs is modulated not only by their molecular composition but also by the collective architecture presented upon their surface, and we discuss fundamental aspects of this surface architecture that are central to successful recognition, such as the mode of biomolecule conjugation and nanomaterial passivation. We also emphasize the need for thorough characterization of engineered bionanoconstructs and highlight the significance of population heterogeneity, which too presents a significant challenge in the interpretation of in vitro and in vivo results. Consideration of such issues together will better define the arena in which bioconjugation, in the future, will deliver functional and clinically relevant bionanoconstructs.

In the present study, we report the synthesis and human growth factor receptor-2 (HER2) specific tumor targeting properties of a dendrimer conjugated to anti-HER2 mAb (monoclonal antibody) conjugate. The polyamidoamine (PAMAM) dendrimer generation five (G5) was labeled with alexaFluor 488 and conjugated to anti-HER2 mAb. The binding and internalization of the antibody-conjugated dendrimer to HER2-expressing cells was evaluated by flow cytometry and confocal microscopy. Uniquely, the conjugate demonstrated cellular uptake and internalization in HER2-expressing cells as compared to free antibody. The time course of internalization and blocking experiments with free antibody suggest that the rapid and efficient cellular internalization of the dendrimer-antibody conjugate was achieved without alterations in specificity of targeting. Animal studies demonstrated that the conjugate targets HER2-expressing tumors.

Poly(ethylene oxide)-b-poly(ǫ-caprolactone) (PEO-b-PCL) block copolymers self-assemble into micelles in aqueous solution. We have examined whether these micelles can internalize into P19 cells in vitro. Fluorescently labeled PEO 45 -b-PCL 23 block copolymer was prepared by conjugating a tetramethylrhodamine molecule to the end of the hydrophobic PCL block. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies yielded 24 ( 2 and 25 ( 2 nm, respectively, for the diameters of the micelles. The studies also showed that chemical labeling did not effect the morphology or size. When the rhodamine-labeled PEO 45 -b-PCL 23 block copolymer micelles were tested in vitro, time-, concentration-, and pH-dependence of the internalization process suggested that internalization proceeded by endocytosis. The results from these studies provide the first direct evidence for the internalization of PEO 45 -b-PCL 23 micelles. Future studies will utilize multiple labeling of these micelles, allowing questions to be addressed related to the fate of internalized micelles as drug carriers, the destination of the incorporated drugs or fluorescent probes released from micelles, and the identification of the subcellular localization of the whole drug-carrier system within cells, both in vitro and in vivo.

Targeted delivery of therapeutics possesses the potential to localize therapeutic agents to a specific tissue as a mechanism to enhance treatment efficacy and abrogate side effects. Antibodies have been used clinically as therapeutic agents and are currently being explored for targeting drug-loaded nanoparticles. Peptides such as RGD peptides are also being developed as an inexpensive and stable alternative to antibodies. In this study, cyclo(1,12)PenITDGEATDSGC (cLABL) peptide was used to target nanoparticles to human umbilical cord vascular endothelial cell (HUVEC) monolayers that have upregulated intercellular cell-adhesion molecule-1 (ICAM-1) expression. The cLABL peptide has been previously demonstrated to possess high avidity for ICAM-1 receptors on the cell surface. Poly(DL-lactic-coglycolic acid) nanoparticles conjugated with polyethylene glycol and cLABL demonstrated rapid binding to HUVEC with upregulated ICAM-1, which was induced by treating cells with the proinflammatory cytokine, interferon-γ. Binding of the nanoparticles could be efficiently blocked by preincubating cells with free peptide suggesting that the binding of the nanoparticles is specifically mediated by surface peptide binding to ICAM-1 on HUVEC. The targeted nanoparticles were rapidly endocytosed and trafficked to lysosomes to a greater extent than the untargeted PLGA-PEG nanoparticles. Verification of peptide-mediated nanoparticle targeting to ICAM-1 may ultimately lead to targeting therapeutic agents to inflammatory sites expressing upregulated ICAM-1.

In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective crosslinking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese Hamster Ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization, and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eucariotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbinding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (>lo0 mg) of conjugates.

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide targeting α v β 3 integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibit high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pKa of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Polyethylenimine (PEI) shows high transfection efficiency and cytoxicity due to its high amine density. The new disulfide cationic polymer, linear poly(ethylenimine sulfide) (l-PEIS), was synthesized for efficient and safe gene delivery. As the amine density of l-PEIS increased, the ...