A fluorescence study of the loading and time stability of doxorubicin in sodium cholate/PEO-PPO-PEO triblock copolymer mixed micelles.

HYPOTHESIS: Doxorubicin hydrochloride (DX) is one of the most powerful anticancer agents though its clinical use is impaired by severe undesired side effects. DX encapsulation in nanocarrier systems has been introduced as a mean to reduce its toxicity. Micelles of the nonionic triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) (PEO-PPO-PEO), are very promising carrier systems. The positive charge of DX confines the drug to the hydrophilic corona region of the micelles. The use of mixed micelles of PEO-PPO-PEO copolymers and a negatively charged bile salt should favour the solubilization of DX in the apolar core region of the micelles. EXPERIMENTS: We studied the DX uptake in the micellar systems formed by sodium cholate (NaC) and the PEO100PPO65PEO100 (F127) copolymer, prepared with different mole ratios (MR = nNaC/nF127) in the range 0 ÷ 1. The systems were characterized by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS); DX encapsulation was followed by steady-state and time-resolved fluorescence spectroscopy. FINDINGS: The successful solubilization of DX in the host micellar systems did not affect their structure, as evidenced by both SAXS and DLS data. In the presence of NaC, DX experiences a more apolar environment as indicated by its characteristic fluorescent behaviour. The almost complete uptake of the drug occurred shortly after the sample preparation; however, time resolved fluorescence revealed a slow partition of DX between corona and core regions of the micelles. DX degradation in the mixed micellar systems was markedly reduced relative to aqueous DX solutions.

A Stereochemically Driven Supramolecular Polymerisation.

Anthracyclines self-assemble in water into dimers. In the presence of sufficiently high salt (NaCl) concentrations, solutions of the antibiotic doxorubicin, but not those of the closely related molecules daunomycin and epirubicin, turn into gels barely compatible with the presence of small oligomers. The use of spectroscopic, scattering, imaging and computational techniques, allowed light to be shed on the self-assembly process that triggered doxorubicin gelification. A complex picture emerged, with doxorubicin molecules assembled into long, highly chiral, supramolecular aggregates made of hundreds of units, showing redshifted fluorescence spectra, very short fluorescence lifetimes and small-angle X-ray scattering profiles compatible with long cylinders. The involvement of specific chemical groups and the need for a specific stereochemistry of the monomers in the formation of a hydrogen-bond network to stabilise the supramolecular aggregates was supported by molecular dynamics calculations. A salt-induced, temperature-dependent, cooperative nucleation-elongation supramolecular polymerisation of the doxorubicin molecules is deduced.

Interaction of doxorubicin with polynucleotides. A spectroscopic study.

The interaction of doxorubicin (DX) with model polynucleotides poly(dG-dC)·poly(dG-dC) (polyGC), poly(dA-dT)·poly(dA-dT) (polyAT), and calf thymus DNA has been studied by several spectroscopic techniques in phosphate buffer aqueous solutions. UV-vis, circular dichroism, and fluorescence spectroscopic data confirm that intercalation is the prevailing mode of interaction, and also reveal that the interaction with AT-rich regions leads to the transfer of excitation energy to DX not previously documented in the literature. Moreover, the DX affinity for AT sites has been found to be on the same order of magnitude as that reported for GC sites.

Effect of the alkaline cations on the stability of the model polynucleotide poly(dG-dC)·poly(dG-dC).

When the model polynucleotide poly(dG-dC)∙poly(dG-dC) [polyGC] is titrated with a strong acid (HCl) in unbuffered aqueous solutions containing the chlorides of the alkali metals in the concentration range 0.010 M-0.600 M, two transitions in the absorbance vs. pH plots are evidenced, characterized by the constants pK(a(1)) and pK(a(2)). The limiting values at infinite saline concentrations of these two constants, namely pK(∞)(a(1)) and pK(∞)(a(2)) obtained making use of the "one site saturation constant" equation or, in turn, of the double logarithmic plot: pK(a) vs. log([salt]⁻¹), exhibit a clear dependence on the nature of the cations. The effects of the different alkali cations on the pK(∞)(a) values follow the Hofmeister series. In fact, the pK(∞)(a(1)) and the pK(∞)(a(2)) values are smaller for Li+ and Na+ than for Rb+ and Cs+, with K+ at the border between the two, showing that the transitions require higher concentrations of protons to occur in the presence of high concentrations of the cosmotropic ions.

The CdCl2 effects on synthetic DNAs encaged in the nanodomains of a cationic water-in-oil microemulsion.

The present work is dedicated to the study of the interactions of CdCl(2) with the synthetic polynucleotides polyAT and polyGC confined in the nanoscopic aqueous compartment of the water-in-oil microemulsion CTAB/pentanol/hexane/water, with the goal to mimic in vitro the situation met by the nucleic acids in vivo. In biological structures, in fact, very long strings of nucleic acids are segregated into very small compartments having a radius exceedingly smaller than the length of the encapsulated macromolecule. For comparison, the behaviour of polyGC was also studied in aqueous solutions of matched composition. The conformational and thermal stabilities of both polynucleotides enclosed in the inner compartment of the microemulsion are scarcely affected by the presence of CdCl(2), whereas in solution immediate and large effects were observed also at room temperature. The lack of effects of CdCl(2) on the properties of the biopolymers entrapped in the aqueous core of the microemulsion has been attributed to the peculiar characteristics of the medium (low dielectric constant, in particular) which cause a total repression of the CdCl(2) dissociation that is not complete even in water. In fact, several of the numerous effects of CdCl(2) observed on the conformational stability of polyGC in aqueous solutions have also been ascribed to the limited dissociation of the cadmium salt.

The interaction of native calf thymus DNA with Fe(III)-dipyrido[3,2-a:2',3'-c]phenazine.

The mono and bis dipyrido[3,2-a:2',3'-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl(3) and [Fe(dppz)(2)]Cl(3), have been synthesized and characterized. The interaction of the Fe(III)dppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex-DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the Fe(III)dppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of Fe(III)dppz, indicate the existence of a rigid metal complex-DNA-binding leading to dominating chiral organization of Fe(III)dppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of Fe(III)dppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA-Fe(III)dppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex-DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the Fe(III)dppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate Fe(II)(phen)(2)dppz complex (phen=1,10-phenantroline).

Circular dichroism of polynucleotides: Interactions of NiCl2 with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) in a water-in-oil microemulsion.

The thermal behavior of the synthetic, high molecular weight, double stranded polynucleotides poly(dA-dT).poly(dA-dT) [polyAT] and poly(dG-dC).poly(dG-dC) [polyGC] solubilized in the aqueous core of the quaternary water-in-oil cationic microemulsion CTAB|n-pentanol|n-hexane|water in the presence of increasing amounts of NiCl(2) at several constant ionic strength values (NaCl) has been studied by means of circular dichroism and electronic absorption spectroscopies. In the microemulsive medium, both polynucleotides show temperature-induced modifications that markedly vary with both Ni(II) concentration and ionic strength. An increase of temperature causes denaturation of the polyAT duplex at low nickel concentrations, while more complex CD spectral modifications are observed at higher nickel concentrations and ionic strengths. By contrast, thermal denaturation is never observed for polyGC. At low Ni(II) concentrations, the increase of temperature induces conformational transitions from B-DNA to Z-DNA form, or, more precisely, to left-handed helical structures. In some cases, at higher nickel concentrations, the CD spectra suggest the presence of Z'-type forms of the polynucleotide.

Interaction of the alternating double stranded copolymer poly(dA-dT) x poly(dA-dT) with NiCl(2) and CdCl(2): solution behavior.

The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT) x poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0 mM; pH 7.8+/-0.2) in the presence of increasing concentrations of either Ni(2+) (borderline cation) or Cd(2+) (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaCl is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni(2+) than for Cd(2+). At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of T(m). In many cases, when denaturation starts, interstrand cross-linking occurs with formation of aggregates that precipitate.

Alkaline titrations of poly(dG-dC).poly(dG-dC): microemulsion versus solution behavior.

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid. In solution, the apparent pK(a) of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK(a) on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R(OH), defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R(OH) has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK(a) on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion.

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.