Flow field-flow fractionation and multi-angle light scattering as a powerful tool for the characterization and stability evaluation of drug-loaded metal-organic framework nanoparticles.

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. Graphical abstract ᅟ.

Supramolecular photochemistry of drugs in biomolecular environments.

In this tutorial review we illustrate how the interaction of photoactive drugs/potential drugs with proteins or DNA in supramolecular complexes can determine the course of the reactions initiated by the drug absorbed photons, evidencing the mechanistic differences with respect to the solution conditions. We focus on photoprocesses, independent of oxygen, that lead to chemical modification of the biomolecules, with formation of new covalent bonds or cleavage of existing bonds. Representative systems are mainly selected from the literature of the last decade. The photoreactivity of some aryl propionic acids, (fluoro)quinolones, furocoumarins, metal coordination complexes, quinine-like compounds, naphthaleneimides and pyrenyl-peptides with proteins or DNA is discussed. The use of light for biomolecule photomodification, historically relevant to biological photosensitization processes and some forms of photochemotherapy, is nowadays becoming more and more important in the development of innovative methods in nanomedicine and biotechnology.

Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a ß-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research.

Water-soluble naphthalene diimides as singlet oxygen sensitizers.

Bromo- and/or alkylamino-substituted and hydrosoluble naphthalene diimides (NDIs) were synthesized to study their multimodal photophysical and photochemical properties. Bromine-containing NDIs (i.e., 11) behaved as both singlet oxygen ((1)O2) photosensitizers and fluorescent molecules upon irradiation at 532 nm. Among the NDIs not containing Br, only 12 exhibited photophysical properties similar to those of Br-NDIs, by irradiation above 610 nm, suggesting that for these NDIs both singlet and triplet excited-state properties are strongly affected by length, structure of the solubilizing moieties, and pH of the solution. Laser flash photolysis confirmed that the NDI lowest triplet excited state was efficiently populated, upon excitation at both 355 and 532 nm, and that free amine moieties quenched both the singlet and triplet excited states by intramolecular electron transfer, with generation of detectable radical anions. Time-resolved experiments, monitoring the 1270 nm (1)O2 phosphorescence decay generated upon laser irradiation at 532 nm, allowed a ranking of the NDIs as sensitizers, based on their (1)O2 quantum yields (ΦΔ). The tetrafunctionalized 12, exhibiting a long-lived triplet state (τ ~ 32 µs) and the most promising absorptivity for photodynamic therapy application, was tested as efficient photosensitizers in the photo-oxidations of 1,5-dihydroxynaphthalene and 9,10-anthracenedipropionic acid in acetonitrile and water.

Citric acid-γ-cyclodextrin crosslinked oligomers as carriers for doxorubicin delivery.

Two citric acid crosslinked γ-cyclodextrin oligomers (pγ-CyD) with a MW of 21-33 kDa and 10-15 γ-CyD units per molecule were prepared by following green chemistry methods and were fully characterized. The non-covalent association of doxorubicin (DOX) with these macromolecules was investigated in neutral aqueous medium by means of circular dichroism (CD), UV-vis absorption and fluorescence. Global analysis of multiwavelength spectroscopic CD and fluorescence titration data, taking into account the DOX monomer-dimer equilibrium, evidenced the formation of 1 : 1 and 1 : 2 pγ-CyD unit-DOX complexes. The binding constants are 1-2 orders of magnitude higher than those obtained for γ-CyD and depend on the characteristics of the oligomer batch used. The concentration profiles of the species in solution evidence the progressive monomerization of DOX with increasing oligomer concentration. Confocal fluorescence imaging and spectral imaging showed a similar drug distribution within the MCF-7 cell line incubated with either DOX complexed to pγ-CyD or free DOX. In both cases DOX is taken up into the cell nucleus without any degradation.

Pyrazinoporphyrazines with externally appended pyridine rings. 13. Structure, UV-visible spectral features, and noncovalent interaction with DNA of a positively charged binuclear (Zn(II)/Pt(II)) macrocycle with multimodal anticancer potentialities.

We investigated with spectroscopic techniques the noncovalent interaction of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation, [(PtCl(2))(CH(3))(6)LZn](6+), and its octacationic analogue [(CH(3))(8)LZn](8+), lacking the cis-platin-like functionality, with a 21-mer double strand (ds) 5'-d[GGG(TTAGGG)(3)]-3'/3'-d[CCC(AATCCC)(3)]-5', as model for B-DNA. Both hexacation and octacation tend to aggregate in water. The structure as well as the ground and excited-state electronic properties of the Zn(II)/Pt(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) in water solution were investigated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods. TDDFT calculations of the lowest excited states of [(PtCl(2))(CH(3))(6)LZn](6+) in water provided an accurate description of the Q-band spectral region. In particular, the calculated optical spectra were in agreement with the experimental ones, obtained in the presence of micelles favoring complete disruption of the aggregates. The model for dsDNA binding that emerges from the analysis of UV-vis absorption and time-resolved fluorescence data shows the presence of complexes of 1 dsDNA molecule with 1, 2, and 4 macrocycles. Comparing the results for the hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with those for the [(CH(3))(8)LZn](8+)octacation, we observed a higher degree of monomerization for the [(PtCl(2))(CH(3))(6)LZn](6+) derivative.

Unravelling molecular mechanisms in the fluorescence spectra of doxorubicin in aqueous solution by femtosecond fluorescence spectroscopy.

Doxorubicin (DOX) is a potent anti-tumoral agent widely used for cancer therapy. Despite numerous studies, the fluorescence properties of DOX, usually exploited for the characterization of the interaction with biological media, have until now led to controversial interpretations, mainly due to self-association of the drug in aqueous solution. We present here the first femtosecond study of DOX based on measurements with the fluorescence up-conversion technique in combination with time-correlated single photon counting using the same laser source. We provide evidence that fluorescence signals of DOX stem from monomers and dimers. DOX dimerization induces a dramatic decrease in the fluorescence quantum yield from 3.9 × 10(-2) to 10(-5) associated with the red shift of the fluorescence spectrum by ~25 nm. While the fluorescence lifetime of the monomer is 1 ns, the dimer fluorescence is found to decay with a lifetime of about 2 ps. In contrast to monomers, the fluorescence anisotropy of dimers is found to be negative. These experimental observations are consistent with an ultrafast internal conversion (<200 fs) between two exciton states, possibly followed by a charge separation process.

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1.

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.

ß-Cyclodextrin polymer nanoparticles as carriers for doxorubicin and artemisinin: a spectroscopic and photophysical study.

The association of doxorubicin (DOX) and artemisinin (ART) to a ß-CyD-epichlorohydrin crosslinked polymer (pß-CyD), organized in nanoparticles of ca. 15 nm size, was investigated in neutral aqueous medium by circular dichroism (CD), UV-vis absorption and fluorescence. The stability constants and the absolute CD spectra of the drug complexes were determined by global analysis of multiwavelength data from spectroscopic titrations. The polymer pß-CyD proved able to disrupt the DOX dimer when the latter is the predominant form of DOX in solution. The spectroscopic and photophysical properties of the complexes evidenced an alcohol-like environment for ART and an improved inherent emission ability for DOX in the nanoparticle frame.

Stereoselective interaction of ketoprofen enantiomers with ß-cyclodextrin: ground state binding and photochemistry.

The chiral recognition ability of ß-cyclodextrin (ß-CyD) vs.S- and R-ketoprofen (KP) enantiomers has been studied by circular dichroism (CD), isothermal titration calorimetry (ITC) and NMR. The association constants of the 1 : 1 complexes obtained from CD and ITC titration experiments resulted to be the same for both enantiomers within the experimental uncertainty. Well differentiated CD spectra were determined for the diastereomeric complexes. Their structure was assessed by molecular mechanics and molecular dynamics calculations combined with quantum mechanical calculation of the induced rotational strengths in the low energy KP:ß-CyD associates, upon comparison of the calculated quantities with the corresponding experimental CD. The inclusion geometry is similar for both enantiomers with the aromatic carbonyl inserted in the CyD cavity, the monosubstituted ring close to the primary CyD rim and the carboxylate group exposed to the solvent close to the secondary rim. NMR spectra fully confirmed the geometry of the diastereomeric complexes. Tiny structural differences were sensibly probed by CD and confirmed by 2D ROESY spectra. Photoproduct studies with UV absorption and MS detection as well as nanosecond laser flash photolysis evidenced lack of chiral discrimination in the photodecarboxylation of KP within the cavity and formation of a photoaddition product to ß-CyD by secondary photochemistry of 3-ethylbenzophenone.

Complexes of the antitumoral drugs Doxorubicin and Sabarubicin with telomeric G-quadruplex in basket conformation: ground and excited state properties.

We studied the binding of two anthracycline drugs, Doxorubicin and Sabarubicin, to a model telomeric sequence 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming the basket G-quadruplex (G4) conformation in Na(+)-rich aqueous solution. We used an approach that combines spectroscopic and microcalorimetric techniques to obtain information about ground and excited state properties of the most stable complexes. Both drugs bind to the 21-mer in basket conformation and complexes of 1:1 and 2:1 drug : 21-mer stoichiometry coexist in solution. Binding constants were determined from fluorescence and isothermal titration calorimetry experiments. For both drugs association is driven by enthalpy and disfavoured by entropy in the case of two sequential binding events to different sites. The drug fluorescence is completely quenched in the 1:1 complex, most likely by electron transfer from the guanine system to the anthraquinone moiety, while part of the emission survives in the 2:1 complex. Circular dichroism (CD) of the individual complexes is dominated by the G-quadruplex signal in the UV and by the anthracycline signal in the near-UV and Vis region. The experimental CD spectra combined with conformational calculations at MM level and quantum mechanical calculation of the rotational strength of the electronic transitions afforded information on the binding geometries.

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 10. A water-soluble bimetallic (Zn(II)/Pt(II)) porphyrazine hexacation as potential plurimodal agent for cancer therapy: exploring the behavior as ligand of telomeric DNA G-quadruplex structures.

The behavior of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation as ligand of G-quadruplex (G4) structures adopted by a human telomeric DNA sequence has been examined with different spectroscopic techniques. In K(+) rich solution the hexacationic Zn(II) porphyrazine ligand bearing a peripheral cis-platin-like functionality changes the G-quadruplex conformational equilibrium of the human telomeric sequence 5'-d[AGGG(TTAGGG)(3)]-3' and drives it exclusively toward a very stable parallel G4 form in the complex with 2:1 stoichiometry. An increase of the melting temperature of more than 20 °C is observed in this complex compared to the G4 alone. On the contrary ligand binding to G-quadruplex of the same telomeric sequence in Na(+) rich solution neither markedly influences the predominant basket conformation nor confers increased thermal stability to the G4 structure.

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 9. Novel heterobimetallic macrocycles and related hydrosoluble hexacations as potentially active photo/chemotherapeutic anticancer agents.

New homo- and heterobimetallic porphyrazine complexes of general formula [(M'Cl(2))LM] (L = tetrakis-2,3-[5,6-di-(2-pyridyl)pyrazino]porphyrazinato dianion), with M = Zn(II), Mg(II)(H(2)O), or Pd(II) in the central cavity and one M'Cl(2) unit (M' = Pd(II), Pt(II)) peripherally coordinated at the pyridine N atoms of one of the dipyridinopyrazine fragments, were prepared and characterized by elemental analyses and IR/UV-visible spectroscopy. Related water-soluble salt-like species, carrying the hexacations [(PtCl(2))(CH(3))(6)LM](6+) (neutralized by I(-) ions), were also prepared and similarly characterized. Retention of clathrated water molecules is a common feature of all the compounds. A detailed (1)H and (13)C NMR investigation in dimethylformamide (DMF-d(7)) and dimethyl sulfoxide (DMSO-d(6)) provided useful information on the type of arrangement in the neutral and hexacationic species of the metalated dipyridinopyrazine fragments, in which the metal centers (Pd(II)/Pt(II)) are bound to the pyridine N atoms ("py-py" coordination) with formation of N(2(pyr))PdCl(2) or N(2(pyr))PtCl(2) coordination sites, the latter one featuring a cis-platin-like functionality. Data obtained in DMF solution of the quantum yield (Φ(Δ)) for the generation of singlet oxygen, (1)O(2), the cytotoxic agent in photodynamic therapy (PDT), indicate that all the neutral and charged complexes, among them particularly those carrying centrally Zn(II) or Pd(II), exhibit excellent photosensitizing properties, this qualifying the externally platinated complexes as potential bimodal PDT/chemotherapeutic anticancer agents. Fluorescence data (Φ(F)) provided additional information on the photoactivity of all the species studied. The following companion paper describes the observed interaction of the Zn(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with a G-quadruplex (G4) structure of the telomeric DNA sequence 5'-d[AGGG(TTAGGG)(3)]-3' in water.

A cationic Zn(II) porphyrazine induces a stable parallel G-quadruplex conformation in human telomeric DNA.

A water soluble Zn(II) porphyrazine drives the conformational equilibrium of the G-quadruplex of a human telomeric sequence exclusively towards a parallel conformation upon complexation.

Combination of spectroscopic and computational methods to get an understanding of supramolecular chemistry of drugs: from simple host systems to biomolecules.

Circular dichroism (CD) spectra of non-covalent ligand : biomolecule couples contain information on the equilibrium geometries of the associated structures that can be retrieved upon comparison of the sign and intensity of the experimental CD bands with the quantum mechanically calculated rotational strengths of low energy supramolecular complexes, obtained from molecular modelling methods. For both chiral and achiral ligands this approach proved useful to reach a structure based rationale of ground and excited state properties of the non-covalent ligand : protein associates. In this Perspective we illustrate the potential of this method focusing on the main achievements of our recent spectroscopic, conformational and photochemical studies on drug-albumin complexes and collocate it in the frame of current methodologies of molecular modelling and spectroscopic investigation of ligand : biomolecule binding.

Affinity of the anthracycline antitumor drugs Doxorubicin and Sabarubicin for human telomeric G-quadruplex structures.

Combining various techniques in solution we proved that Doxorubicin, also called Adriamycin, and Sabarubicin, also known as MEN 10755, bind to the human telomeric sequence, 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+). Complexes of drugs with the 21-mer in 1 : 1 and 2 : 1 stoichiometry coexist in solution. Association constants were obtained from titration experiments and confirmed by isothermal titration calorimetry. The fluorescence of the drugs was quenched upon complexation. UV circular dichroism (CD) spectra of the complexes were characterized by the G-quadruplex signal and indicated that drug binding influences the equilibrium between quadruplex conformations. The visible CD spectra were exclusively due to the drug and show differences in the complexation modes of the two drugs. Spectroscopic and thermodynamic parameters of the 1 : 1 complexes point to drug stacking with the G-quadruplex top or bottom tetrad. Thermodynamic data suggests that the binding of the second drug molecule in the 2 : 1 complex may occur in a groove. Complexation caused a small increase in the thermal stability of the G-quadruplex main conformation, shifting T(m) from 62 to 67 °C.

Fluoroquinolones as potential photochemotherapeutic agents: covalent addition to guanosine monophosphate.

The triplet aryl cation photochemically generated from fluoroquinolones bearing a fluoro atom at position 8 attacks guanosine monophosphate (k(r) > 10(9) M(-1)s(-1)) and forms covalent adducts. The reaction is a model for the implementation of oxygen-independent photochemotherapy.

Chiral recognition of 2-(3-benzoylphenyl)propionic acid (ketoprofen) by serum albumin: an investigation with microcalorimetry, circular dichroism and molecular modelling.

The interaction of enantiomeric ketoprofen (KP) with BSA and HSA was studied by isothermal titration calorimetry (ITC). Affinity constants and thermodynamic parameters for complexation in two main protein sites were determined. Affinity constants for both proteins are generally lower for S(+)- than for R(-)-KP. Large enthalpic contributions to Gibbs free energy are compensated by large negative entropic terms for S(+) in the BSA-subdomain IIIA and HSA-subdomain IIA. The lowest energy BSA complexes of both enantiomers were structurally characterized by combining molecular mechanics (MM) and molecular dynamics (MD) with circular dichroism (CD). Comparison of quantum mechanically calculated rotational strengths with the CD signals of the complexes supported the structures. These allowed to identify the main interactions of the KP enantiomers with surrounding amino acids at short distances, that limit significantly KP mobility in both sites. In the primary binding site S(+) is close to Tyr 409 in subdomain IIIA (Sudlow site II), and R(-) is close to Trp 212 and His 240 in subdomain IIA (Sudlow site I). The same sites are involved in the formation of 2:1 complexes. The equilibrium structures are characterized by marked geometrical distortion of KP.

Gaining an insight into the photoreactivity of a drug in a protein environment: a case study on nalidixic acid and serum albumin.

The binding of nalidixic acid (NA) with human and bovine serum albumin (HSA and BSA) in buffer solution at pH 7.4 was investigated using circular dichroism (CD), UV absorption and fluorescence spectroscopy. Global analysis of multiwavelength spectroscopic data afforded the equilibrium constants of the most stable noncovalent drug/protein adducts of 1:1 and 2:1 stoichiometry and their individual CD, UV absorption, and fluorescence spectra. The primary binding site of the drug was located in subdomain IIIA (Sudlow Site II), whereas the secondary one was assigned to subdomain IIA. Conformational and CD calculations afforded the binding geometries. In the complexes, the fluorescence of the protein was strongly quenched by energy transfer and that of the drug was suppressed by electron transfer. Laser flash photolysis at 355 nm evidenced the formation of a radical pair consisting of a tyroxyl radical (lambdamax = 410 nm) and a reduced nalidixate anion radical NA(2-)* (lambdamax = 640 nm) with quantum yield of 0.4-0.5. Strong evidence was obtained that the process that involves Tyr411 in HSA (Tyr409 in BSA). A further transient with lambdamax approximately 780 nm observed in HSA was attributed to oxidation of the -(S200-S246)- bridge upon electron transfer to NA(-)*. Decay of the confined radical pairs occurred with rates approximately 10(7) s(-1). Formation of covalent drug-protein adducts in mixtures irradiated at lambdairr> 324 nm was proved using HPLC with fluorescence detection.

Diastereoselectivity and site dependency in the photochemistry of ketoprofen in the bovine serum albumin matrix.

The photodegradation of the S(+)- and R(-)-ketoprofen (KP) enantiomers in the bovine serum albumin matrix was studied by steady-state photolysis with the use of lambda(irr) > 320 nm and transient absorption spectroscopy with lambda(exc) = 355 nm, at 1/1 and 2/1 KP/BSA molar ratios. R(-)-KP was found to be more labile than S(+). Triplet ketoprofen species were evidenced with lifetimes of 400 ns for S(+) and 600 ns for R(-)-KP. Further longer-lived transients with lifetimes of 2.6 and 6.0 mus for S(+) and R(-), respectively, were detected. On the basis of the binding constants of the drug enantiomers to the two main binding sites of the protein, obtained from circular dichroism experiments, the individual disappearance quantum yields of the 1:1 and 2:1 diastereomeric KP:BSA complexes could be estimated. The photoreactivity in the BSA matrix was rationalized on the basis of diastereoselective photodecarboxylation in the two main protein sites.