SYNTHESIS OF NOVEL POLYDIAMIDOPROPANOATE DENDRIMER PNA-PEPTIDE CHIMERAS FOR NON-INVASIVE MAGNETIC RESONANCE IMAGING OF CANCER

A variety of dendrimers can be conjugated to oligonucleotides to increase the number of contrast paramagnetic atoms (e.g., gadolinium or dysprosium) per probe. Thus, it was of interest to test a route for assembly of chelating dendrimer branches directly on the N-termini of peptide nucleic acid (PNA)-peptide chimeras by continuous solid-phase coupling on polymer supports. Dendrimer-PNA-peptides complementary to 12 nt of mutant KRAS mRNA have been prepared with a C-terminal insulin-like growth factor 1 (IGF1) analog d(Cys-Ser-Lys-Cys) and N-terminal polydiamidopropanoate (PDAP) dendrimers with different numbers of diaminopropanoate residues. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating moieties were then coupled to PDAP dendrimer-PNA-peptide chimeras before cleavage from the polymer supports. The DOTA-PDAP-PNA-peptide probes with 1, 2, 4, 8, or 16 amino (or DOTA) moieties were cleaved, purified by RP-HPLC, and characterized by MALDI-TOF mass spectroscopy.     

Synthesis of novel polydiamidopropanoate dendrimer PNA-peptide chimeras for non-invasive magnetic resonance imaging of cancer

A variety of dendrimers can be conjugated to oligonucleotides to increase the number of contrast paramagnetic atoms (e.g., gadolinium or dysprosium) per probe. Thus, it was of interest to test a route for assembly of chelating dendrimer branches directly on the N-termini of peptide nucleic acid (PNA)-peptide chimeras by continuous solid-phase coupling on polymer supports. Dendrimer-PNA-peptides complementary to 12 nt of mutant KRAS mRNA have been prepared with a C-terminal insulin-like growth factor 1 (IGF1) analog d(Cys-Ser-Lys-Cys) and N-terminal polydiamidopropanoate (PDAP) dendrimers with different numbers of diaminopropanoate residues. 1,4, 7, 10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating moieties were then coupled to PDAP dendrimer-PNA-peptide chimeras before cleavage from the polymer supports. The DOTA-PDAP-PNA-peptide probes with 1, 2, 4, 8, or 16 amino (or DOTA) moieties were cleaved, purified by RP-HPLC, and characterized by MALDI-TOF mass spectroscopy.     

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.     

PEGylated dendrimers with core functionality for biological applications

The synthesis of a variety of core functionalized PEGylated polyester dendrimers and their in vitro and in vivo properties are described in this report. These water-soluble dendrimers have been designed to carry eight functional groups on their dendritic core for a variety of biological applications such as drug delivery and in vivo imaging as well as eight solubilizing groups. Using a common symmetrical aliphatic ester dendritic core and trifunctional amino acid moieties, a library of dendrimers with phenols, alkyl alcohols, alkynes, ketones, and carboxylic acid functionalities has been synthesized without the need for column chromatography. The amines were PEGylated, leaving the other functionality of the amino acid available for further manipulation such as the attachment of drugs and/or labels. Radiolabeling experiments with the PEGylated dendrimers showed that they had a long circulation half-life in mice, confirming the potential of this class of dendrimers for therapeutic and/or diagnostic applications. A carboxylic acid functionalized dendrimer was elaborated to carry doxorubicin bound via a hydrazone bond. The drug-loaded carrier accumulated more in tumors and less in healthy organs than the clinically used PEGylated liposomal formulation Doxil. The efficient synthesis, high versatility, and favorable biological properties make these PEGylated polyester dendrimers promising structures for therapeutic and/or imaging applications.     

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.     

Synthesis and evaluation of novel dendrimers with a hydrophilic interior as nanocarriers for drug delivery

Novel polyester-co-polyether dendrimers consisting of a hydrophilic core were synthesized by a combination of convergent and divergent syntheses. The core was synthesized from biocompatible moieties, butanetetracarboxylic acid and aspartic acid, and the dendrons from PEO (poly(ethylene oxide)), dihydroxybenzoic acid or gallic acid, and PEG monomethacrylate. The dendrimers, Den-1-(G 2) (second generation dendrimer-1) and Den-2-(G 2) (second generation dendrimer-2) consisting of 16 and 24 allyl surface groups, respectively, were obtained by coupling the dendrons to the core. The dendrimer (Den-1-(G 2)-OH) with hydroxyl groups at the surface was synthesized by oxidation of the allyl functional groups of Den-1-(G 2), which was divergently coupled to the dendrons to obtain the third generation dendrimer Den-1-(G 3) consisting of 32 surface groups. The modifications in surface groups and generation of dendrimers were shown to influence the shape of dendrimers in the AFM studies. The aggregation as well as self-assembly of dendrimers was observed at high concentration in water by light scattering studies; however, it was reduced on dilution and in the presence of sodium chloride. Dendrimers demonstrated good ability to encapsulate the guest molecule, with loading of 15.80 and 6.47% w/w for rhodamine and beta-carotene, respectively. UV spectroscopy proved the absence of any pi-pi complexation between the dendrimer and encapsulated compounds. (1)H NMR and FTIR studies showed that the physical entrapment and/or hydrogen bonding by PEO in the interior and branch of the dendrimer are the mechanisms of encapsulation. The release of the encapsulated compounds was found to be slow and sustained, suggesting that these dendrimers can serve as potential drug delivery vehicles.     

Dendritic iodinated contrast agents with PEG-cores for CT imaging: synthesis and preliminary characterization

The purpose of this study was to design, synthesize, and initially characterize a representative set of novel constructs for large-molecular radiographic/computed tomography (CT) contrast agents, intended for a primarily intravascular distribution. A new assembly of well-known and biocompatible components consists of paired, symmetrical dendritic polylysines initiated from both ends of a poly(ethylene glycol) (PEG) core, yielding an array of multiple free amino groups to which were conjugated highly soluble and stable triiodophthalamide ("triiodo") moieties. An array of six dendritic contrast agents was synthesized originally, using three different PEG cores (3, 6, 12 kDa) with t-Boc lysine-generated dendrimer "amplifiers" (from three to five generations) containing 16 to 64 amino groups for conjugation with reactive triiodo moieties. A clinically used, nonionic, small molecular CT contrast agent, iobitridol, was derivatized via a hydroxyl protection/deprotection strategy, introducing a new carboxyl group available for conjugation to the lysine amino groups of dendrimers. Final products were purified by size exclusion chromatography and characterized by NMR, UV, HPLC, and elemental analysis. Preliminary evaluations were conducted for physicochemical characterization and in vivo CT contrast enhancement in a rat model. All six iodinated PEG-core dendrimer conjugates were synthesized in good yields, with a high degree of size monodispersity, large apparent molecular weight, favored physicochemical properties. A representative compound, PEG12000-carbamate-Gen4-IOB conjugate, 27% (w%) rich in iodine, demonstrated a desirable strong and persistent intravascular enhancement with a monoexponential blood half-life of approximately 35 min assayed by dynamic CT imaging and also showed high water solubility (>550 mg/mL at 25 degrees C), large apparent molecular size (comparable to a 143-kDa protein), high hydrophilicity (butanol-water partition coefficient 0.015), and stability to autoclaving conditions. This study showed the synthetic feasibility, desired basic characteristics, and potential utility for CT contrast enhancement achieved with a new type of iodinated, large-molecular PEG-core dendritic construct. Further development of this class of macromolecular contrast agents will be required to define the optimal formulation, pharmacology, safety profile, and the full range of diagnostic applications including tumor microvascular quantitative characterization by CT imaging.     

Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle

Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.     

Introduction of monosaccharides having functional groups onto a carbosilane dendrimer: a broadly applicable one-pot reaction in liquid ammonia involving Birch reduction and subsequent SN2 reaction

Benzylthioalkyl glycosides of D-glucuronic acid, N-acetyl-D-glucosamine, and N-acetylneuraminic acid (common monosaccharide constituents of natural oligosaccharide chains) have been prepared as sulfide precursors for the carbohydrate coating of dendric carbosilane cores and used in a generally applicable one-pot reaction (Birch reduction in liquid ammonia and subsequent SN2 reaction) to generate a thioether linkage between the monosaccharide moieties and a carbosilane dendrimer. The dendrimers were uniformly functionalized with the monosaccharides in good yields.     

Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121

Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.     

Genotoxicity of poly(propylene imine) dendrimers

Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers.     

Acidic glycolipids from dolphin kidney

The composition and contents of acidic glycolipids in the kidney of a striped dolphin (Stenella coeruleoalba, the order Cetacea, whales) were determined. The following eight acidic glycolipids were isolated and characterized: SM4s (124.2 nmol/g tissue), SM3 (8.7), GM3 (NeuAc) (12.3), GM3 (NeuGc) (31.6), GD3 (NeuAc-NeuAc) (14.7), GD3 (NeuAc-NeuGc) (II3 alpha(NeuAc alpha 2-8NeuGc)-LacCer) (9.8), GD3 (NeuGc-NeuAc) (II3 alpha(NeuGc alpha 2-8NeuAc)-LacCer) (5.3), and GD3 (NeuGc-NeuGc) (15.8). The assignment of the four types of GD3 was further confirmed as described below. Evidence indicating 2-8 linkages of the disialosyl residues of GD3 was obtained on methylation analysis of sialic acid. GD3 (NeuAc-NeuAc) and GD3 (NeuGc-NeuAc) were degraded to GM3 (NeuAc), and GD3 (NeuAc-NeuGc) and GD3 (NeuGc-NeuGc) yielded GM3 (NeuGc) on mild acid hydrolysis. Fragment ions characteristic of the carbohydrate and lipophilic moieties of the permethylated GD3 were observed in direct inlet-electron impact-mass spectra. The presence of these four types of GD3 in a tissue has not been reported previously. GD3 contained non-hydroxy (69-84%) and hydroxy fatty acids (16-31%) with 16-24 carbons. The long chain base of all GD3, except GD3 (NeuGc-NeuAc) (not analyzed), consisted of 4-sphingenine (d18:1) and 4-hydroxysphinganine (t18:0) in almost equal amounts. The total amount of renal lipid-bound acidic groups (sulfate and sialic acid) of the dolphin (190 mumol/animal) is considerably higher than that of a terrestrial mammal (88 mumol/animal) with a body weight comparable to that of the dolphin. This deviation suggests that the amount of renal acidic amphiphiles required to maintain the osmotic balance of body fluids in marine mammals might be higher than that in terrestrial ones.     

Oligosaccharide-derivatized dendrimers: defined multivalent inhibitors of the adherence of the cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1

Poly(propylene imine) dendrimers having four or eight primary amino groups and a Starburst^TM (PAMAM) dendrimer having eight primary amino groups were used as core molecules, to which phenylisothiocyanate derivatized (PITC) galβ1-3galNAcβ1-4[sialic acidβ2-3]-galβ1-4glc (oligo-GM1) residues were covalently attached to yield multivalent oligosaccharides. The synthesis of the oligo-GM1-PITC derivatized dendrimers was monitored using high performance thin layer chromatography, infrared spectroscopy, sialic acid content, and mass spectroscopy. The ability of multivalent oligo-GM1-PITC dendrimers to inhibit the binding of ¹²⁵I-labeled cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1-coated microtiter wells was determined. IC₅₀s obtained for the oligo-GM1-PITC dendrimers, GM1, and the oligosaccharide moiety of GM1 indicated that the derivatized dendrimers inhibited binding of the choleragenoid and the heat labile enterotoxin to GM1-coated wells at a molar concentration five- to 15-fold lower than native GM1 and more than 1,000-fold lower than that of the free oligosaccharide. This revised version was published online in November 2006 with corrections to the Cover Date.     

Self-assembled monolayer of light-harvesting core complexes from photosynthetic bacteria on a gold electrode modified with alkanethiols

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodoseudomonas palustris were self-assembled on a gold electrode modified with self-assembled monolayers (SAMs) of the alkanethiols NH2(CH2)nSH, n = 2, 6, 8, 11; HOOC(CH2)7SH; and CH3(CH2)7SH, respectively. Adsorption of the LH1-RC complexes on the SAMs depended on the terminating group of the alkanethiols, where the adsoption increased in the following order for the terminating groups: amino groups > carboxylic acid groups > methyl groups. Further, the adsorption on a gold electrode modified with SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, depended on the methylene chain length, where the adsorption increased with increasing the methylene chain length. The presence of the well-known light-harvesting and reaction center peaks of the near infrared (NIR) absorption spectra of the LH1-RC complexes indicated that these complexes were only fully stable on the SAM gold electrodes modified with the amino group. In the case of modification with the carboxyl group, the complexes were partially stable, while in the presence of the terminal methyl group the complexes were extensively denatured. An efficient photocurrent response of these complexes on the SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, was observed upon illumination at 880 nm. The photocurrent depended on the methylene chain length (n), where the maximum photocurrent response was observed at n = 6, which corresponds to a distance between the amino terminal group in NH2(CH2)6SH and the gold surface of 1.0 nm.     

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.     

Modification of corn cob meal with quarternary ammonium groups

Corn cob meal was modified with quarternary ammonium groups and subsequently extracted with 80% ethanol, water, and 5% NaOH. The fractions obtained had lower polydispersities, values, and yields than unmodified material. The yields are lower than those obtained on bagasse under the same conditions. The modification caused the drastic degradation of the ethanol-lignin (EL) fraction. The one-step extraction with NaOH/H₂O₂ gave 28·8% yield of material (calculated on the starting material) which contained 12·0% Klason lignin, and had the highest polydispersity (4·3, ). The water-soluble fractions consisted of arabinoglucuronoxylan and alkali-soluble fractions of xylan without other sugar moieties. The water-soluble fraction from NaOH/H₂O₂ extraction contained arabinoglucuronoxylan with modified arabinose and acid units. By this method higher yields could be obtained than on bagasse treated by the sequential extraction.     

Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.     

Therapeutic anti-methamphetamine antibody fragment-nanoparticle conjugates: synthesis and in vitro characterization

Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Toward this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG cross-linker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting the PEG cross-linker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH binding function. The dendribodies were found to have affinity for METH identical to that of the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.     

Dendrimers based lysine and their "starburst" polymeric derivatives: prospects of use in compacting DNA and in vitro delivery of genetic constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Modification of sialyl residues of glycoconjugates by reductive amination. Characterization of the modified sialic acids

The sialic acid residues of alpha 1-acid glycoprotein and fetuin were modified by introduction of an amino residue, such as glycine and [3H]glycine. This modification involved (a) the selective periodate oxidation of the exocyclic carbon atoms of the sialic acid residue generating an aldehyde group at C-7, and (b) the reduction of the Schiff base formed with an amino compound by use of sodium cyanoborohydride. Thin layer chromatography, high pressure liquid chromatography, and amino acid composition data of the modified glycoprotein showed that the conversion was essentially quantitative. The glycine-modified sialic acids were isolated by mild acid hydrolysis and identified by g.l.c.-m.s. and n.m.r. spectroscopy, thus confirming that the quantitative modification produced a glycine-aminated C-7 sialic acid analog. Strong acid hydrolysis of the glycine-modified sialic acid yielded a fragment that had chromatographic characteristics similar to those of glycine.