Anionic PAMAM dendrimers as drug delivery vehicles for transition metal-based anticancer drugs

The use of anionic half-generation poly(amidoamine) dendrimers as drug delivery vehicles for [Pt(S,S-dach)(5,6-Me₂phen)]²⁺ (56MESS) (where S,S-dach = 1S,2S-diaminocyclohexane; 5,6-Me₂phen = 5,6-dimethyl-1,10-phenanthroline) and [{Δ,Δ-Ru(phen)₂}₂(μ-bb7)]⁴⁺ (Rubb₇) (where phen = 1,10-phenanthroline; bb7 = 1,7-bis[4-(4′-methyl-2,2′-bipyridyl)heptane]) has been studied by nuclear magnetic resonance spectroscopy. From one- and two-dimensional ¹H NMR spectra both 56MESS and Rubb₇ were found to bind to the surface of generation 3.5, 4.5, 5.5 and 6.5 dendrimers through electrostatic interactions. The higher charge and larger size of Rubb₇ resulted in stronger binding to all dendrimer generations (K_b ? 2 × 10⁵ M⁻¹) compared with 56MESS (K_b ? 1 × 10⁴ M⁻¹). Interestingly, there appeared to be no observable trend between dendrimer size and binding constant strength. The size of the free and 56MESS-bound dendrimers were examined using pulsed-gradient spin-echo NMR. The dendrimers ranged in hydrodynamic diameter from 11 to 20 nm and in all cases were larger than their corresponding full-generation dendrimer. Upon the addition of 56MESS the diameter of the dendrimers increased, consistent with surface binding.     

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.     

Copper bis(thiosemicarbazone) complexes as hypoxia imaging agents: structure-activity relationships

Copper(II) bis(thiosemicarbazone) complexes labelled with Cu-60/62/64 are useful radiopharmaceuticals for imaging blood flow and hypoxic tissues in vivo. The aim of this study was to identify structure-activity relationships within a series of analogues with different alkyl substitution patterns in the ligand, in order to design improved hypoxia imaging agents and elucidate hypoxia selectivity mechanisms. Thirteen such complexes were synthesised and characterised spectroscopically and electrochemically. The uptake of each (labelled with Cu-64) in EMT6 tumour cells in vitro under normoxic and hypoxic conditions was studied. All complexes were taken up efficiently into cells, and some showed strong hypoxia selectivity, which was highly correlated with the Cu(II/I) redox potential. Redox potentials at the low end of the range were found to be essential for hypoxia selectivity. In turn, the redox potential was strongly correlated with alkyl substitution pattern, and the most important determinant of the redox potential was the number of alkyl groups on the diimine backbone of the ligand. Several complexes in the series warrant further evaluation as hypoxia imaging agents. The radioactivity uptake/release behaviour in the cells provides insight into possible mechanisms, and a model for hypoxia-selective intracellular trapping is discussed.     

Surface modified dendrimers: synthesis and characterization for cancer targeted drug delivery

Dendrimers represents a highly branched three-dimensional structure that provides a high degree of surface functionality and versatility. PAMAM dendrimers are used as well-defined nanocontainers to conjugate, complex or encapsulate therapeutic drugs or imaging moieties. Star-burst [PAMAM] dendrimers represent a superior carrier platform for drug delivery. The present study was aimed at synthesis of a surface modified dendrimer for cancer targeted drug delivery system. For this 4.0 G PAMAM dendrimer was conjugated with Gallic acid [GA] and characterized through UV, IR, 1H NMR and mass spectroscopy. Cytotoxicity study of dendrimer conjugate was carried out against MCF-7 cell line using MTT assay. The study revealed that the conjugate is active against MCF-7 cell line and might act synergistically with anti-cancer drug and gallic acid-dendrimer conjugate might be a promising nano-platform for cancer targeting and cancer diagnosis.     

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.     

Mammaglobin as a potential molecular target for breast cancer drug delivery

Background

Mammaglobin (MAM) has been used as a specific molecular marker for breast cancer diagnosis. Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule. Some of the strategies are based upon an essential but not demonstrated hypothesis – mammaglobin is associated with the surface of breast cancer cells, which strongly disputes the therapeutic strategies.

Results

We conducted a computer-based predictive analysis and identified a small fragment at the N-end of MAM as a potential transmembrane domain. We provided several evidences to demonstrate the presence of the membrane-associated MAM. We isolated the membrane protein components from known MAM positive breast cancer cells (MDA-MB361 and MDA-MB415). We showed that about 22–64% of MAM proteins, depending upon the types of the cancer cells, directly attached on the membrane of breast cancer cells, by Western blotting assays. To directly visualize the presence of the membrane-bound MAM protein, we incubated the MAM positive cancer cells with FITC labeled anti-MAM antibody, and observed clear fluorescent signals on the surface of the cells. In studying the MAM protein distribution in human breast cancer tissues, we first identified two immunostain patterns that are associated with the membrane-bound MAM: the membrane stain pattern and luminary surface stain pattern. To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL. Specific binding and cytotoxicity of the MAM targeted and Dox loaded LDL was tested in the MAM positive breast cancer cells in vitro.

Conclusion

We first showed that some of MAM protein directly associated with the surface of breast cancer cells. The membrane-associated MAM protein may be utilized as a useful molecular marker for breast cancer targeted drug delivery.     

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.     

Minimum analogue peptide sets (MAPS) for quantitative structure-activity relationships

The information contents in previously published peptide sets was compared with smaller sets of peptides selected according to statistical designs. It was found that minimum analogue peptide sets (MAPS) constructed by factorial or fractional factorial designs in physiochemical properties contained substantial structure-activity information. Although five to six times smaller than the originally published peptide sets the MAPS resulted in QSAR models able to predict biological activity. The QSARs derived from a MAPS of nine dipeptides, and from a set of 58 dipeptides inhibiting angiotensin converting enzyme were compared and found to be of equal strength. Furthermore, for a set of bitter tasting dipeptides it was found that an incomplete MAPS of 10 dipeptides gave just as good a model as the model based on a set of 48 dipeptides. By comparison other non-designed sets of peptides gave QSARs with poor predictive power. It was also demonstrated how MAPS centered on a lead peptide can be constructed as to specifically explore the physiochemical and biological properties in the vicinity of the lead. It was concluded that small information-rich peptide sets MAPS can be constructed on the basis of statistical designs with principal properties of amino acids as design variables.     

Phylogenetic relationships in Blumea (Asteraceae: Inuleae) as evidenced by molecular and morphological data

The present study, based on sequences of cpDNA (trnL-F & psbA-trnH) and nrDNA (ITS) and morphology, examined the evolutionary relationships in Blumea and its position among related genera. The results confirmed that the closest relatives of Blumea are Caesulia, Duhaldea and Pentanema p.p., and showed that the monotypic genera Blumeopsis and Merrittia are nested within Blumea. In Blumea s.l., two major, well-supported clades were recognised and a single species, the widespread Blumea balsamifera, that could not be placed with certainty relative to the two main clades. The two main clades differ in habit, ecology and distribution. The Blumea densiflora clade contains shrubs and subshrubs of evergreen forests, distributed from continental Asia to New Guinea and Polynesia, whereas the Blumea lacera clade is a widespread paleotropical group that comprises mostly annual, weedy herbs of open forests and fields.     

Poly(fumaric-co-sebacic) microspheres as oral drug delivery systems

The current study focuses on the development of bioadhesive oral delivery systems based on bioerodible polyanhydrides. The polymers were studied and characterized using a novel tensiometer based on a very sensitive electrobalance. The system was designed to mimic in vivo interactions, thus all experiments were conducted with freshly excised tissue immersed in physiological saline at 37 degrees C. Poly(fumaric-co-sebacic) [P(FA:SA)] was found to be the most bioadhesive polymer from a series of different thermoplastic materials evaluated. Correlation with in vivo performance was investigated by determining gastrointestinal (GI) residence time of barium-loaded microspheres. Residence times of 24 to 36 h provided a strong indication that these microspheres were good candidates for bioadhesive drug delivery systems. To evaluate the effect of these materials on bioavailability, the anticoagulant drug, dicumarol, was encapsulated. Systemic blood levels demonstrated increased bioavailability for the encapsulated dicumarol formulation as compared with unencapsulated drug. (c) 1996 John Wiley & Sons, Inc.     

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%.     

PEGylated rosin derivatives: Novel microencapsulating materials for sustained drug delivery

The aim of this study was to investigate PEGylated rosin derivatives (PRDs) as microencapsulating materials for sustained drug delivery. PRDs (D1, D2, and D3) composed of a constant weight of rosin and varied amounts of polyethylene glycol (PEG) 400 and maleic anhydride were synthesized in the laboratory. Microparticles were prepared by the O/O solvent evaporation technique using the acetone/paraffin system. Diclofenac sodium (DFS) and diltiazem hydrochloride (DLTZ) were used as model drugs. The effect of the type of PRD, drug, PRD:drug ratio, viscosity of external phase, stirring speed, concentration of magnesium stearate (droplet stabilizer), and method of preparation on particle size, drug loading, and drug release profiles of microparticles was investigated. PRDs could produce discrete and spherical microspheres (with DFS) and microcapsules (with DLTZ). The drug loading value for microparticles was found to be in the range of 37.21% to 87.90%. The microparticle size range was 14 to 36 μm. The particle size and drug loadings of microparticles were substantially affected by the concentration of magnesium stearate and the type of drug, respectively. Most of the formulations could sustain the DFS and DLTZ release for 20 hours. DFS and DLTZ release from PRD microparticles followed Hixson-Crowell and first-order kinetics, respectively. The results suggest that PRDs can be used successfully to prepare discrete and spherical microparticles with DFS and DLTZ for sustained drug delivery. Published: June 22, 2007     

Poly(amidoamine) (PAMAM) dendritic nanostructures for controlled sitespecific delivery of acidic anti-inflammatory active ingredient

The purpose of the investigation was to evaluate the potential of polyamidoamine (PAMAM) dendrimer as nanoscale drug delivery units for controlled release of water insoluble and acidic anti-inflammatory drug. Flurbiprofen (FB) was selected as a model acidic anti-inflammatory drug. The aqueous solutions of 4.0 generation (G) PAMAM dendrimer in different concentrations were prepared and used further for solubilizing FB. Formation of dendrimer complex was characterized by Fourier transform infrared spectroscopy. The effect of pH on the solubility of FB in dendrimer was evaluated. Dendrimer formulations were further evaluated for in vitro release study and hemolytic toxicity. Pharmacokinetic and biodistribution were studied in male albino rats. Efficacy of dendrimer formulation was tested by carrageenan induced paw edema model. It was observed that the loaded drug displayed initial rapid release (more than 40% till 3rd hour) followed by rather slow release. Pharmacodynamic study revealed 75% inhibition at 4th hour that was maintained above 50% till 8th hour. The mean residence time (MRT) and terminal half-life (THF) of the dendritic formulation increased by 2-fold and 3-fold, respectively, compared with free drug. Hence, with dendritic system the drug is retained for longer duration in the biosystem with 5-fold greater distribution. It may be concluded that the drug-loaded dendrimers not only enhanced the solubility but also controlled the delivery of the bioactive with localized action at the site of inflammation. Published: October 27, 2005     

Synthesis and structure-activity relationships of guanine analogues as phosphodiesterase 7 (PDE7) inhibitors

The synthesis of a novel series of guanine analogues is reported. The compounds have been assessed in vitro and some analogues have been found to be inhibitors of phosphodiesterase type 7 (PDE7).     

Acidic hydrolysis of N-Ethoxybenzylimidazoles (NEBIs): potential applications as pH-sensitive linkers for drug delivery

This paper describes the development of a new class of N-linked imidazoles as potential pH-sensitive, cleavable linkers for use in cancer drug delivery systems. Kinetic analysis of eight derivatives of N-ethoxybenzylimidazoles (NEBIs) showed that their rates of hydrolysis are accelerated in mild aqueous acidic solutions compared to in solutions at normal, physiological pH. Incorporation of electron donating or electron withdrawing substituents on the phenyl ring of the NEBI resulted in the ability to tune the rates of hydrolysis under mild acidic conditions with half-lives ranging from minutes to months. A derivative of NEBI carrying doxorubicin, a widely used anticancer agent, also showed an increased rate of hydrolysis under mild acid compared to that at normal physiological pH. The doxorubicin analogue resulting from hydrolysis from the NEBI exhibited good cytotoxic activity when exposed to human ovarian cancer cells. These results demonstrate a potentially useful, general strategy for conjugating a wide range of drugs to imidazole-containing delivery vessels via NEBI functionalities for controlled release of therapeutics for drug delivery applications.     

Synthesis and structure-activity relationships of 3-cyano-4-(phenoxyanilino)quinolines as MEK (MAPKK) inhibitors

A series of 3-cyano-4-(phenoxyanilino)cyanoquinolines has been prepared as MEK (MAP kinase kinase) inhibitors. The best activity is seen with alkoxy groups at both the 6- and 7-positions. The lead compounds show low nanomolar IC₅₀'s against MAP kinase kinase, and have potent inhibitory activity in tumor cells.     

Boron-bridged poly(ferrocenylene)s as promising materials for nanoscale molecular wires

The ferrocene-containing polymer [-fc-B(Br)-]_n (6) with an average chain length of ca. 20 repeat units (n = 20) is readily accessible from fc(BBr₂)₂ and HSiEt₃ [fc = (C₅H₄)₂Fe] via a novel polycondensation reaction. Treatment of 6 with excess THF yields the highly soluble alkoxy-substituted derivative [-fc-B(O(CH₂)₄Br)-]_n (10; n = 20).     

Small-molecule glucosylation by sucrose phosphorylase: structure-activity relationships for acceptor substrates revisited

Sucrose phosphorylase catalyzes the O-glucosylation of a wide range of acceptor substrates. Acceptors presenting a suitable 1,2-diol moiety are glucosylated exclusively at the secondary hydroxyl. Production of the naturally occurring compatible solute, 2-O-α-d-glucopyranosyl-sn-glycerol, from sucrose and glycerol is a notable industrial realization of the regio- and stereoselective biotransformation promoted by sucrose phosphorylase. The acceptor substrate specificity of sucrose phosphorylase was analyzed on the basis of recent high-resolution crystal structures of the enzyme. Interactions at the acceptor binding site, observed in the crystal (d-fructosyl) and suggested by results of docking experiments (glycerol), are used to rationalize experimentally determined efficiencies and regioselectivities of enzymatic glucosyl transfer.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.