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The aquatic ecotoxicity of three α-amino acid-derived polyamidoamines (PAAs) was studied using zebrafish embryos as a viable vertebrate model organism. The PAAs examined were water-soluble amphoteric polyelectrolytes with a primarily negative charge, which were efficient flame retardants for cotton. The fish embryo acute toxicity test performed with PAA water solutions using 1.5-500 mg L-1 concentrations showed that toxicity did not statistically differ from the control. The survival rates were indeed >90%, even at the highest concentration; the hatching rates were >80%; and the numbers of morphological defects were comparable to those of the control. Tests using transgenic zebrafish lines indicated that the numbers of microscopic vascular and musculoskeletal defects were comparable to the control, with one random concentration showing doubled alterations. Sensory-motor tests in response to visual and tactile stimuli were also performed. In the presence of PAAs, embryos exposed to alternating light/dark cycles showed an insignificant mobility reduction during the dark phase. Touch-evoked response tests revealed a mild effect of PAAs on the neuromotor system at concentrations > 10 mg L-1. The cystine/glycine copolymer at 100 mg L-1 exhibited the greatest effect. Overall, the studied PAAs showed a minimal impact on aquatic systems and should be further considered as promising ecofriendly materials.
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Six differently charged amphoteric polyamidoamines, synthesized by the polyaddition of N,N'-methylenebisacrylamide to alanine, leucine, serine, arginine (M-ARG), glutamic acid (M-GLU) and a glycine/cystine mixture, were screened for their short-term phytotoxicity using a seed germination test. Lepidium sativum L. seeds were incubated in polyamidoamine water solutions with concentrations ranging from 0.156 to 2.5 mg mL-1 at 25 ± 1 °C for 120 h. The seed germination percentage (SG%), an indicator of acute toxicity, and both root and shoot elongation, related to plant maturation, were the considered endpoints. The germination index (GI) was calculated as the product of relative seed germination times relative radical growth. The SG% values were in all cases comparable to those obtained in water, indicating no detectable acute phytotoxicity of the polyamidoamines. In the short term, the predominantly positively charged M-ARG proved to be phytotoxic at all concentrations (GI < 0.8), whereas the predominantly negatively charged M-GLU proved to be biostimulating at intermediate concentrations (GI > 1) and slightly inhibitory at 2.5 mg mL-1 (0.8 < GI < 1). Overall, polyamidoamine phytotoxicity could be correlated to charge distribution, demonstrating the potential of the test for predicting and interpreting the eco-toxicological behavior of water-soluble polyelectrolytes.
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A novel polyamidoamine (M-PCASS) bearing a disulfide group and two phosphonate groups per repeat unit was obtained by reacting N,N'-methylenebisacrylamide with a purposely designed bis-sec-amine monomer, namely, tetraethyl(((disulfanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(phosphonate) (PCASS). The aim was to ascertain whether the introduction of phosphonate groups, well-known for inducing cotton charring in the repeat unit of a disulfide-containing PAA, increased its already remarkable flame retardant efficacy for cotton. The performance of M-PCASS was evaluated by different combustion tests, choosing M-CYSS, a polyamidoamine containing a disulfide group but no phosphonate groups, as a benchmark. In horizontal flame spread tests (HFSTs), M-PCASS was a more effective flame retardant than M-CYSS at lower add-ons with no afterglow. In vertical flame spread tests, the only effect was afterglow suppression with no self-extinguishment even at add-ons higher than in HFSTs. In oxygen-consumption cone calorimetry tests, M-PCASS decreased the heat release rate peak of cotton by 16%, the CO2 emission by 50%, and the smoke release by 83%, leaving a 10% residue to be compared with a negligible residue for untreated cotton. Overall, the set of results obtained envisage that the newly synthesized phosphonate-containing PAA M-PCASS may be suitable for specific applications as flame retardant, where smoke suppression or reduction of total gas released is a key requirement.
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Polyamidoamine hydrogels prepared by the radical post-polymerization of α,ω-bisacrylamide-terminated M-AGM oligomers, in turn obtained by the polyaddition of 4-aminobutylguanidine with N,N'-methylenebisacrylamide, were reinforced with raw silk fibers, which can establish covalent bonds with the polyamidoamine matrix via reaction of the amine groups in the lysine residues with the acrylamide terminals of the M-AGM oligomer. Silk/M-AGM membranes were prepared by impregnating silk mats with M-AGM aqueous solutions and subsequent crosslinking by UV irradiation. The guanidine pendants of the M-AGM units imparted the ability to form strong but reversible interactions with oxyanions, including the highly toxic chromate ions. The potential of the silk/M-AGM membranes to purify Cr(VI)-contaminated water down to the drinkability level, that is, below 50 ppb, was tested by performing sorption experiments both in static (Cr(VI) concentration 20-2.5 ppm) and flow conditions (Cr(VI) concentration 10-1 ppm). After static sorption experiments, the Cr(VI)-loaded silk/M-AGM membranes could easily be regenerated via treatment with a 1 M sodium hydroxide solution. Dynamic tests performed using two stacked membranes and a 1 ppm Cr(VI) aqueous solution reduced Cr(VI) concentration down to 4 ppb. Remarkably, the use of renewable sources, the environmentally friendly preparation process, and the goal achieved meet eco-design requirements.
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Hydrophilic cyclodextrin nanosponges were prepared by the oxo-Michael polyaddition in an aqueous solution at pH > 10 of α-, ß-, and γ-cyclodextrin with 1,4-bisacryloylpiperazine or 2,2-bisacrylamidoacetic acid. These nanosponges and, for comparison purposes, their precursor cyclodextrins were tested as sorbents of o-toluidine, a carcinogenic wastewater contaminant, by monitoring the depletion of o-toluidine from a 10-4 M (10 ppm) aqueous solutions. To this aim, an innovative analytical procedure was used: The voltammetric peak currents of o-toluidine in linear sweep voltammetry experiments were registered using multi-walled carbon nanotubes-modified glassy carbon electrodes. The experimental sorption curves fitted a mono-exponential kinetic model, and the residual o-toluidine was 0.16 ppm, one order of magnitude lower than those of all other sorbents reported so far. The sorption capacities ranged from 88 to 199 µmol g-1 (10-21.3 mg g-1), equal to or higher than those of the parent cyclodextrins. All nanosponges were completely regenerated by extracting with methanol. After regeneration, the sorption capacity slightly improved, suggesting a rearrangement of the nanosponge network. Overall, it may be reasonably concluded that the cyclodextrin nanosponges reported in this paper warrant potential as o-toluidine exhaustive sorbents.
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Ciclodextrinas , Nanotubos de Carbono , Contaminantes del Agua , ToluidinasRESUMEN
Linear amphoteric polyamidoamines (PAAs) are usually water-soluble, biodegradable and biocompatible. Crosslinked PAAs form in water hydrogels, retaining most of the favorable properties of their linear counterparts. The hydrogels prepared by the radical post-polymerization of the oligo-α,ω-bisacrylamido-terminated PAA called AGMA1, obtained by the polyaddition of 4-aminobutylguanidine (agmatine) with 2,2-bis(acrylamido)acetic acid, exhibit excellent cell-adhesion properties both in vitro and in vivo. However, due to their low mechanical strength, AGMA1 hydrogels cannot be sewn to biological tissues and need to be reinforced with fibrous materials. In this work, short silk fibers gave excellent results in this sense, proving capable of establishing covalent bonds with the PAA matrix, thanks to their lysine content, which provided amino groups capable of reacting with the terminal acrylamide groups of the AGMA1 precursor in the final crosslinking phase. Morphological analyses demonstrated that the AGMA1 matrix was intimately interconnected and adherent to the silk fibers, with neither visible holes nor empty volumes. The silk/H-AGMA1 composites were still reversibly swellable in water. In the swollen state, they could be sewn and showed no detachment between fibers and matrix and exhibited significantly improved mechanical properties compared with the plain hydrogels, particularly as regards their Young's modulus and elongation at break.
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Hidrogeles , Seda , Seda/química , Hidrogeles/química , Poliaminas/química , AguaRESUMEN
Polyamidoamines (PAAs) are biocompatible and biodegradable polymers with a huge potential as biomaterials for pharmaceutical applications. They are obtained by the step-wise aza-Michael polyaddition of bifunctional or multifunctional amines with bisacrylamides in water. To the best of our knowledge, no synthetic protocols leading to hyperbranched PAAs as well as PAA microgels have been published so far. To fill this gap, a statistical approach was established in this work to fine-tune the aza-Michael polyaddition stoichiometry when a multifunctional co-monomer (bf) is added to a mixture of bifunctional monomers with complementary functions (a2 + b2), possibly even in presence of a monofunctional co-monomer (b1), for obtaining either microgels or hyperbranched polymers by a one-pot reaction. For this purpose, two new equations, obtained by reworking the classic Flory-Stockmayer equations, were successfully applied to the synthesis of different model systems, obtaining biocompatible microgels with tunable size distribution (200-500 nm) and properly designed end-chains in a simple and straightforward way. The same mathematical approach allowed us to empirically evaluate the actual number of active reactive functions of the co-monomers. A number of selected systems, being evaluated for their cytotoxicity in vitro, proved highly cytocompatible and, therefore, endowed with great potential for pharmaceutical and medical applications.
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The aza-Michael polyaddition of L-arginine and N,N'-methylene-bis-acrylamide gives the biocompatible and easily cell-internalized polyamidoamine ARGO7. By controlled synthesis, two ARGO7 oligomers, namely a trimer and a pentamer, bearing acrylamide terminal units, were obtained as precursors of the ß-cyclodextrin-end-terminated oligomers P3 and P5, which have been shown to encapsulate curcumin at both pH 7.4 and 4.5. After lyophilization, P3- and P5-curcumin complexes gave stable water solutions. The apparent solubility of encapsulated curcumin was in the range 20-51 µg mL-1, that is, three orders of magnitude higher than the water solubility of free curcumin (0.011 µg mL-1). The drug release profiles showed induction periods both at pH levels 4.5 and 7.4, suggesting a diffusive release mechanism, as confirmed by kinetic studies. The release rate of curcumin was higher at pH 7.4 than at pH 4.5 and, in both cases, it was higher for the P5 complex. Encapsulated curcumin was more photostable than the free drug. Molecular mechanics and molecular dynamics simulations explain at atomistic level the formation of aggregates due to favorable van der Waals interactions. The drug molecules interact with the external surface of carriers or form inclusion complexes with the ß-cyclodextrin cavities. The aggregate stability is higher at pH 4.5.
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Linear polyamidoamines (PAAs) derived from the polyaddition of natural α-amino acids and N,N'-methylene bis(acrylamide) are intumescent flame retardants for cotton. Among them, the glycine-derived M-GLY extinguished the flame in horizontal flame spread tests at 4% by weight add-on. This paper reports on an extensive study aimed at understanding the molecular-level transformations of M-GLY-treated cotton upon heating in air at 300 °C, 350 °C and 420 °C. Thermogravimetric analysis (TGA) identified different thermal-oxidative decomposition stages and, coupled to Fourier transform infrared spectroscopy, allowed the volatile species released upon heating to be determined, revealing differences in the decomposition pattern of treated and untreated cotton. XPS analysis of the char residues of M-GLY-treated cotton revealed the formation of aromatic nanographitic char at lower temperature with respect to untreated cotton. Raman spectroscopy of the char residues provided indications on the degree of graphitization of treated and untreated cotton at the three reference temperatures. Solid state 13C nuclear magnetic resonance spectroscopy (NMR) provided information on the char structure as a function of the treatment temperature, clearly indicating that M-GLY favors the carbonization of cotton with the formation of more highly condensed aromatic structures.
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In this paper, bioinspired polyamidoamines (PAAs) were synthesized from N,N'-methylenebisacrylamide and nine natural α-amino acids: L-alanine, L-valine, L-leucine (M-LEU), L-histidine, L-serine, L-asparagine, L-glutamine (M-GLN), L-aspartic acid and L-glutamic acid (M-GLU) and their performance as flame retardants (FRs) for cotton were determined. The aim was to ascertain if the ability to protect cotton from fire by the process of intumescing, previously found for the glycine-derived M-GLY, was a general feature of α-amino acid-derived PAAs. None of the PAAs ignited by flame impingement, apart from M-LEU, which burned for a few seconds leaving 93% of residue. All of them formed carbon- and oxygen-rich, porous chars with a graphitic structure in the air at 350 °C, as revealed by X-ray photoelectron spectroscopy. All samples were tested as FRs for cotton by horizontal flame spread tests. At a 5% add-on, M-GLU and M-GLN extinguished the flame. The same results were obtained with all the other PAAs at a 7% add-on. The α-amino acid residues influenced the FR performance. The most effective were those that, by heating, were most suitable for producing thermally stable cyclic aromatic structures. All PAA-treated cotton samples, even when burning, left significant residues, which, according to scanning electron microscopy analysis, maintained the original cotton texture.
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The hitherto known polyamidoamines (PAAs) are not suitable as structural materials because they are usually water-soluble or swellable in water. This paper deals with the synthesis and characterization of semi-crystalline hydrophobic PAAs (H-PAAs) by combining different bis-sec-amines with bis-acrylamides obtained from C6-C12 bis-prim-amines. H-PAAs were initially obtained in a solution of benzyl alcohol, a solvent suitable for both monomers and polymers. Their number average molecular weights, M¯n, which were determined with 1H-NMR by evaluating the percentage of their terminal units, varied from 6000 to >10,000. The solubility, thermal properties, ignitability and water resistance of H-PAAs were determined. They were soluble in organic solvents, semi-crystalline and thermally stable. The most promising ones were also prepared using a bulk process, which has never been previously reported for PAA synthesis. In the form of films, these H-PAAs were apparently unaffected by water. The films underwent tensile and wettability tests. They showed similar Young moduli (260-263 MPa), whereas the maximum stress and the stress at break depended on the number of methylene groups of the starting bis-acrylamides. Their wettability was somewhat higher than that of common Nylons. Interestingly, none of the H-PAAs considered, either as films or powders, ignited after prolonged exposure to a methane flame.
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A linear polyamidoamine (PAA) named BAC-EDDS, containing metal chelating repeat units composed of two tert-amines and four carboxylic groups, has been prepared by the aza-Michael polyaddition of ethylendiaminodisuccinic (EDDS) with 2,2-bis(acrylamido)acetic acid (BAC). It was characterized by size exclusion chromatography (SEC), FTIR, UV-Vis and NMR spectroscopies. The pKa values of the ionizable groups of the repeat unit were estimated by potentiometric titration, using a purposely synthesized molecular ligand (Agly-EDDS) mimicking the structure of the BAC-EDDS repeat unit. Dynamic light scattering (DLS) and ζ-potential analyses revealed the propensity of BAC-EDDS to form stable nanoaggregates with a diameter of approximately 150 nm at pH 5 and a net negative charge at physiological pH, in line with an isoelectric point <2. BAC-EDDS stably chelated Gd (III) ions with a molar ratio of 0.5:1 Gd (III)/repeat unit. The stability constant of the molecular model Gd-Agly-EDDS (log K = 17.43) was determined as well, by simulating the potentiometric titration through the use of Hyperquad software. In order to comprehend the efficiency of Gd-BAC-EDDS in contrasting magnetic resonance images, the nuclear longitudinal (r1) and transverse (r2) relaxivities as a function of the externally applied static magnetic field were investigated and compared to the ones of commercial contrast agents. Furthermore, a model derived from the Solomon-Bloembergen-Morgan theory for the field dependence of the NMR relaxivity curves was applied and allowed us to evaluate the rotational correlation time of the complex (τ = 0.66 ns). This relatively high value is due to the dimensions of Gd-BAC-EDDS, and the associated rotational motion causes a peak in the longitudinal relaxivity at ca. 75 MHz, which is close to the frequencies used in clinics. The good performances of Gd-BAC-EDDS as a contrast agent were also confirmed through in vitro magnetic resonance imaging experiments with a 0.2 T magnetic field.
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Medios de Contraste/química , Gadolinio/química , Imagen por Resonancia Magnética , Poliaminas/química , Quelantes/química , Técnicas de Química Sintética , Ligandos , Imagen por Resonancia Magnética/métodos , Estructura Molecular , Nanopartículas , Fenoles/química , Poliaminas/síntesis química , Sulfóxidos/química , TermodinámicaRESUMEN
Strategies for endosomal escape and access to the cell nucleus are highly sought for nanocarriers to deliver their load efficiently following endocytosis. In this work, we have studied the uptake and intracellular trafficking of a polycationic polyamidoamine (PAA) endowed with a luminescent Ru complex, Ru-PhenAN, that shows unique trafficking to the cell nucleus. Live cell imaging confirmed the capacity of this polymer to access the nucleus, excluding artifacts due to cell fixation, and clarified that the mechanism of escape is light-triggered and relies on the presence of the Ru complexes and their capacity to absorb light and act as photosensitizers for singlet oxygen production. These results open up the possibility to use PAA-ruthenium complexes for targeted light-triggered delivery of genetic material or drugs to the cytosol and nucleus.
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Núcleo Celular/efectos de los fármacos , Complejos de Coordinación/farmacología , Luz , Fármacos Fotosensibilizantes/farmacología , Poliaminas/farmacología , Rutenio/farmacología , Cationes/química , Cationes/farmacología , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Células HeLa , Humanos , Estructura Molecular , Imagen Óptica , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Poliaminas/química , Rutenio/química , Oxígeno Singlete/metabolismo , Células Tumorales CultivadasRESUMEN
This paper aims at elucidating the degradation mechanism of linear polyamidoamines (PAAs) in water. PAAs are synthesized by the aza-Michael polyaddition of prim-monoamines or bis-sec-amines with bisacrylamides. Many PAAs are water-soluble and have potential for biotechnological applications and as flame-retardants. PAAs have long been known to degrade in water at pH ≥ 7, but their degradation mechanism has never been explored in detail. Filling this gap was necessary to assess the suitability of PAAs for the above applications. To this aim, a small library of nine PAAs was expressly synthesized and their degradation mechanism in aqueous solution studied by 1H-NMR in different conditions of pH and temperature. The main degradation mechanism was in all cases the retro-aza-Michael reaction triggered by dilution but, in some cases, hints were detected of concurrent hydrolytic degradation. Most PAAs were stable at pH 4.0; all degraded at pH 7.0 and 9.0. Initially, the degradation rate was faster at pH 9.0 than at pH 7.0, but the percent degradation after 97 days was mostly lower. In most cases, at pH 7.0 the degradation followed first order kinetics. The degradation rates mainly depended on the basicity of the amine monomers. More basic amines acted as better leaving groups.
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D- and L-arginine-based polyamidoamino acids, called D- and L-ARGO7, retain the chirality and acid/base properties of the parent ï¡-amino acids and show pH-dependent self-structuring in water. The ability of the ARGO7 chiral isomers to selectively interact with chiral biomolecules and/or surfaces was studied by choosing sodium deoxycholate (NaDC) as a model chiral biomolecule for its ability to self-assembly into globular micelles, showing enantio-selectivity. To this purpose, mixtures of NaDC with D-, L- or D,L-ARGO7, respectively, in water were analysed by circular dichroism (CD) spectroscopy and small-angle neutron scattering (SANS) at different levels of acidity expressed in terms of pD and concentrations. Differences in the CD spectra indicated chiral discrimination for NaDC/ARGO7 mixtures in the gel phase (pD 7.30) but not in the solution phase (pD 9.06). SANS measurements confirmed large scale structural perturbation induced by this chiral discrimination in the gel phase yet no modulation of the structure in the solution phase. Together, these techniques shed light on the mechanism by which ARGO7 stereoisomers modify the morphology of NaDC micelles as a function of pH. This work demonstrates chirality-dependent interactions that drive structural evolution and phase behaviour of NaDC, opening the way for designing novel smart drug delivery systems.
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This paper reports on synthesis, acid-base properties, and self-structuring in water of a chiral polyamidoamino acid, M-l-Gln, obtained from the polyaddition of N,N'-methylenebisacrylamide with l-glutamine, with the potential of establishing hydrogen bonds through its prim-amide pendants. The M-l-Gln showed pH-responsive circular dichroism spectra, revealing ordered conformations. Structuring was nearly insensitive to ionic strength but sensitive to denaturing agents. The NMR diffusion studies were consistent with a population of unimolecular nanoparticles thus excluding aggregation. The M-l-Gln had the highest molecular weight and hydrodynamic radius among all polyamidoamino acids described. Possibly, transient hydrogen bonds between l-glutamine molecules and M-l-Gln growing chains facilitated the polyaddition reaction. Theoretical modeling showed that M-l-Gln assumed pH-dependent self-ordered coil conformations with main chain transoid arrangements reminiscent of the protein hairpin motif owing to intramolecular dipole moments and hydrogen bonds. The latter were most numerous at the isoelectric point (pH 4.5), where they mainly involved even topologically distant main chain amide N-H and side chain amide C=O brought to proximity by structuring. Hydrogen bonds at pH 4.5 were also suggested by variable temperature NMR. The 2D NOESY experiments at pH 4.5 confirmed the formation of compact structures through the analysis of the main chain/side chain hydrogen contacts, in line with MD simulations.
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Gold nanoparticles are elective candidate for cancer therapy. Current efforts are devoted to developing innovative methods for their synthesis. Besides, understanding their interaction with cells have become increasingly important for their clinical application. This work aims to describe a simple approach for the synthesis of extra-small gold nanoparticles for breast cancer therapy. In brief, a biocompatible and biodegradable polyamidoamine (named AGMA1-SH), bearing 20%, on a molar basis, thiol-functionalized repeat units, is employed to stabilize and coat extra-small gold nanospheres of different sizes (2.5, 3.5, and 5 nm in gold core), and to generate a nanoplatform for the link with Trastuzumab monoclonal antibody for HER2-positive breast cancer targeting. Dynamic light scattering, transmission electron microscopy, ultraviolet visible spectroscopy, X-ray powder diffraction, circular dichroism, protein quantification assays are used for the characterization. The targeting properties of the nanosystems are explored to achieve enhanced and selective uptake of AGMA1-SH-gold nanoparticles by in vitro studies against HER-2 overexpressing cells, SKBR-3 and compared to HER-2 low expressing cells, MCF-7, and normal fibroblast cell line, NIH-3T3. In vitro physicochemical characterization demonstrates that gold nanoparticles modified with AGMA1-SH are more stable in aqueous solution than the unmodified ones. Additionally, the greater gold nanoparticles size (5-nm) is associated with a higher stability and conjugation efficiency with Trastuzumab, which retains its folding and anticancer activity after the conjugation. In particular, the larger Trastuzumab functionalized nanoparticles displays the highest efficacy (via the pro-apoptotic protein increase, anti-apoptotic components decrease, survival-proliferation pathways downregulation) and internalization (via the activation of the classical clathrin-mediated endocytosis) in HER-2 overexpressing SKBR-3 cells, without eliciting significant effects on the other cell lines. The use of biocompatible AGMA1-SH for producing covalently stabilized gold nanoparticles to achieve selective targeting, cytotoxicity and uptake is completely novel, offering an important advancement for developing new anticancer conjugated-gold nanoparticles.
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The polyamidoamine derived from N,N'-methylenebisacrylamide (M) and glycine (G), M-G, has been shown to be an effective flame-retardant (FR) for cotton in horizontal flame spread tests (HFST), extinguishing the flame at 5% add-on. Its activity was attributed to its intrinsic intumescence. In vertical flame spread tests (VFST), M-G failed to extinguish the flame even at 30% add-on. Conversely, in VFST, the polyamidoamine derived from M and cystine (C), M-C, inhibited cotton combustion at 16% add-on, but in HFST failed to extinguish the flame below 12% add-on. Its activity was ascribed to the release of sulfur-containing volatiles acting as radical scavengers. In this work, the FR effectiveness of M-Gm-Cn copolymers with different G/C ratio was compared with that of the M-G and M-C homopolymers and of M-G/M-C blends of the same compositions. In HFST, both copolymers and blends extinguished the flame. In particular, M-G50-C50 and (M-G/M-C)50/50 extinguished the flame, even at 7% add-on. In VFST, the copolymers with ≥50% M-C units, similar to M-C, inhibited cotton combustion at 16% add-on. At the same add-on, the M-G/M-C blends failed to extinguish the flame. It may be concluded that, in contrast to blends, copolymers combined the merits of both homopolymers in all tests.
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In this work, we report the synthesis of [Ru(phen)32+]-based complexes and their use as photosensitizers for photodynamic therapy (PDT), a treatment of pathological conditions based on the photoactivation of bioactive compounds, which are not harmful in the absence of light irradiation. Of these complexes, Ru-PhenISA and Ru-PhenAN are polymer conjugates containing less than 5%, (on a molar basis), photoactive units. Their performance is compared with that of a small [Ru(phen)32+] compound, [Ru(phen)2BAP](OTf)2 (BAP = 4-(4'-aminobutyl)-1,10-phenanthroline, OTf = triflate anion), used as a model of the photoactive units. The polymer ligands, PhenISA and PhenAN, are polyamidoamines with different acid-base properties. At physiological pH, the former is zwitterionic, the latter moderately cationic, and both intrinsically cytocompatible. The photophysical characterizations show that the complexation to macromolecules does not hamper the Ru(phen)32+ ability to generate toxic singlet oxygen upon irradiation, and phosphorescence lifetimes and quantum yields are similar in all cases. All three compounds are internalized by HeLa cells and can induce cell death upon visible light irradiation. However, their relative PDT efficiency is different: the zwitterionic PhenISA endowed with the Ru-complex lowers the PDT efficiency of the free complex, while conversely, the cationic PhenAN boosts it. Flow cytometry demonstrates that the uptake efficiency of the three agents reflects the observed differences in PDT efficacy. Additionally, intracellular localization studies show that while [Ru(phen)2BAP](OTf)2 remains confined in vesicular structures, Ru-PhenISA localization is hard to determine due to the very low uptake efficiency. Very interestingly, instead, the cationic Ru-PhenAN accumulates inside the nucleus in all treated cells. Overall, the results indicate that the complexation of [Ru(phen)2BAP](OTf)2 with a cationic polyamidoamine to give the Ru-PhenAN complex is an excellent strategy to increase the Ru-complex cell uptake and, additionally, to achieve accumulation at the nuclear level. These unique features together make this compound an excellent photosensitizer with very high PDT efficiency.
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Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Fotoquimioterapia , Poliaminas/farmacología , Rutenio/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Estructura Molecular , Tamaño de la Partícula , Poliaminas/química , Rutenio/química , Células Tumorales CultivadasRESUMEN
The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer d,l-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 Å thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the Å discreteness.