Polarity studies of single polyelectrolyte layers in polyelectrolyte multilayers probed by steady state and life time doxorubicin fluorescence

Hypothesis: Polarity in polyelectrolyte multilayers (PEMs) may vary from the inner to the top layers of the film as the charge compensation of the layers is more effective inside the PEMs than in outer layers. Doxorubicin hydrochloride (DX) is used here to sense polarity at the single polyelectrolyte level inside PEMS. Experimental: DX is complexed electrostatically to a polyanion, either polystyrene sulfonate (PSS) or polyacrylic acid (PAA) and assembled at selected positions in a multilayer of the polyanion and polyally lamine hydrochloride (PAH) as polycation. Local polarity in the layer domain is evaluated through changes in the intensity ratio of the first to second band of spectra of DX (I1/I2 ratio) by steady state flu orescence, and by Lifetime fluorescence. Findings: PAH/PSS multilayers, show a polarity similar to water with DX/PSS as top layer, decreasing to I1/ I2 ratios similar to organic solvents as the number of polyelectrolyte layers assembled on top increases. For PAH/PAA multilayers, polarity values reflect a more polar environment than water when DX/PAA is the top layer, remaining unaltered by the assembly of polyelectrolyte layers on top. Results show that different polar environments may be present in a PEM when considering polarity at the single layer level.Fil: Martinelli, Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; Argentina. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Tasca, Elisamaria. Centro de Investigación Cooperativa en Biomateriales; España. Università degli Studi di Roma "La Sapienza"; ItaliaFil: Andreozzi, Patrizia. Università degli Studi di Firenze; Dipartimento di Chimica “Ugo Schiff”; Italia. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Libertone, Sara. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Ritacco, Hernán Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; ArgentinaFil: Giustini, Mauro. Università degli Studi di Roma "La Sapienza"; Italia. Università degli Studi di Firenze; Dipartimento di Chimica “Ugo Schiff” ; ItaliaFil: Moya, Sergio Enrique. Centro de Investigación Cooperativa en Biomateriales; España. Università degli Studi di Roma "La Sapienza"; Itali

F127 pluronic in coformulation with sodium cholate as hosting system for doxorubicin

F127 pluronic in coformulation with sodium cholate as hosting system for doxorubicin Elisamaria Tasca, Mauro Giustini, Luciano Galantini, Karin Schillén* Chemistry Department, University “La Sapienza”, 00185 Rome (Italy), * Division of Physical Chemistry, Department of Chemistry, Lund University, Lund (Sweden) Nonionic pluronic triblock copolymers received considerable attention as modern drug delivery carriers [1]. They have the general formula PEOx-PPOy-PEOx, and are composed of a hydrophobic poly(propylene oxide) (PPO) block and two units of a hydrophilic poly(ethylene oxide) (PEO) block [2]. Their amphiphilic character results into surfactant properties, which includes the ability to interact with hydrophobic surfaces and biological membranes. Above their critical micelle concentration (cmc), these copolymers self-assemble into micelles. Due to their unique core−shell structure, polymeric micelles such as F127 have the ability to solubilize hydrophobic drugs in the PPO core, thereby enhancing their solubility in water media. In addition they are suitable for drug delivery in medicine [3] as they are non-toxic and stabilized to aggregation, protein adsorption and deactivation because of the presence of the PEO corona [4]. The stability of the loaded pluronic micelles is however low due to their high cmc (∼1 mM for F127) resulting in their disaggregation by dilution or interaction with the blood components. The mixture of pluronic micelles with other polymers/surfactants [5,6] enhances the stability of the resulting micelles thus increasing the bioavailability of the encapsulated drugs. To this end, the loading efficiency of bile salt/pluronic coformulation toward the fluorescent anticancer antibiotic doxorubicin (DX) has been studied. DX is administered as chlorohydrate to enhance its solubility in water. This limits its solubilization into F127 micelles to the corona region. To promote its solubility in the hydrophobic core of F127 micelles the coformulation with the cationic bile salt sodium cholate (NaC) has been used. Indeed, in the presence of NaC the DX experience a more apolar environment, as indicated by its fluorescence spectra. The presence of DX seems also to alter the F127 micellar structure, as deduced by DLS experiments. [1] Roesler et al., Adv. Drug Delivery Rev. 2012, 64, 270 [2] S. Ghosh et al., J. Phys. Chem. B 2014, 118, 11437 [3] W. Zhang et al., Biomaterials 2011, 32, 2894 [4] Z. Sezgin et al., Eur. J. Pharm. Biopharm. 2006, 64, 261 [5] P. Singla et al., Spectrochim. Acta A 2018, 191, 143 [6] S. Bayati et al., Langmuir, 2015, 31, 1351

Doxorubicin and self-assembly: from main character to guest actor

The anticancer antibiotic doxorubicin (DX) is one of the most active drugs against both solid and systemic tumours. In water solutions, even at a 10-5M concentration, it is present for more than 50% as dimers. By rising the DX concentration to 10-2M and by adding a critical amount of NaCl, the solution turns into a thixotropic gel. Gel formation is due to the ability of doxorubicin to form, in the presence of salt, supramolecular helical chiral structures, similar to long fibers. One of the main concerns in the clinical use of DX is related to the severe side effects (cardiotoxicity being the principal) that impair its use. One strategy to overcome this problem is to host DX into suitable carriers (liposomes, mainly). Because of their self-assembly ability as well as to their intrinsically stealth nature, poly(ethylene oxide) (PEO)/poly(propylene oxide) (PPO) copolymers in general and F127 in particular, they can be used for DX vehiculation. To increase the solubility of DX in water, it is administered in the form of hydrochloride. Its cationic nature, however, limits its solubilization to the polar corona region of the F127 micelles. The cosolubilization with an anionic bile salt has been attempted to be used to drive the DX solubilization into the hydrophobic core of the F127 micelles. The NaC/F127 hydrophobic core provides a safe environment for DX, slowing down its degradation thus potentially increasing its latency time once injected into an organism. This should result into a reduction of the DX therapeutic dose thus leading to an attenuation of the undesired side-effects

Sodium cholate/PEO-PPO-PEO triblock copolymer mixed micelles as stealth nanocarrier for doxorubicin

Polymer nanomaterials have received a great deal of interest as vehicles used for diagnostic and therapeutic agents [1]. The loading efficiency of a bile salt/block copolymer coformulation toward the fluorescent anticancer antibiotic doxorubicin has been studied. The coformulation is based on the anionic bile salt sodium cholate (NaC) and a nonionic triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) denoted EO100-PO65-EO100 (F127) that itself forms micelles in water with a core composed mostly of PPO and a PEO corona. Doxorubicin is usually administered as the hydrochloride (DX) to increase its solubility in water. This limits its partitioning to the corona region of F127 micelles. To promote its solubility in the hydrophobic core, NaC was introduced in the system. The resulting systems obtained by varying the NaC/F127 mole ratio were characterized by small angle X-ray and dynamic light scattering (SAXS and DLS) in combination with spectroscopic fluorescence techniques (steady state and time-resolved). The host structure is not affected by the guest presence as deduced by SAXS and DLS data while in the presence of NaC, DX experiences a more apolar environment as indicated by its characteristic fluorescence behaviour (Figure 1). The stability against degradation of DX in the mixed micellar system was markedly enhanced relative to aqueous solutions without the coformulation [2]. The DX increased time stability in the NaC/pluronic mixed micellar systems is a promising characteristic that could lead to an increase of the drug latency and protection against hydrolytic degradation. In addition, the PEO hydrophilic corona could provide a certain level of biocompatibility and stealth characteristics to the mixed system, thus being attractive from an applicative point of view

Self-assembly of doxorubicin into gels

Self-assembly of doxorubicin into gels Elisamaria Tasca1*, Mauro Giustini1,2, Marco D’Abramo1, Luciano Galantini1,2, Gerardo Palazzo2,3 1 Chemistry Dept., “La Sapienza” University, P.le Aldo Moro 5, 00185-Rome, [Italy] 2 CSGI (O.U. of Bari), Chemistry Dept., University of Bari, 70126 Bari [Italy] 3 Chemistry Dept., University of Bari, Via Orabona 4, 70126-Bari, [Italy] *[email protected] The closely related anthracyclines doxorubicin (DX), daunomycin (DN) and epirubicin (EPI) in aqueous solution are present essentially as dimers at concentrations above or equal to 10-5M. However, DX, but not DN and EPI, in the presence of NaCl, at concentrations above a threshold value that depends also on the DX concentration, gives rise to gels [1-2]. The properties of these gels have been studied by UV-Vis, circular dichroism (Figure 1-A), steady-state and time resolved fluorescence (TCSPC - Figure 1-B) spectroscopy and SAXS (Figure 1-C), supported by MD simulations and fluorescence microscopy. The experimental results and the simulations suggest self-assembly of the somewhat distorted dimers in supramolecular aggregates, possibly in the form of fibrils, that would aggregate into larger structures to give the gels [3]. References [1] M. Giomini, A.M. Giuliani, M. Giustini and E. Trotta, Biophys. Chem., 1992 (45) 31. [2] E. Hayakawa, K. Furuya, T. Kuroda, M. Moriyama and A. Kondo, Chem. Pharm. Bull., 1991 (39) 1282. [3] M. Giustini, A.M. Giuliani, E. Tasca, M. D’Abramo, L. Galantini and G. Palazzo, manuscript in preparation

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

HypothesisDoxorubicin 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.ExperimentsWe 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.FindingsThe 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

The self-association equilibria of doxorubicin at high concentration and ionic strength characterized by fluorescence spectroscopy and molecular dynamics simulations

The self-association equilibria of doxorubicin hydrochloride (DX), at high drug and NaCl concentrations, are studied by temperature scan fluorescence spectroscopy, with the support of molecular dynamics (MD) calculations. Even though all anthracyclines show dimerization equilibria, DX only can further associate into long polymeric chains according to DXmon ⇄ DXdim ⇄ DXpol. This is reflected not only in the mechanical properties of DXpol solutions (behaving as thixotropic gels) but also in their spectroscopic behaviour. Fluorescence, in particular, is the technique of election to study this complex set of equilibria. Upon increasing the temperature, DXpol melts into DXdim, which in turn is in equilibrium with DXmon. Since DXdim is non fluorescent, with a fluorescence temperature scan experiment the DXpol⇄ DXmon equilibrium is probed. However, also information on the DX dimerization equilibrium can be derived together with the relevant thermodynamic parameters ruling the dimerization process (ΔHdim °= -56 kJ mol-1; ΔSdim °= -97 J mol-1 K-1). The residence time of DX molecules in the dimer (74.7 μs), as well as the monomers mutual orientation in the dimer, are characterized by means of theoretical and computational modelling. © 2019 Elsevier B.V

Antibacterial Layer‐by‐Layer Films of Poly(acrylic acid)–Gentamicin Complexes with a Combined Burst and Sustainable Release of Gentamicin

There is an urgent need for the development of effective antibacterial coatings to cope with more and more resistant bacterial strains in medical environments, and particularly to prevent nosocomial infections following bone implant surgery. Polyelectrolyte multilayers (PEMs) based on poly-llysine (PLL) and complexes of poly(acrylic acid) (PAA) and gentamicin have been fabricated here applying the layer-by-layer (LbL) technique. Complexes are prepared by mixing PAA and gentamicin solutions in 500 × 10−3 m NaCl at pH 4.5. The assembly of PLL and the complexes follows an exponential growth allowing a high loading of gentamicin in a four bilayer PEM. Although PEMs are stable and do not degrade at physiological pH, there is a continuous release of gentamicin at pH 7.4. PEMs show an initial burst release of gentamicin in the first 6 h, which liberates 58% of the total gentamicin released during the experiment, followed by a sustainable release lasting over weeks. This release profile makes the coating appealing for the surface modification of bone implants as a high concentration of antibiotics is necessary during implant surgery while a lower antibiotic concentration is needed until tissue is regenerated. PEMs are effective in preventing the proliferation of the Staphylococcus aureus strain.Fil: Escobar, Ane. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Muzzio, Nicolás Eduardo. Centro de Investigacion Cooperativa En Biomateriales.; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Andreozzi, Patrizia. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Libertone, Sara. Centro de Investigacion Cooperativa En Biomateriales.; EspañaFil: Tasca, Elisamaria. Università degli studi di Roma "La Sapienza"; ItaliaFil: Azzaroni, Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Grzelczak, Marek. Fundación Vasca para la Ciencia; España. Donostia International Physics Center; EspañaFil: Moya, Sergio Eduardo. Centro de Investigacion Cooperativa En Biomateriales.; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Antibacterial Coatings: Antibacterial Layer‐by‐Layer Films of Poly(acrylic acid)–Gentamicin Complexes with a Combined Burst and Sustainable Release of Gentamicin (Adv. Mater. Interfaces 22/2019)

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Poloxamer/sodium cholate co-formulation for micellar encapsulation of doxorubicin with high efficiency for intracellular delivery: an in-vitro bioavailability study

Hypothesis: Doxorubicin hydrochloride (DX) is widely used as a chemotherapeutic agent, though its severe side-effects limit its clinical use. A way to overcome these limitations is to increase DX latency through encapsulation in suitable carriers. However, DX has a high solubility in water, hindering encapsulation. The formulation of DX with sodium cholate (NaC) will reduce aqueous solubility through charge neutralization and hydrophobic interactions thus facilitating DX encapsulation into poloxamer (F127) micelles, increasing drug latency. Experiments: DX/NaC/PEO-PPO-PEO triblock copolymer (F127) formulations with high DX content (DX-PMs) have been prepared and characterized by scattering techniques, transmission electron microscopy and fluorescence spectroscopy. Cell proliferation has been evaluated after DX-PMs uptake in three cell lines (A549, Hela, 4T1). Cell uptake of DX has been studied by means of confocal laser scanning microscopy and flow cytometry. Findings: DX-PMs formulations result in small and stable pluronic micelles, with the drug located in the apolar core of the polymeric micelles. Cell proliferation assays show a delayed cell toxicity for the encapsulated DX compared with the free drug. Data show a good correlation between cytotoxic response and slow DX delivery to nuclei. DX-PMs offer the means to restrict DX delivery to the cell interior in a highly stable and biocompatible formulation, suitable for cancer therapy.Fil: Tasca, Elisamaria. Università degli studi di Roma "La Sapienza"; ItaliaFil: Andreozzi, Patrizia. Basque Research and Technology Alliance; España. Università degli Studi di Firenze; ItaliaFil: Del Giudice, Alessandra. Università degli studi di Roma "La Sapienza"; ItaliaFil: Galantini, Luciano. Università degli studi di Roma "La Sapienza"; Italia. Università degli Studi di Bari; ItaliaFil: Schillén, Karin. Lund University; SueciaFil: Giuliani, Anna Maria. Università degli Studi di Palermo; ItaliaFil: Ramirez, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Basque Research and Technology Alliance; ArgentinaFil: Moya, Sergio Enrique. Basque Research and Technology Alliance; ArgentinaFil: Giustini, Mauro. Università degli studi di Roma "La Sapienza"; Italia. Università degli Studi di Bari; Itali