Streptokinase-polyethylene glycol conjugates with increased stability and reduced side effects

Covalent SK-PEG2 and SK-PEG5 conjugates with various degrees of modification of the protein amino groups were obtained by variation of the duration of streptokinase (SK) incubation with activated polyethylene glycol (M 2 and 5 kDa, PEG2 and PEG5); their properties were studied in comparison with the properties of unmodified SK in vitro. SK-PEG2 and SK-PEG5 conjugates with the highest stability in plasma retaining 80% of initial fibrinolytic activity were formed at modification degrees of 54 and 52%, respectively. Interaction of the conjugates with equimolar plasminogen resulted in the formation of plasmin (Pm) activator complexes Pm·SK-PEG2 and Pm·SK-PEG5 with the maximum amidase activity being the same as that of Pm complex with native SK. Catalytic efficiency of plasminogen activation (k _Pg/K _Pg) was found to be slightly higher (2.84 min^?1 μM^?1) in case of Pm·SK-PEG2 complex and slightly lower, in case of the Pm·SK-PEG5 complex (1.17 min^?1 μM^?1), if compared to that of the unmodified complex Pm·SK (2.1 min^?1 μM^?1). Investigation of lysis kinetics of human plasma clot and depletion of plasminogen and fibrinogen plasma levels under the effect of equal doses of SK in free and conjugated forms demonstrated that SK-PEG2 and SK-PEG5 conjugates possess high thrombolytic activity (89 and 72% to the activity of free SK, respectively) and cause 3.5–4-fold lower side effects than free SK. The SK-PEG2 and SK-PEG5 conjugates with increased stability in plasma and reduced side effects may be used in therapy of thrombotic disorders.     

Synthesis, characterization, and in vitro activity of dendrimer-streptokinase conjugates

Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention ( approximately 80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease []. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes []. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest.

Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM–5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes.

End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through π–π stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

Evaluation of anionic half generation 3.5-6.5 poly(amidoamine) dendrimers as delivery vehicles for the active component of the anticancer drug cisplatin

Aquated cisplatin was added to half-generation PAMAM dendrimers and the resultant complexes were purified by centrifuge. The drug-dendrimer complexes were then characterised by 1-D and diffusion ¹H NMR and ICP-AES. The amount of drug bound was found to increase in proportion with dendrimer size: G3.5, 22 cis-{Pt(NH₃)₂} molecules per dendrimer; G4.5, 37; G5.5, 54; and G6.5, 94, which represent only a fraction of the available binding sites on each dendrimer (68, 58, 42 and 37%, respectively). Drug release studies showed that some drug remains bound to the dendrimer even after prolonged incubation with 5′—GMP at temperatures of 60 °C for over a week (percentage of drug released 18, 30, 35 and 63%, respectively). Attachment of the drug was found to decrease the radius of the dendrimers. Finally, the effect of the dendrimer on drug cytotoxicity was determined using in vitro assays with the A2780, A2780cis and A2780cp ovarian cancer cell lines. The free dendrimers display no cytotoxicity whilst the drug-dendrimer complexes showed moderate activity. In vivo activity was examined using an A2780 tumour xenograft. Cisplatin, at its maximum tolerated dose of 6 mg/kg, reduced tumour size by 33% compared to an untreated control group. The G6.5 cisplatin-dendrimer complex was administered at two doses (6 and 8 mg/kg equivalent of cisplatin). Both were well tolerated by the mice. The lower dose displayed comparable activity to cisplatin with a tumour volume reduction of 32%, but the higher dose was significantly more active than free cisplatin with a tumour reduction of 45%.     

Dendrimers as carrier protein mimetics for IgE antibody recognition. Synthesis and characterization of densely penicilloylated dendrimers

The synthesis of benzylpenicilloyl-containing dendrimers has been achieved by a convenient procedure involving quantitative functionalization of the terminal amino groups of the three Starbust PAMAM generations used (G(n); n = 0, 1, 2). All these densely penicilloylated dendrimers (G(n)P) exhibit similar, simple NMR spectroscopic data suggesting highly symmetric structures and a monodisperse nature, and the results obtained from MALDI-TOF-MS demonstrate their exact chemical composition. The use of PAMAM dendrimers has allowed us to synthesize, for the first time, carrier benzylpenicilloyl conjugates (G(n)P) of precisely defined chemical structure. The attempts to synthesize G(2)P show that forced experimental conditions are not always useful for the functionalization of the dendrimer, especially in introducing bulky groups. The initial results with sera from patients with different RAST levels were positive and thus suggestive that inhibition occurs, so recognition exists; we can therefore conclude that the hapten-carrier (dendrimer) conjugates studied mimic recognition with natural hapten-carrier (protein) conjugates.     

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest. Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM-5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes. End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through pi-pi stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH₂), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure.     

Evaluation of poly(amidoamine) dendrimers as potential carriers of iminodiacetic derivatives using solubility studies and 2D-NOESY NMR spectroscopy

The interactions between dendrimers and different types of drugs are nowadays one of the most actively investigated areas of the pharmaceutical sciences. The interactions between dendrimers and drugs can be divided into: internal encapsulation, external electrostatic interaction, and covalent conjugation. In the present study, we investigated the potential of poly(amidoamine) (PAMAM) dendrimers for solubility of four iminodiacetic acid derivatives. We reported that PAMAM dendrimers contribute to significant solubility enhancement of iminodiacetic acid analogues. The nature of the dendrimer–drug complexes was investigated by ¹H NMR and 2D-NOESY spectroscopy. The ¹H NMR analysis proved that the water-soluble supramolecular structure of the complex was formed on the basis of ionic interactions between terminal amine groups of dendrimers and carboxyl groups of drug molecules, as well as internal encapsulation. The 2D-NOESY analysis revealed interactions between the primary amine groups of PAMAM dendrimers and the analogues of iminodiacetic acid. The results of solubility studies together with ¹H NMR and 2D-NOESY experiments suggest that the interactions between PAMAM dendrimers of generation 1–4 and derivatives of iminodiacetic acid are based on electrostatic interactions and internal encapsulation.     

Polymeric and dendrimeric pyridoxal enzyme mimics

Pyridoxal was covalently attached to polyethylenimine polymers, but the resulting materials were found to degrade rapidly. In comparison, the dendrimeric pyridoxals, which possess only one pyridoxal unit at the core of every dendrimer molecule were found to be relatively stable compounds. A total of 12 poly(amidoamine) type dendrimers were synthesized. They range from G1 to G6 with either NMe(2) or NHAc termini. The NMe(2)-terminated pyridoxal dendrimers racemize alpha-amino acids 50-100 times faster than does simple pyridoxal, while the NHAc-terminated pyridoxal dendrimers racemize alpha-amino acids only 3-5 times faster than does simple pyridoxal. Both the NMe(2)- and NHAc-terminated pyridoxal dendrimers decarboxylate 2-amino-2-phenyl-propionic acid 1-3 times faster than simple pyridoxal. The interior polarity in the pyridoxal dendrimers is similar to that of 85:15 water-DMF solution. Furthermore, we successfully incorporated eight lauryl groups to the G5 pyridoxal dendrimer at known positions. The laurylated dendrimer exhibits lower racemization and decarboxylation rates than do the unlaurylated ones, in contrast to the positive rate effects of laurylation in polyethylenimine-pyridoxamines in our previous transamination studies.     

Engineering streptokinase for generation of active site-labeled plasminogen analogs

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH₂-terminal His₆ tags. The NH₂-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile¹ residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys⁴¹⁴ residue and residues Arg253–Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg^∗/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253–L260)ΔK414–His₆ mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry.     

Paramagnetic NMR Investigation of Dendrimer-Based Host-Guest Interactions

In this study, the host-guest behavior of poly(amidoamine) (PAMAM) dendrimers bearing amine, hydroxyl, or carboxylate surface functionalities were investigated by paramagnetic NMR studies. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) derivatives were used as paramagnetic guest molecules. The results showed that TEMPO-COOH significantly broaden the ¹H NMR peaks of amine- and hydroxyl-terminated PAMAM dendrimers. In comparison, no paramagnetic relaxation enhancement (PRE) was observed between TEMPO-NH₂, TEMPO-OH and the three types of PAMAM dendrimers. The PRE phenomenon observed is correlated with the encapsulation of TEMPO-COOH within dendrimer pockets. Protonation of the tertiary amine groups within PAMAM dendrimers plays an important role during this process. Interestingly, the absence of TEMPO-COOH encapsulation within carboxylate-terminated PAMAM dendrimer is observed due to the repulsion of TEMPO-COO- anion and anionic dendrimer surface. The combination of paramagnetic probes and ¹H NMR linewidth analysis can be used as a powerful tool in the analysis of dendrimer-based host-guest systems.     

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.     

Catalytic solid substrate-room-temperature phosphorimetry detection for trace cadmium with Cd(2+) -3.5-generation polyamidoamine dendrimer-Tween-80 complex

3.5‐Generation polyamidoamine dendrimers (3.5‐G‐D) emitted strong and stable room‐temperature phosphorescence (RTP) on filter paper when Pb²⁺ was used as a heavy atom perturber. The RTP signal of 3.5‐G‐D was sharply enhanced upon the formation of 3.5‐G‐D–Tween‐80 micelle compound. The complex Cd²⁺–3.5‐G‐D–Tween‐80, generated in the coordination reaction between Cd²⁺ and the tertiary amidocyanogen on the outer layer of 3.5‐G‐D in 3.5‐G‐D–Tween‐80 micelle compound, could catalyze KBrO₃ to oxidize 3.5‐G‐D in 3.5‐G‐D–Tween‐80, which caused the sharp quenching of the RTP signal of the system. The phosphorescence intensity change (ΔI_p) of the system had a linear relationship with the content of Cd²⁺. Thus a new catalytic solid substrate–room‐temperature phosphorimetry (SS‐RTP) for the determination of trace cadmium has been established. This highly selective and sensitive method has been applied to determine trace cadmium in biological samples with a limit of detection (LD) of 1.2 ag per spot (when the sample volume was 0.4 μL per spot, the corresponding concentration was 3.0 × 10^?15 g mL^?1), the results agreeing with those obtained by atomic absorption spectrometry. The mechanism of catalytic SS‐RTP for the determination of trace cadmium was also discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.     

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.     

Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity

Surface modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG 550 and PEG 750 and longer PEG 2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by (1)H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D 2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (< or =1 microM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG 2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 muM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.     

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

Structural studies of biologically active glycosylated polyamidoamine (PAMAM) dendrimers

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer’s surface for interaction with the biological target.     

Comparison of MRI properties between derivatized DTPA and DOTA gadolinium–dendrimer conjugates

In this report we directly compare the in vivo and in vitro MRI properties of gadolinium–dendrimer conjugates of derivatized acyclic diethylenetriamine-N,N′,N′,N″,N″-pentaacetic acid (1B4M-DTPA) and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (C-DOTA). The metal–ligand chelates were pre-formed in alcohol prior to conjugation to the generation 4 PAMAM dendrimer (G4D), and the dendrimer-based agents were purified by Sephadex® G-25 column. The analysis and SE-HPLC data indicated chelate to dendrimer ratios of 30:1 and 28:1, respectively. Molar relaxivity measured at pH 7.4, 22 °C, and 3T are comparable (29.5 vs 26.9 mM^?1 s^?1), and both conjugates are equally viable as MRI contrast agents based on the images obtained. The macrocyclic agent however exhibits a faster rate of clearance in vivo (t_1/2 = 16 vs 29 min). Our conclusion is that the macrocyclic-based agent is the more suitable agent for in vivo use for these reasons combined with kinetic inertness associated with the Gd(III) DOTA complex stability properties.