Probing the EumelaninâSilica Interface in Chemically Engineered

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Probing the Eumelanin-Silica Interface in Chemically Engineered Bulk Hybrid Nanoparticles for Targeted Subcellular Antioxidant Protection Brigida Silvestri, Giuseppe Vitiello, Giuseppina Luciani, Vincenzo Calcagno, Aniello Costantini, Maria Gallo, Silvia Parisi, Simona Paladino, Mariagrazia Iacomino, Gerardino D'Errico, Maria Federica Caso, Alessandro Pezzella, and Marco d'Ischia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11839 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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ACS Applied Materials & Interfaces

Probing the Eumelanin-Silica Interface in Chemically Engineered Bulk Hybrid Nanoparticles for Targeted Subcellular Antioxidant Protection

Brigida Silvestri, Costantini,

†

†

Giuseppe Vitiello,

†∫

Giuseppina Luciani,

†

Vincenzo Calcagno,

Maria Gallo,ǁ Silvia Parisi,*,ǁ,◊ Simona Paladino,

ǁ,◊

♦

Aniello

Mariagrazia Iacomino,

∆

Gerardino D'Errico, ∆∫ M. Federica Caso,≈ Alessandro Pezzella* ᴨ,#,∆, and Marco d'Ischia ∆ † Department of Chemical, Materials and Production Engineering, University of Naples “Federico II”, p.le V. Tecchio 80, 80125 Naples, Italy ∫CSGI, Consorzio interuniversitario per lo sviluppo dei Sistemi a Grande Interfase, Sesto Fiorentino, via della Lastruccia 3, Firenze, Italy. ♦Institute of Biophysics and Medical Physic, University of Leipzig, Härtelstraße 16-18, D – 04107 Leipzig, Germany ǁ Department of Molecular Medicine and Medical Biotechnology, University of Naples ‘‘Federico II’’, Naples, Via Pansini, 5- 80131- Napoli, Italy - E-mail [email protected] ◊ Ceinge Biotecnologie Avanzate Via Gaetano Salvatore 486, 80145 Naples, Italy

ACS Paragon Plus Environment

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∆ Department of Chemical Sciences, University of Naples “Federico II” Via Cintia 4, I-80126 Naples Italy ≈

Nanofaber Spin-off at Italian National Agency for New Technologies, Energy and Sustainable

Economic Development (ENEA), Casaccia Research Centre, Via Anguillarese 301, 00123 Rome, Italy ᴨ

National Interuniversity Consortium of Materials Science and Technology (INSTM), Florence,

Italy ♯ Institute for Polymers, Composites and Biomaterials (IPCB), CNR, Via Campi Flegrei 34, I80078 Pozzuoli (NA), Italy E-mail [email protected]

KEYWORDS. Silica nanoparticles; Eumelanins; Cytoprotection; Antioxidant; Hybrid nanomaterial; Oxidative stress; Lysosomal localization

ABSTRACT

We disclose herein the first example of stable monodispersed hybrid nanoparticles (termed MelaSil-NPs) made up of eumelanin biopolymer intimately integrated into a silica nanoscaffold matrix and endowed with high antioxidant and cytoprotective effects associated with a specific subcellular localization. MelaSil-NPs have been fabricated by an optimized sol-gel methodology


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involving ammonia-induced oxidative polymerization of a covalent conjugate of the eumelanin building

block

5,6-dihydroxyindole-2-carboxylic

acid

(DHICA)

with

3-

aminopropyltriethoxysilanes (APTS). They displayed a round-shaped (ca. 50 - 80 nm) morphology, exhibited the typical EPR signal of eumelanin biopolymers and proved effective in promoting decomposition of hydrogen peroxide under physiologically relevant conditions. When administered to human ovarian cancer cells (A2780) or cervical cancer cells (HeLa), MelaSilNPs were rapidly internalized and co-localized with lysosomes, and exerted efficient protecting effects against hydrogen peroxide-induced oxidative stress and cytotoxicity.

Introduction.

Bioinspired nanoparticles (NPs) endowed with antioxidant and cytoprotective properties associated with specific subcellular targeting mechanisms are currently an active research focus in

state-of-the-art

nanomedicine.

Besides

serving

as

biocompatible

platforms

for

functionalization or loading with active components and localization to specific compartments or organelles of target cells for both diagnostic and therapeutic applications, antioxidant NPs hold promise for treating specific conditions and disease states associated with oxidative stress and excessive production of reactive oxygen species (ROS).1 2 A representative example is provided by ceria (CeO2) NPs which exhibit ROS-scavenging activity via redox shuttling mechanisms and can protect cells against superoxide and hydrogen peroxide-induced injury.3 Although diverse nanomaterials, chiefly inorganic, exhibit sufficient antioxidant capacity coupled with greater stability than natural antioxidant defense systems, including enzymes, none of them seems to


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meet critical requisites of complete biocompatibility and stability to manipulation for further functionalization. In an attempt to expand the scope of ROS scavenging nanomaterials for biomedical applications, we recently focused our attention to the design of hybrid nanosystems integrating a soft bioinspired eumelanin-based antioxidant component into a non-toxic inorganic nanoplatform combining stability to biological environments with tunable permeability and porosity. Eumelanins, the characteristic black insoluble biopolymers of human skin and hair and cephalopod ink,4

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display unique physicochemical and radical scavenging6 properties which

make them a most attractive bioinspired functional material for nanotechnological and nanomedical applications. Eumelanin-based NPs are currently exploited for antioxidant activity,7 8 9 10 11

drug delivery,12

13

photoacoustic (PA) imaging,14 multimodal imaging of tumors,15 skin

photoprotection,16 determination of antioxidant capacity of biological fluids,17 polymer nanocomposite reinforcement7 and radioprotection.18 Very recently, PEG-modified melanin NPs have been shown to display anti-inflammatory properties mediating effective protection of ischemic brains.1 19 Despite the increasing impact of eumelanin research in nanotechnology and biomedicine, several gaps and technical issues are still to be settled. These include difficulties in the preparation of size-controlled melanin-like nanoparticles with good dispersibility in water and biological media, in the preservation of major eumelanin properties, such as free radical scavenging, during the process of fabrication, and in the chemical functionalization of eumelanin biopolymers. Promising strategies to overcome these issues are based on the construction of hybrid NPs in which crucial properties for in vivo applications, such as size, reactivity and aggregation are finely controlled by the balance of inorganic and organic components.20


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In support of this, we recently demonstrated that the biofunctional properties of eumelanin can be markedly enhanced if its formation occurs through templated polymerization in the presence of a ceramic phase, leading to hybrid nanostructures.21 Coated and hybrid functional nanoarchitectures were fabricated, integrating a eumelanin-like 5,6-dihydroxyindole-2carboxylic acid (DHICA) polymer with TiO2 via LMCT (ligand to metal charge transfer)-based photo-oxidative process.22 Herein, to probe and expand the scope of eumelanin-based hybrid NPs, we investigated a novel prototype, termed MelaSil, which combines the potent antioxidant activity of DHICA melanin with the convenient properties of silica nanoparticles. Silica, which has been approved by FDA for manifold diagnostic and therapeutic applications, represents a most valuable option for hybrid NPs. It may ensure active targeting as well as sufficient stability for imaging23 and drug delivery applications24,25 because of tunable structural and surface properties, as well as good biocompatibility and bioactivity. To increase NP stability, cytocompatibility and ease of internalization into cultured cells for protection against oxidative stress injury, preparation of a bulk hybrid was preferred over a core-shell architecture, for which limited stability to chemical degradation of the outer organic component and a higher tendency to aggregation was predicted. Optimization of the synthetic strategy allowed to obtain stable MelaSil-NPs smaller than 100 nm, which could be tagged at the silica component with a fluorescent marker to track the carrier by fluorescent imaging. Efficient cytoprotective properties of MelaSil-NPs against ROS damage could be demonstrated following internalization and subcellular localization in mammalian cells.

Results and Discussion.


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MelaSil-NPs were obtained by a sol-gel methodology which relies on the oxidative polymerization of DHICA in TEOS-APTS (0.25% mol) mixtures. The rationale and the procedure developed to prepare hybrid MelaSil-NPs are illustrated in Figure 1. The synthetic approach relied on the formation of an APTS-DHICA conjugate that was obtained through EDC/NHS coupling reaction between the amino group of APTS and the carboxyl group of DHICA. Reaction conditions were set up based on previous literature20 and temperature was kept low to avoid undesirable side reactions.26 Interestingly, covalent conjugation of DHICA to APTS proved to be critical to obtain stable hybrid NPs, since omission of the EDC-promoted conjugation step led to broad distribution of NP size, micrometric aggregates (SI, Figure S1) and apparent lack of incorporation of melanin, as judged by the light color of the nanostructures (SI, Figure S2) Conjugation of APTS with rhodamine B isothiocyanate (RBITC) allowed to prepare a fluorescent RBITC-APTS monomer. Both hybrid (DHICA-APTS) and fluorescent (RBITCAPTS) monomers were used to produce fluorescent hybrid MelaSil-NPs by an in situ synthesis process, using a modified Stöber sol–gel method (Figure 1). Careful optimization of the synthetic procedure was also important to produce stable monodisperse hybrid nanostructures since DHICA polymerization as well as hydrolysis and condensation of TEOS are fast processes depending on experimental conditions: in particular, higher concentrations of precursors as well as higher APTS/TEOS ratios led to a bimodal distribution of large NP aggregates, as evidenced by SEM analysis (SI, Figure S3a, b)


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Figure 1 – Schematic view of design and preparation of MelaSil nanoparticles. The final detour involving rhodamine B tagging is also reported (2’). Pictures of the reaction time course are reported in SI Figure S4.

NP formation was assessed by both dynamic light scattering (DLS) measurements and scanning electron microscopy (SEM) images. DLS revealed a single broad population (Figure 2A) with a hydrodynamic radius of 76.0 ± 5.0 nm and a negative surface charge, as indicated by ζ-potential measurements (-43.4 ± 1.2mV). SEM analysis of MelaSil-NPs revealed remarkably regular round-shaped nanostructures (Figure 2B). Moreover, as the fabrication conditions are not compatible with sole silica as well as melanin NPs, the MelaSil NPs are not e mixture of MelaSil NPs and other NPs (i.e. silica NPs, and melanin NPs). Pore size analysis by nitrogen adsorption gave results that were consistent with largely compact, non-porous NP structures with a specific surface area of about 72.0 m2·g-1. All morphological properties are summarized in Table 1.


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A Hydrodynamic radius distribution (a.u.)

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A

10

100

1000

10000

RH (nm)

Figure 2– Panel A: Intensity weighed hydrodynamic radius distribution of MelaSil-NPs_0.25%. The distribution was obtained from one of the DLS measurements performed with the instrumental configuration corresponding to a scattering angle of 173°. This distribution showed the existence of a single population of aggregates within the suspension. Panel B: SEM micrograph of MelaSil-NPs_0.25%.

MelaSil-NPs_0.25%

RH (nm)

ζ-potential (mV)

Surface area (m2g-1)

76.0 ± 5.0

-43.4 ± 1.2

72.0 ± 5.0

Table 1 – Morfological properties of MelaSil-NPs at 0.25% APTS.

Overall DLS, SEM and TEM data (SI Figure S5) indicated that integrating DHICA melanin into silica NPs resulted in a higher stability with efficient control of morphology, and a low tendency to aggregate, an important achievement to improve biodistribution and cellular uptake and a convenient alternative to surface functionalization with bioactive molecules.27 28 29


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To characterize the eumelanin component of MelaSil-NPs, electron paramagnetic resonance (EPR) spectra were recorded on samples obtained under different conditions and analyzed against DHICA-melanin (Figure 3A and Table 2) to gain an insight into the nature of the paramagnetic centers and the supramolecular organization of eumelanins within the hybrid NPs.

A

1.0

B

(a)

0.8

a

0.6

(I/I0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


∆B

(b)

0.4

b

0.2

3440 3460 3480 3500 3520 3540 3560

0.0 0.0

0.2

B(G )

0.4

0.6

0.8

1.0

1/2

(P/P ) 0

Figure 3 – EPR spectra (A) and plot of normalized amplitude vspower intensities of free radicals (B) for MelaSil-NPs (a) and DHICA-melanin (b) samples.

MelaSil-NPs showed an intense peak at a g value of 2.0035 ± 0.0004, consistent with the presence of carbon-centered radicals derived from DHICA polymerization.30

21

The asymmetric

lineshape reveals the superposition of signals due to different radical centers. Notably, however, the signal from MelaSil-NPs (Figure 3A, spectrum a) exhibited lower amplitude (∆B) than that of pure DHICA melanin (Figure 2A, spectrum b), indicating a larger distance between the radical centers within the nanostructures, probably due to the presence of the silica matrix controlling aggregation and organization of the melanin component. Moreover, a marked difference was observed in the normalized power saturation profiles (Figure 3B): the heterogeneous trend


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observed in the case of MelaSil-NPs suggests a non-uniform spatial distribution of free-radicals relative to the melanin.30

21

EPR data showed a marked influence of chemical composition (e.g.

amount of APTS) on signal amplitude, suggesting marked changes in NP structure and properties in line with morphological analysis (see SI, Figure. S6 and Table S1).

∆B (G)

Spin-density (spin×g-1)

DHICA-melanin

5.6 ± 0.2

4.5 × 1021

MelaSil -NPs

4.2 ± 0.2

6.4 × 1019

Samples

Table 2 - EPR parameter obtained from spectra of DHICA-melanin and MelaSil nanoparticles.

Based on previous studies31

32 33

indicating efficient endocytosis of silica mesoporous NPs by

various cells without toxic effects, in a final series of experiments we investigated the antioxidant and cytoprotective effects of MelaSil-NPs toward different cell lines. Initially, the time and efficiency of MelaSil-NPs cellular uptake were determined on human ovarian cancer cells (A2780). Cells were incubated with 100 µg/ml of MelaSil-NPs conjugated with rhodamine and the uptake was monitored at different time points by fluorescence microscopy. MelaSil-NPs were found to be quickly internalized in the cells (as early as 1 h) attaining maximum values between 8 and 24 hrs incubation (Figure 4, other data not shown). No evident effects of cytotoxicity were detected by morphological inspection and appearance of stained nuclei after 24 hours of MelaSil-NP treatment (Figure 4). Comparable results were obtained using human cervical cancer cells (HeLa) (SI, Figure S7). Interestingly, MelaSil-NPs persisted in the cells at


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least over 6 days (SI, Figure S8) indicating good stability and compatibility with cell life (see also below). In further experiments, human epitheloid cervix carcinoma cells (HeLa) were incubated with rhodamine-conjugated MelaSil-NPs for 24 hours, chased for 24 h and then stained with specific antibodies for cellular compartments. As shown in Figure 5, the cells were stained with both Lamp1 and the lysotracker Dye showing that MelaSil-NPs were co-localized with lysosomes, being excluded from other cellular compartments (even in very close proximity to the Golgi complex). It has been reported that negative surface charges would produce migration of NPs from endosomal/lysosomal compartments to the cytosol31 and analogous mechanisms could operate in the case of MelaSil-NPs because of the negative charge imparted by the eumelanin component.


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Figure 4: Time course analysis of the MelaSil NPs uptake in mammalian cells. A2780 (ovarian cancer) cells were incubated with rhodamine-conjugated MelaSil NPs (red) at the indicated time points. After incubation, the cells were extensively washed to remove the residual particles in the medium and then, the cells were fixed and nuclei were stained with DAPI (blue). The presence of rhodamine-conjugated MelaSil NPs was analysed by fluorescent microscopy. The particles showed a specific fluorescent signal compared to the negative control (ctrl, cells


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untreated with the particles). Corresponding bright fields images (bottom) did not show any evident cytotoxicity effects upon MelaSil NP uptake.


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Figure 5: MelaSil NPs localization in the cells. HeLa cells were incubated with rhodamineconjugated MelaSil NPs (red) for 24 hours and then the residual particles were washed out. After further 24 hours cells were fixed and stained with specific antibodies against organelle markers (giantin for Golgi, TOM 20 for mitochondria, Lamp1 for lysosomes) revealed with a secondary antibody Alexa-488 conjugated (green). Lysotracker (red) was used to stain the lysosomes in vivo upon uptake of MelaSil NPs conjugated with fluorescein (green). Images were acquired with a confocal microscope from the top to the bottom of cells. Single Z-section and 3D reconstruction of all Z-slices (left panels) are shown. Scale bars: 10 µm.

To assess the protective effects of MelaSil-NPs against hydrogen peroxide-induced apoptosis, HeLa and A2780 human cells were incubated for 24 hours with MelaSil-NPs and then treated with 1 mM H2O2 for 30 min to induce cell death after NPs had been washed out. Determination of apoptosis after 24 h by annexin V staining through FACS did not indicate any significant effect caused by uptake of MelaSil-NPs, supporting full biocompatibility (Figure 6, untreated samples with or without particles). Actually, a marked protective effect of MelaSil-NPs against hydrogen peroxide cytotoxicity was demonstrated by the drop in the apoptosis rate in MelaSilNP-containing cells (Figure 6).


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Figure 6. Protective effect of MelaSil NPs in mammalian cells. Two human cell lines A2780, Panel A (ovarian cancer) and HeLa Panel B (cervical cancer) were treated with 1mM H2O2 for 30 min after 24 hours of incubation with MelaSil NPs followed by wash out of the particles. After 24 hours from treatment the cells were stained with annexin V and the apoptosis was evaluated by FACS. NT: untreated with H2O2. Data are expressed as means ± SEM of three independent experiments. *p

Probing the EumelaninâSilica Interface in Chemically Engineered

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