Evaluation of nanoparticle drug-delivery systems used in preclinical studies.

Multifunctional nanoparticles have been identified as a promising drug-delivery system for sustainable drug release. The structural and size tunability and disease-targeting ability of nanoparticles have made them more suitable for multiple drug loading and delivery, thereby enhancing therapeutic results through synergistic effects. Nanoparticulate carriers with specific features such as target specificity and stimuli-responsiveness enable selective drug delivery with lower potential side effects. In this review we have classified the recently published articles on polymeric and inorganic nanoparticle-mediated drug delivery into three different categories based on functionality and discussed their efficiency for drug delivery and their therapeutic outcomes in preclinical models. Most of the drug-loaded nanodelivery systems discussed have demonstrated negligible or very low systemic toxicity throughout the experimental period in animal models compared with free drug administration. In addition, some challenges associated with the translation of nanoparticle-based drug carrier responses to clinical application are highlighted.

Engineering of cell microenvironment-responsive polypeptide nanovehicle co-encapsulating a synergistic combination of small molecules for effective chemotherapy in solid tumors.

In this study, we report a facile method to construct a bioactive (poly(phenylalanine)-b-poly(l-histidine)-b-poly(ethylene glycol) polypeptide nanoconstruct to co-load doxorubicin (DOX) and quercetin (QUR) (DQ-NV). The smart pH-sensitive nanovehicle was fabricated with precisely tailored drug-to-carrier ratio that resulted in accelerated, sequential drug release. As a result of ratiometric loading, QUR could significantly enhance the cytotoxic potential of DOX, induced marked cell apoptosis; change cell cycle patterns, inhibit the migratory capacity of sensitive and resistant cancer cells. In particular, pro-oxidant QUR from DQ-NV remarkably reduced the GSH/GSSG ratio, indicating high oxidative stress and damage to cellular components. DQ-NV induced tumor shrinkage more effectively than the single drugs in mice carrying subcutaneous SCC-7 xenografts. DQ-NV consistently induced high expression of caspase-3 and PARP and low expression of Ki67 and CD31 immunomarkers. In summary, we demonstrate the development of a robust polypeptide-based intracellular nanovehicle for synergistic delivery of DOX/QUR in cancer chemotherapy. STATEMENT OF SIGNIFICANCE: In this study, we report a facile method to construct bioactive and biodegradable polypeptide nanovehicles as an advanced platform technology for application in cancer therapy. We designed a robust (poly(phenylalanine)-b-poly(l-histidine)-b-poly(ethylene glycol) nanoconstruct to co-load doxorubicin (DOX) and quercetin (QUR) (DQ-NV). The conformational changes of the histidine block at tumor pH resulted in accelerated, sequential drug release. QUR could significantly enhance the cytotoxic potential of DOX, induce marked cell apoptosis, change cell cycle patterns, and inhibit the migratory capacity of sensitive and resistant cancer cells. DQ-NV induced tumor shrinkage more effectively than the single drugs and the 2-drug cocktail in tumor xenografts. In summary, we demonstrate the development of an intracellular nanovehicle for synergistic delivery of DOX/QUR in cancer chemotherapy.

Ligand effect and cooperative role of metal ions on the DNA cleavage efficiency of mono and binuclear Cu(II) macrocyclic ligands complexes.

Two binuclear Cu(II) complexes of N-functionalized macrocycle ligands, namely 1,3-bis(1,4,7-triaza-1-cyclonomyl)propane and 1-(3-(1,4,7-triazonan-1-yl)propyl)-1,4,7,10-tetraazacyclo-dodecane, were synthesized and their ability to hydrolyze the cleavage of supercoiled plasmid DNA (pBR322) was compared with that of structurally related non-functionalized mononuclear Cu(II) complexes. The former, binuclear Cu(II) complex with the symmetrical ligand exhibited enhanced double-strand cleavage activity compared to the other three complexes at the same [Cu(2+)] concentration. In contrast, the latter binuclear complex with unsymmetrical macrocylic ligand did not give rise to double-strand DNA cleavage. The linear DNA formation induced by the mononuclear Cu(II) 1,4,7,10-tetraazacyclo-dodecane complex was realized via a non-random double-stranded scission process. The differential cleavage activity is discussed in relation to dimer formation, effective cooperation and coordination environment of the metal center. The hydrolytic cleavage by the copper complexes without H2O2 is supported by evidence from an anaerobic reaction, free radical quenching, and nitro blue tetrazolium assay. In contrast, both the binuclear complexes cleaved supercoiled DNA efficiently to Form III (linearized DNA) in the presence of H2O2, indicating that nuclearity is a crucial parameter in oxidative cleavage. The radical scavenger inhibition study and nitro blue tetrazolium assay suggested the involvement of H2O2 and superoxide ions in the oxidative cleavage of DNA by the binuclear complexes.

DNA cleavage induced by [Cu(L)x(NO3)2] (L=2,2'-dipyridylamine, 2,2'-bipyridine, dipicolylamine, x=1 or 2): Effect of the ligand structure.

The efficiency of [Cu(2,2'-bipyridine)2(NO3)]NO3, [Cu(2,2'-dipyridylamine)2(NO3)2], and [Cu(dipicolylamine)2(NO3)2] complexes (complex 1, 2 and 3, respectively) in oxidative DNA cleavage was examined by electrophoresis and linear dichroism (LD). Among the three Cu complexes, complex 1 showed the highest efficiency in super-coiled DNA (scDNA) cleavage in electrophoresis. The presence of tiron, a superoxide radical scavenger, suppressed the reaction almost completely. The LD signal at 260 nm decreased gradually as the time passed. The decrease in LD magnitude was explained best by the sum of the two single exponential curves. This suggests that the cleavage reaction involves two first order kinetic processes; an increase in flexibility due to scission of one of the strands and a shortening in the DNA stem due to cut of both strands of double stranded DNA (dsDNA). In agreement with the electrophoresis data, complex 1 exhibited the highest efficiency with the superoxide radical found to be the essential reactive oxygen species. The order of efficiency in both scDNA and dsDNA was as follows: complex 1>complex 2>complex 3. The electrochemical properties alone were insufficient to explain the observed efficiencies, even though reduction of the central Cu ion is essential for the oxidative DNA cleavage. This highlights the importance of an ability to ligate the molecular oxygen (or hydrogen peroxide) to the central Cu ion to produce the superoxide radical, in addition to the reduction of Cu ion, in oxidative DNA cleavage.

Synthesis, characterization, DNA interaction, antioxidant and anticancer activity of new ruthenium(II) complexes of thiosemicarbazone/semicarbazone bearing 9,10-phenanthrenequinone.

A new series of octahedral ruthenium(II) complexes supported by tridentate ligands derived from phenanthrenequinone and derivatives of thiosemicarbazide/semicarbazide and other co-ligands have been synthesized and characterized. DNA binding experiments indicated that ruthenium(II) complexes can interact with DNA through non-intercalation and the apparent binding constant value (Kb) of [RuCl(CO)(PPh3)(L3)] (3) at room temperature was calculated to be 2.27 × 10(3)M(-1). The DNA cleavage studies showed that the complexes have better cleavage of pBR 322 DNA. Antioxidative activity proved that the complexes have significant radical scavenging activity against free radicals. Cytotoxic activities showed that the ruthenium(II) complexes exhibited more effective cytotoxic activity against selected cancer cells.

Influence of carboxylic acid functionalities in ruthenium (II) polypyridyl complexes on DNA binding, cytotoxicity and antioxidant activity: synthesis, structure and in vitro anticancer activity.

Four new Ru(II) complexes [RuHCl(bpy)(PPh(3))(CO)] (1), [RuHCl(bpy)(AsPh(3))(CO)] (2) (bpy = 2,2'-bipyridine), [RuCl(HL)(PPh(3))(2)(CO)] (3) and [RuCl(HL)(AsPh(3))(2)(CO)] (4) (HL = 2,2'-bipyridine-4,4'-dicarboxylic acid) were synthesized and characterized. X-ray diffraction was used to characterize 3 in solid state. The interactions of these complexes with DNA were explored by different techniques which revealed that the complexes could bind to CT-DNA through non-intercalation. The in vitro cytotoxic and antioxidant activities of the complexes validated against a panel of cancer cell lines and free radicals showed that 3 and 4 possess quite high anticancer and antioxidant activities over 1, 2 and standard drugs. An apparent dependence of biological activities on incorporation of COOH in bipyridine moiety was noticed: Inclusion of COOH caused significant differences in DNA binding, cytotoxicity and antioxidant activity.

Photo-induced DNA scission by Cu(ii)-meso-tetrakis(n-N-methylpyridiniumyl)porphyrins (n = 2, 3, 4) and their binding modes to supercoiled DNA.

The photo-induced cleavage of pGEM-7zf-NIS super-coiled DNA by Cu(ii)-meso-tetrakis(n-N-methylpyridiniumyl)porphyrins (n = 2, 3, 4 referred to as o-, m- and p-CuTMPyP, respectively) and their binding mode were investigated in this study. m-CuTMPyP was most efficient in cleavage than o- and p-CuTMPyP isomers. Cleavage was suppressed by N(2) bubbling, suggesting that the cleavage occurred by an oxidative cleavage mechanism. Sodium azide, an (1)O(2) quencher, and DMSO, a hydroxyl radical scavenger, inhibited cleavage, indicating that hydroxyl radicals and singlet oxygen were likely reactive species responsible for the cleavage. Reduced linear dichroism spectroscopy showed angles of o-CuTMPyP's electric transition moments, in which the periphery pyridinium ring was prevented from free rotation, of 59° and 61° with respect to the local DNA helix axis. The spectra of m- and p-CuTMPyP complexed with pGEM-7zf-NIS DNA were characterized by large signals in the Soret band, coincident with those of known intercalated porphyrins.

Ruthenium(II)/(III) complexes of 4-hydroxy-pyridine-2,6-dicarboxylic acid with PPh3/AsPh3 as co-ligand: impact of oxidation state and co-ligands on anticancer activity in vitro.

With the aim to develop more efficient, less toxic, target specific metal drugs and evaluate their anticancer properties in terms of oxidation state and co-ligand sphere, a sequence of Ru(II), Ru(III) complexes bearing 4-hydroxy-pyridine-2,6-dicarboxylic acid and PPh(3)/AsPh(3) were synthesized and structurally characterized. Biological studies such as DNA binding, antioxidant assays and cytotoxic activity were carried out and their anticancer activities were evaluated. Interactions of the complexes with calf thymus DNA revealed that the triphenylphosphine complexes could bind more strongly than the triphenylarsine complexes. The free radical scavenging ability, assessed by a series of in vitro antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion radical, hydrogen peroxide and metal chelating assay, showed that the Ru(III) complexes possess excellent radical scavenging properties compared to those of Ru(II). Cytotoxicity studies using three cancer lines viz HeLa, HepG2, HEp-2 and a normal cell line NIH 3T3 showed that Ru(II) complexes exhibited substantial cytotoxic specificity towards cancer cells. Furthermore, the Ru(II) complexes were found to be superior to Ru(III) complexes in inhibiting the growth of cancer cells.

Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA.

A pair of chiral binuclear ruthenium(II) complexes were prepared and their binding affinities towards double stranded native DNA were assessed by observing isotropic absorption, polarized light spectra - circular and linear dichroism (CD and LD), fluorescence quenching and DNA thermal denaturation. Upon binding to DNA, the complexes produced LD signals consisting of positive and negative signals in the absorption region, although they exhibited red shift and hypochromism in the absorption spectrum. These contrasting observations indicated that the binding modes of the complexes are largely deviated from classical intercalative binding. Groove binding of the complexes to DNA was found to be more likely than intercalative binding. The small increase of DNA melting temperature in the presence of the complexes indicated a predominance of DNA groove binding. The absence of "molecular light switch effect" further supported non-intercalative binding. The groove binding propensity of complexes was also supported by comparison of the resulting data with the [Ru(phen)(2)(dppz)](2+).

Synthesis, spectroscopic, crystal structure and DNA binding of Ru(II) complexes with 2-hydroxy-benzoic acid [1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-ethylidene]-hydrazide.

Reactions of 2-hydroxy-benzoic acid [1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-ethylidene]-hydrazide (H(2)L) with [RuHCl(CO)(EPh(3))(3)] (E = P or As) were carried out and the new complexes obtained were characterized by elemental analysis, electronic, IR, (1)H NMR and (13)C NMR spectroscopic techniques and single crystal X-ray diffraction studies. Complex (1) crystallizes in the monoclinic space group P2(1)/c with unit cell dimensions a=18.6236(17) Å, b=12.8627(12) Å, c=21.683(2) Å, α=90.00, ß=114.626(2), γ=90.00 V=4721.8(8) Å, Z=4. The crystal structure of the complex shows Ru(II) atom is six-coordinated, forming a slightly distorted octahedral geometry with two P atoms in axial positions, and three chelating donor atoms of the tridentate Schiff base ligand and one carbonyl group located in the equatorial plane. The molecular structure is stabilized by intramolecular O-H···N interactions. No intermolecular hydrogen bond was observed. The intramolecular hydrogen bond exists between the oxygen atom from salicylic acid moiety and nitrogen from the same moiety. A variety of solution studies were carried out for the determination of DNA binding mode of the complexes. The results suggest that both complexes bind to Herring sperm DNA via non intercalative mode.

Bioconjugation of L-3,4-dihydroxyphenylalanine containing protein with a polysaccharide.

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.