Functionalizing the Mesoporous Silica Shell of Upconversion

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Functionalizing the mesoporous silica shell of upconversion nanoparticles to enhance bacterial targeting and killing via photosensitizer induced aPDT Malte Christian Grüner, Marylyn Arai, Mariana Carreira, Natalia Inada, and Andrea Simone Stucchi de Camargo ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00224 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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ACS Applied Bio Materials

Functionalizing the mesoporous silica shell of upconversion nanoparticles to enhance bacterial targeting and killing via photosensitizer induced aPDT Malte C. Grüner, [a],ỻ,* Marylyn Setsuko Arai, [a], ỻ Mariana Carreira, [a,b] Natalia Inada, [a] Andrea S. S. de Camargo [a]* [a] University of São Paulo, São Carlos Institute of Physics, Av. Trabalhador Sãocarlense 400, 13566-590, São Carlos-SP (Brasil); [b] Universidade Brasil, Estrada projetada F1, 15600-000, Fernandópolis-SP (Brasil) E-mail: [email protected], [email protected] ỻ

Co-first authors that equally contributed to this work.

Keywords: UCNP, aPDT, bacterial targeting, theranostic, mesoporous silica.

Abstract Core-shell nanoparticles operating by infrared-to-visible energy upconversion (UCNPs) have been proposed as theranostic carriers for photosensitizers to increase deep tissue penetration of photodynamic therapy against tumors and bacterial infections (aPDT). Herein we present a series of core-shell mesoporous silica coated NaYF4:Yb:Er UCNPs (mSiO2@UCNP), with different surface functionalizations to enhance bacterial targeting, and loaded with the hydrophobic photosensitizer SiPc (silicon 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide) to boost the bactericidal effect against Gram (+) and Gram (-) bacteria upon near infrared irradiation. Förster resonance energy transfer (FRET) from the UCNP core to loaded SiPc was facilitated while its efficiency depended on UCNP shell functionalization influencing the SiPc penetration depth into the mesoporous silica, constituting a convenient tool to modify FRET intensity. Functionalized UCNPs displayed dark toxicity towards Gram (-) E. coli of up to five orders of magnitude, while Gram (+) S. aureus viability was not decreased in the dark, offering practical means for discriminating between the two bacterial strains. Directly exciting SiPc on the UNCP led to complete eradication of E. coli and a drastic decrease of colony forming units of S. aureus of up to seven orders of magnitude. With this study, we demonstrate strategies to potentiate aPDT on nanoparticular structures that can lead to next generation photosensitizing systems based on UCNPs to help encounter and eradicate resistant bacteria, as well as for theranostics and future in vivo applications.

Introduction Antimicrobial photodynamic therapy (aPDT) has the potential to supplement antibiotics in times when resistant bacteria are on the rise.1–3 In aPDT a photosensitizer (PS) is delivered to the bacterial target and excited with light of appropriate wavelength to generate reactive singlet oxygen (1O2) in a diffusion controlled Type II mechanism, in which energy transfer occurs from the excited PS onto

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molecular triplet oxygen (3O2) rendering the PS available for another excitation cycle.4 Due to its high reactivity, 1O2 has a short lifetime and diffusion range in aqueous media, which is why the PS needs to be in close vicinity to the target bacteria in order to effectively inactivate it.5,6 The generated 1O2 unselectively reacts with components of the membrane, leaving the bacteria inactivated and making it very unlikely for bacterial resistances to develop.7 Non-linear optical imaging via upconversion luminescence (UCL) has been the center of attention for biological imaging, labeling and sensing in recent years due to its beneficial properties including reduced autofluorescence, tissue penetration depth and enhanced photostability.8 Recently, there have been promising attempts to use upconverting nanoparticles (UCNPs) as carriers for PS, where the PS can either be excited by energy transfer from the IR activated UCNP or by its own absorption band. Tree-Udom et al. designed the PS meso-tetraphenyltetrabenzoporphyrinatozinc matching its absorbance to UCNP emission to enhance FRET efficiency.9 Ye et al. conjugated UCNPs with curcumin to photo-inhibit S. aureus bacteria in lung tissue in vivo with NIR light excitation.10 Joshi et al. proved the near-infrared activation of UCNPs loaded with chlorin e6 through beef tissue.11 Yin et al. doped a dense silica shell with methylene blue around the UCNP core to carry out aPDT on E. coli and S. aureus.

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encapsulation of CuS nanoparticles within chitosan wrapped around the UCNPs for simultaneous photothermal therapy (PTT). Moreover, Qu and coworkers recently used UCNPs with TiO2 shell to produce reactive oxygen species (ROS) and to facilitate release of D-amino acids for dual action against bacteria and biofilms.13 And Dong et al. applied mesoporous silica and chitosan covered UCNPs loaded with Roussin’s black salt as NO donor molecule against resistant bacteria-based biofilms.14 Furthermore, phthalocyanines are ideal PSs due to stability towards self-oxidation, ability to absorb near infrared light and high hydrophobicity, which prevents leaking from the nanomaterial. Consequently, they have been applied in combination with UCNPs: Watkins et al. covalently attached aluminum phthalocyanines onto the silica shell of UCNPs, which prevented aggregation of the PS to some extent and allowed for FRET between UCNP and Pc to occur.15 Li et al. presented a chitosanmodified UCNP loaded with zinc phthalocyanine for dual antibacterial activity.16 For our study we used hydrophobic silicon 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide (SiPc), which has been proven to be a good candidate for adsorption onto nanoparticles due to its four bulky tert-butyl moieties and its affinity to silica, helping to avoid aggregation on the nanoparticles surface and preventing leakage into aqueous media.17,18 We herein present a series of differently functionalized UCNPs to study the correlation of surface properties, targeting and photo-inactivation of Gram (+) E. coli and Gram (-) S. aureus bacteria as model strains.

UCNP Synthesis and Properties


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The UCNP core was synthesized as previously described by Li et al. with a monodisperse size distribution and around 45 nm in diameter.16 The crystals belong to the cubic phase of NaYF4:Yb:Er UCNP as reported in the JCPDS card No. 77-2042 (Figure 1A – D). Although the hexagonal phase of NaYF4:Yb:Er UCNPs has been reported to have enhanced fluorescence properties, the cubic phase has sufficiently high UCL efficiency for the purposes of this proof-of-principle study.19 A mesoporous silica shell was directly formed on the polyvinylpyrrolidone (PVP) stabilized UCNP core with a thickness of 30-40 nm, increasing the average particle size to 100-120 nm (Figure 1) and the BET surface area from 35 m2/g to 486 m2/g with a pore diameter of 2.4 nm (Figure S1).

Figure 1. Characterization of UCNPs. A) SEM and B) TEM images of α-NaYF4:Yb, Er; C) XRD pattern of UCNP sample compared to α-NaYF4:Yb, Er, D) particle diameter distribution; E-F) TEM images of UCNP@mSiO2. The UCNPs have an absorption maximum at 980 nm as expected from the Er3+/Yb3+ co-doping. Although Er3+ is the ion responsible for UCL, Yb3+ acts as an efficient sensitizer with higher absorption cross section in this region of the electromagnetic spectrum (Figure 2A). UCL emissions can be observed in powder (Figure 2B) and in aqueous dispersion in the green (520 – 560 nm) as well as in the red (640 – 680 nm), while emissions are enhanced for the core-shell particles (UCNP@mSiO2) in dispersion due to reduced solvent quenching (Figure 2C).20



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Figure 2. A) Normalized absorbance of UCNPs in ethanol (0.5 mg/mL), B) UCL emission of UNCPs in powder and C) Comparison of UCL emission of UCNPs and UCNP@mSiO2 in aqueous dispersion. UCL excitation with a 976 nm diode laser with power density of 2 W/ cm². Additional surface decorations were introduced through reaction of (3-aminopropyl)triethoxysilane (APTES) with the mesoporous silica shell and further chemical reactions including permethylation with iodomethane, or EDC/NHS coupling with HOOC-TEG-COOH or HOOC-TEG-NH2 and permethylation according to Scheme 1, respectively. To assure the same amount of PS loading on the nanocrystals, namely 1 mg SiPc / 10 mg of UCNP, adsorption followed in a second step, after the surface decorations were completed, via sonication in methanol solution of the PS (c = 1mg/mL, Scheme 1). Careful washing steps ensured the removal of undesired byproducts and unreacted chemicals.

Scheme 1. Schematic diagram of mesoporous silica shell synthesis and surface reactions from PVP stabilized UCNP 1 towards the loaded UCNPs 2 – 6 including the zeta potentials in parentheses.


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The successful functionalizations were followed by the corresponding change in zeta potential: in water, the value decreased from +33.5 mV to -6.6 mV after mesoporous silica shell formation and increased to +32.3 mV after surface reaction with APTES (Scheme 1, UCNPs 1-3). It further increased after permethylation with iodomethane to +39.0 mV (UCNP 4), while UCNPs 5 and 6 have a zeta potential of around +20 mV. It is noteworthy that the zeta potential is significantly shifted towards positive values, which could be a consequence of the mesoporous silica shell formation in the presence of PVP (Figure S2). Dynamic light scattering (DLS) data showed different tendencies to form aggregates, which grew from 105 nm (UCNP 1) to 190 nm (UCNP 2) in diameter after the mesoporous silica shell formation, and being in good agreement with the values obtained from TEM measurements (Figures 1E-F, S3). Further functionalization slightly increased aggregate size to 220 nm (UCNP 3) and 255 nm (UCNP 4) respectively. The largest-sized aggregates were found for UCNP 5 with 460 – 2669 nm and for UCNP 6 with 342 nm. An explanation can be the formation of hydrogen bonds between tetraethylene glycol (TEG) and amino groups with neighboring particles causing the formation of larger aggregates (Figure S3). Furthermore, FT-IR and Raman measurements were carried out to detect the presence of the desired functional groups (Figures S6 – S8). To confirm the silica shell on the nanocrystals, they were analyzed by FT-IR spectroscopy. The spectrum of the starting NaYF4:Yb3+/Er nanocrystals stabilized by PVP shows characteristic bands at 1664 and 1442 cm-1 that can be assigned to C=O as well as C-N stretching vibrations of PVP. A large band at 3400 cm-1 can be assigned to water due to the hygroscopic PVP encapsulating UCNP 1. After the formation of the mesoporous silica shell, the C-N stretching band at 1442 cm-1 decreased in intensity (samples UCNPs 2-6). A new strong broad and characteristic band was observed around 1090 cm-1, which can be attributed to symmetrical Si-O-Si vibrations confirming the formation of the silica shell.21 The loading of the photosensitizer SiPc also can be followed in the spectra as a broad band at 3450 cm-1 with a shoulder at 3200 cm-1 (stretching vibrations of O-H axially associated to the central Si atom) and a medium intense band at 2956 cm-1 (deformation vibration of C-H associated to the tert-butyl moieties of SiPc) for samples UCNP 2-6 appear (Figure S6). To investigate the presence of the desired functional groups, the particles were further analyzed by Raman spectroscopy (Figures S7 and S8). UCNP 1 shows a band at 2936 cm-1, which was assigned to C-H vibrations within the PVP around the particles. After silica shell formation, this signal disappears, and three other signals could be observed (1611, 1550 and 1404 cm-1) corresponding to vibrations of the loaded SiPc respectively (ν(C-H), ν(C-Caromatic) and δ(C-H)) and present for samples UCNPs 2-6. When functionalized with APTES, another band at 1335 cm-1 showed, attributed to C-N stretching vibrations of the introduced amino groups. Sample UCNP 4 showed a strong band at 2920 cm-1 (C-H vibrations) and the ratio of signals corresponding to C-H (1404 cm-1) and C-N (1335 cm-1) stretching increased as expected after permethylation of the amino groups. The functionalization with carboxylic groups (UCNP 5) led to a



broad O-H stretching vibration in the region of 3200 – 3519 cm-1 and a higher ratio of C-H (1404 cm-1) to C-N (1335 cm-1) band, which points towards the presence of the anticipated functionalities after reaction with HOOC-TEG-COOH. A C-H stretching band at 2920 cm-1 was observable for UCNP 6 as well as a lower C-H (1404 cm-1) to C-N (1335 cm-1) signal ratio indicating introduction of additional amino groups by HOOC-TEG-NH2 following their permethylation. Combining the information of zeta potential, FT-IR and Raman bands, the presence of the functional groups can be undoubtedly confirmed.

Photophysics SiPc as PS was selected due to its bulky tetra-tert butyl substituents to decrease molecular aggregation on the UCNP@mSiO2. The relative amount of SiPc adsorbed at the surface is equal for each of the loaded samples and the characteristic Soret- and Q-absorption bands can be clearly seen for samples UCNP 2-6 (Figure 3B). However, the aggregates on the particles surface result in distinct changes in the Q-band of the absorption as compared to the monomeric species. Predominantly Jaggregates of SiPc are formed within the mesoporous shell as it can be observed from the red-shifted absorption bands of the samples.22 This corresponds to side-by-side aggregates for the rigid disc-like SiPc molecule and points to a favorable interaction of the silica surface with SiPc, possibly by formation of H-bonds between the surface and the axial Si-OH groups of the PS, as previously observed for other nanoparticular systems.17,23 With further surface functionalization, an additional broadening of the H-band occurs, which can be assigned to formation of stacked aggregates of SiPc as seen in the baseline-corrected Q-band spectra (Figure 3C). Additional surface groups confine the available surface space and pore size so that SiPc molecules are partially stacked on top of each other causing a blue-shifted absorption.

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Figure 3. A) Comparison between UCL emission of UCNP@mSiO2 and absorbance of the photosensitizer SiPc. B) Absorbance spectra of functionalized particles normalized at the Q-band. C) Baseline-corrected Q-band absorbance of functionalized particles. UCL excitation with a 976 nm laser at a power density of 2 W/ cm². The Q-band of SiPc superimposes with the red emission of the UCNPs around 660 nm, allowing for a possible Förster resonance energy transfer (FRET) between the UCNP core and SiPc within the ACS Paragon Plus Environment

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mesoporous silica when the core is excited by a 976 nm laser (Figure 3A). Intensity of FRET strongly depends on the distance of donor and acceptor.24 Usual FRET distances lay between 1–10 nm, which is why FRET between the UCNP core and the PS only takes place when the PS thoroughly penetrates the mesoporous 40 nm silica shell of the particles. A significant reduction of the red UCNP emission intensity after SiPc loading (UCNP 2) proves the occurrence of an energy transfer from the core to the SiPc and indicates a deep penetration of the mesoporous silica shell by the photosensitizer (Figure 4A). 20

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Figure 4. A) Comparison between UCL emission spectra of UCNP@mSiO2 and UCNP@mSiO2 encapsulated with SiPc (UCNP 2) indicating FRET. B) Fluorescence emission of 1O2 sensitive dye ABMDMA (λexc = 370nm) against irradiation time for the studied UCNP samples in aqueous dispersions (0.25 mg/mL). Laser power density 2 W/cm2 (λexc = 976 nm). Moreover, excited SiPc produces singlet oxygen (1O2), which can be indirectly detected by the 1O2 sensitive monitor dye ABMDMA (Figure 4B). Since FRET to SiPc is ultimately responsible for 1O2 generation, decay in fluorescence intensity of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) can be interpreted as a measure of FRET efficiency. It decreased with the degree and type of functionalization: TEGylated samples (UCNP 5 and 6) showed lower FRET than the precursor particles (UCNP 2 and 3), while UCNPs with permethylated APTES (UCNP 4) did not exhibit FRET at all (Figure 4B). This can be ascribed to narrowing or partially blocking of the pores of the mesoporous shell by the surface modifications, which affects the depth of SiPc adsorption. Channel blocking is therefore more efficient with permethylation of APTES than with the introduction of TEGylated carboxyl and permethylated amino groups under the given experimental parameters. Surface modifications hence are a straightforward strategy to alter the degree of FRET when the PS loading occurs in a second step. Furthermore, SiPc can also be directly excited by absorption of light in its near-infrared Q-band from 600 – 690 nm (Figure S4). The fluorescence spectra of adsorbed SiPc (samples UCNPs 2 – 6) were unaffected compared to the monomer in dilute solution, which indicates negligible trivial energy transfer (self-absorption) for aqueous dispersions of the UCNPs. For reference, the fluorescence and 1O2 quantum yields (ΦF and Φ∆ respectively) of UCNP 2 were measured in aqueous dispersion (Figures 5 and S5) with SiPc in CH2Cl2 as reference for fluorescence ACS Paragon Plus Environment


(ΦF = 0.47) and methylene blue (MB) in water as reference for 1O2 quantum yield (Φ∆ = 0.52).25,26 With Φ∆ = 0.11 (± 0.03) in water around ¼ of the chromophores are available for 1O2 generation, which is a reasonable value considering the hydrophobicity of SiPc. The considerably lower fluorescence quantum yield (ΦF < 0.01) can be explained by homo-FRET and inactive surface aggregates of SiPc - as previously observed for SiPc on nanoclays and in aqueous solution.17,27,28

Figure 5. Fluorescence emission of ABMDMA (λexc = 380 nm) monitored over time for (A) UCNP2 and (B) reference MB. (C) ABMDMA emission decay for UCNP2 and reference MB caused by the formation of 1O2 after irradiation with a 660 nm LED represented as ln(F/F0) vs different irradiation time. Photobiology The interaction of the differently decorated UCNPs was investigated via confocal fluorescence microscopy for both bacterial strains. The bacteria were stained with DAPI while the particles were visualized by addressing the adsorbed SiPc’s fluorescence and the UCL of the core. All samples exhibit UCL properties and show aggregates of different size throughout the samples. UCNPs 3 to 6 effectively colocalize with E. coli, while sample UCNP 2 is slightly less attracted to the bacteria and UCNP 1 did not show binding interaction at all (Figure 6A). The increase of surface area through mesoporous silica shell formation already enhances interaction potential with E. coli, even though zeta potential of UCNP 2 is slightly negative and a positive charge is more favorable for an interaction with E. coli.28,29 The entire set of samples is capable of interaction with S. aureus (Figure 6B). Though it is noteworthy that interaction of UCNP 2 (negative zeta potential) is not as strong as of the other samples. Hence, a positive zeta potential is the key for inducing a strong binding between the UCNPs and Gram (-) as well as Gram (+) bacteria likewise. Also, organic surface modifications offer additional binding sites that can enhance specific interactions with moieties of bacterial membranes. There is a great number of factors that can play a role for successful bacterial binding: electrostatic interaction, local interactions between surface molecules and membrane proteins, as well as phospholipids, hydrogen bonding, degree of particle aggregation in aqueous media, the solution pH, dissolved substances and porosity of the material that can be altered to enhance targeting of bacteria and their binding.


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Figure 6. Confocal fluorescence images of samples incubated with A) E. coli and B) S. aureus. Bacterial dispersions (CFU = 1.5 x 107) were incubated with DAPI (c = 5 µg/mL) for 10 min followed by mixing with 20 µL of UCNP sample dispersions (2 mg/mL). Lane 1: DAPI (λexc = 400 nm, λem = 405 - 500 nm); Lane 2: SiPc (λexc = 633 nm, λem = 680 - 750 nm); Lane 3: UCL (λexc = 980 nm, λem = 520 - 600 nm); Lane 4: merged images; Lane 5: magnification of indicated part of merged image. The scale bar represents 20 µm and in the magnification 5 µm. To study the phototoxicity of the UCNPs towards the tested bacterial strains, 100 µL of bacterial suspension (~ 108 bacteria) were mixed with 100 µL of UCNPs 1- 6 (c = 2 mg/mL) and irradiated with a 660 nm LED device for 1 hour (radiant exposure of 108 J/ cm²) and compared to untreated as well as dark controls. Already the PVP stabilized particles exhibited dark toxicity towards E. coli and reduced colony forming units (CFUs) up to two orders of magnitude after one hour of incubation (Figure 7A). The dark toxicity increased to five orders of magnitude in CFU reduction when particles had a mesoporous silica shell and were functionalized with -NH2 and -NMe3+I- (UCNPs 2, 3 and 4) and decreased again to three orders of magnitude for TEGylated chemical moieties carrying -COOH or NMe3+I- (UCNP 5 and 6). This decrease in toxicity is already known for PEGylated nanoparticles and is likely due to a shielding of positive charges (lower zeta potentials) by the TEG-COOH and TEG-NMe3+Imoieties.30 Moreover, TEGylated molecules can act as a spacer between bacteria and particles, decreasing direct contact and therefore toxicity, which is a reason why UCNP 2 with its unfunctionalized silica shell shows higher dark toxicity than TEGylated samples. However, the larger particle aggregates of UCNP 5 and 6 can also decrease the degree of interaction with the bacteria, rendering these samples less cytotoxic. No dark toxicity towards S. aureus was detected, which allows for a practical discrimination between the two bacterial strains (Figure 7B). When irradiated with NIR light (660 nm) to directly excite SiPc on the UCNPs, E. coli CFUs were completely eradicated by all but one UCNP. Interestingly, TEGylated carboxylic acid decorated UCNP 5 was less photocytotoxic but still inactivated CFUs by seven orders of magnitude. Larger particle aggregation, a reduced contact as well as the negatively charged carboxylic groups decrease to some degree the interaction with the Gram (-) E. coli bacteria and thus the toxicity, which is also in accordance with the dark toxicity results. The combinatory effect of intrinsic and photoinduced toxicity makes UCNP 2 - 4 and UCNP 6 very strong agents against E. coli bacteria. All SiPc loaded UCNP samples presented efficient aPDT property against S. aureus, with CFU inactivation of six to seven orders of magnitude. The most effective particle is UCNP 4, most likely due to the highly positive zeta potential that enables interaction with negative membrane charges. Also, the limited penetration depth of the loaded SiPc could be beneficial, as it results in shorter diffusion pathways of the photo generated reactive oxygen species (ROS) towards the bacterial target.


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976 nm laser excitation was only studied for UCNP 2, which showed the highest rate of 1O2 generation via FRET. For this purpose, bacterial suspensions were treated with UCNP 2 (c = 1 mg/mL) and irradiated with a 976 nm continuous laser (power density: 400 mW/cm2) for 30 min to induce upconversion, which then excites the loaded photosensitizer to produce 1O2.

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Figure 8. Number of bacterial CFUs per mL (mean values) of A) E. coli and B) S. aureus treated with UCNP 2 (c = 1 mg/mL) and irradiated with a 976 nm continuous laser (power density: 400 mW/cm2) for 30 min including dark controls. The number of E. coli CFUs was decreased by five orders of magnitude by the UCNPs dark toxicity while upconversion and FRET to the PS additionally inactivated two orders of magnitude in CFUs after 30 min of laser irradiation (Figure 8A). Interestingly, there was no reducing effect on S. aureus when SiPc was excited through FRET (Figure 8B). S. aureus is also less sensitive towards the photooxidation by the UCNP particles when SiPc is directly excited (between 6-7 orders of magnitude reduction instead of complete eradication as for E. coli), indicating a higher threshold towards 1O2 under the tested experimental conditions. Gram (+) bacteria, such as S. aureus, have an additional peptidoglycan layer surrounding the membrane, which could provide protection from smaller amounts of 1O2 damaging the membrane integrity and inactivating the bacteria. Because the FRET is


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more efficient when the distance between donor and acceptor is short, most 1O2 and deriving ROS species are generated deep within the mesoporous shell of UCNP 2 making it more difficult to exit the shell by diffusion to reach the bacterial target. Also, less SiPc chromophores are addressed to produce 1O2 due to limitations of the FRET distance.

Conclusion The development of theranostic agents for studying and aPDT treatment of diseases caused by antibiotic resistant bacteria is of utmost importance and UCNPs are ideal candidates for that purpose. UCL of these particles can be either applied to excite an encapsulated PS via FRET or as a diagnostic tool to visualize the particles. Positively charged UCNPs had promising targeting capability for both Gram (-) E. coli and Gram (+) S. aureus bacterial strains, which can guarantee the PS delivery. Mesoporous shell surface functionalization had an influence on the adsorption of PS onto the particle and we demonstrated that FRET intensity via core excitation varied depending on adsorption depth of SiPc. The degree of FRET can be adjusted in this way, offering a tool to separate detection and therapy as shown for UCNP 4 - 6, for cases in which the generation of 1O2 is undesired when exciting the UCNP core. However, if an application for 1O2 photogeneration with FRET is desired, a thin mesoporous silica shell warrants an efficient FRET while allowing the 1O2 to quickly diffuse from the particle to the target. Deep tissue penetrating NIR light excitation (633 nm) directly addressed the Qband of SiPc encapsulated with UCNP, which rapidly inactivated bacteria in vitro. The complete eradication of E. coli and a reduction of seven orders of magnitude of S. aureus CFUs make the studied particles a versatile platform, where especially UCNP 4 showed the most potential. The highly positive zeta potential (+39 mV) caused by permethylated amino groups and the large surface area of the mesoporous silica shell effectively collocate with Gram (-) and Gram (+) bacteria likewise while the shallow loading of the photosensitizer promotes the facile release of 1O2 and derived ROS from the particle to efficiently reach the bacterial target. The effect of the chemical groups on particle characteristic greatly influenced the aPDT efficiency: moieties that facilitate stable particle dispersion as well as a close proximity to the bacterial target are desirable decorations for PS loaded UCNPs. The demonstrated strategies help to further investigate UCNP-based aPDT with emerging bacterial resistances against antibiotics.

Experimental Reagents. Yttrium (III) oxide (99,99%), ytterbium (III) oxide (99,99%), polyvinylpyrrolidone (PVP40), tetraethoxysilane

(TEOS),

triethylamine

(Et3N),

N,N-diisopropylethylamine

(DIPEA),

(3-

aminopropyl)triethoxysilane (APTES), ammonium fluoride (P.A.), sodium hydroxide, ammonium



hydroxide

solution

(25%),

morpholino)ethanesulfonic

N-hydroxysulfosuccinimide acid

(MES),

Page 14 of 30

sodium

salt

(Sulfo-NHS),

2-(N-

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride (EDC), hexadecyltrimethylammonium bromide (CTAB), iodomethane and 9,10anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) were purchased from Sigma Aldrich (www.sigmaaldrich.com), erbium (III) oxide (99,99%) from Alfa Aesar (www.alfa.com), ethylene glycol (p.a.), ethanol (p.a.), toluene (p.a.) and nitric acid from Synth (www.labsynth.com.br) and tetraethyleneglycol-bis

propionic acid (COOH-TEG-COOH)

and

15-amino-4,7,10,13-tetraoxa-

pentadecanoic acid (COOH-TEG-NH2) were purchased from Iris Biotech GmbH (www.iris-biotech.de), Germany. All the reagents were used without further purification if not stated otherwise. Synthesis of UCNPs. The preparation of NaYF4:Yb, Er core nanoparticles was based on a previously published synthesis procedure.31 The procedure was slightly modified as given below: Y2O3 (88 mg, 0.78 mmol), Yb2O3 (39.4 mg, 0.2mmol), and Er2O3 (3.9 mg, 0.02mmol) were dissolved in 10% HNO3 (10 mL), and the solution was heated to completely evaporate water. Ethylene glycol (10 mL) was added to dissolve the obtained LnNO3 (Ln=Y 78%, Yb 20%, Er 2%). (PVP40, 0.5560 g) and NaCl (0.0588 g, 1 mmol) were subsequently added and the solution was heated to 80 °C until a homogeneous solution was formed. NH4F (0.1482g, 4 mmol) was dissolved in ethylene glycol (10 mL) at 80 °C and added dropwise to the LnNO3 solution, which was maintained at 80 °C for 10 min under stirring. The solution was heated to 140 °C for 2h and then cooled to room temperature. The product was isolated by centrifugation, washed twice with absolute ethanol and dried. Synthesis of UCNP@mSiO2. A previous established method with some modifications was used for the synthesis of the mesoporous silica shell around de upconverting nanoparticles.32 2 mL ethanol solution containing 10 mg of UCNPs were mixed with 0.1 g CTAB and 20 mL water. The mixture was vigorously stirred at room temperature for 12 h, resulting in a transparent and clear UCNPs–CTAB water solution. For the formation on the shell, 10 mL of the aqueous CTAB stabilized UCNPs solution was added to a mixture of 20 mL water, 3 mL ethanol and 150 μL NaOH (2 M) solution. The resulting mixture was heated up to 70 °C under stirring. 200 μL tetraethylorthosilicate (TEOS) was added dropwise and the reaction was allowed to proceed for 10 min. The as-synthesized materials were centrifuged and washed with ethanol three times. The surfactant CTAB was removed via a fast and efficient ion exchange method, where the as-synthesized UCNPs@mSiO2 (20 mg) are transferred to 50 mL ethanol containing 0.3 g NH4NO3 (3,75 mmol) and kept at 60 °C for 2 h. The product was isolated by centrifugation, washed twice with absolute ethanol and dried. Functionalization with amino groups. Amino groups were introduced to the mesoporous silica shell surface through the use of APTES. 40 mg of UCNP@mSiO2 were dispersed in 50 mL of ethanol and stirred for 30 min. Subsequently, 650 μL of triethylamine and 1 mL of APTES were added to the


Page 15 of 30 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


mixture which was refluxed for 12 h at 80 °C. The obtained particles were collected by centrifugation, washed three times with ethanol and dried. Functionalization with HOOC-TEG-NH2 and HOOC-TEG-COOH. In a first step 6,5 mg (0,034 mmol) of EDC, 8,85 mg of sulfo-NHS (0,0407 mmol) and 10 mg (0,034 mmol) of HOOC-TEG-COOH or HOOCTEG-NH2 respectively were dissolved in 10 mL of 0,1 M MES-buffer (pH = 6) and the activation was allowed for 8 h. This mixture was then added dropwise into a PBS solution containing 30 mg of amino functionalized particles and the pH adjusted to 7,0 using 10 M NaOH solution. The reaction was kept at 4 °C for 48 h under continuous stirring and the particles were isolated by centrifugation and washed three times with water. Permethylation. Methyl iodide (MeI) was used to permethylate the amino groups on the mesoporous silica shell. 20 mg of the amino functionalized nanoparticles were dispersed in 50 mL of ethanol and sonicated 30 min. 500 μL of N,N-diisopropylethylamine and 5 mL of MeI were added to the solution and stirred for 12 h. To remove the remaining methyl iodide, the mixture was heated to 65 °C for 3h. The final particles were collected by centrifugation, washed three times with ethanol and dried. Loading

the

photosensitizer

onto

the

mesoporous

silica

shell.

The

photosensitizer

silicon(IV)2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide (SiPc) was loaded into the mesoporous shell via adsorption. 20 mg of UCNP@mSiO2 were dispersed in 20 mL of methanol and stirred for 30 min; subsequently 2 mL of methanol SiPc solution (1 mg/ mL) was added. The mixture was kept in the sonication bath for 2 h and the solvent was evaporated under vigorous stirring at 60 °C until blue powder was obtained. Characterization. The size and shape of the UCNPs were studied with a FEI TECNAI20 transmission electron microscope and a JEOL JEM-2100 scanning electron microscope. The powder’s phase composition was determined by X-ray powder diffraction (XRPD) using a Philips X’PERT (λ = 1.5406 Å). The patterns were recorded in the range of 10–80 ° with a step scan of 0.02 ° and accounting time of 5 s per step. The zeta potential and DLS were evaluated in aqueous dispersions at pH 7.2, using a Malvern Zetasizer Nano ZS equipment. Fourier transform infrared spectroscopy (FTIR) measurements were performed using a Nicolet iS50 spectrometer from Thermo Scientific with 4 cm-1 resolution in transmittance mode. The spectra of the powder samples were obtained in KBr pellets containing approximately 1 wt% of each sample. The Raman measurements were performed with a Horiba HR800 Evolution micro-Raman spectrometer using a 488 nm laser of 8 mW at 100% power on the sample and a 50 x distance objective. The integration time was 10 s and each spectrum measurement took about 3 min involving multiple scans to eliminate cosmic ray peaks and reduce the noise. The upconversion spectra and the fluorescence spectra of the photosensitizer and the monitor dye (ABMDMA) were recorded on a HORIBA FluoroLog TCSPC spectrofluorometer; for the upconversion



an external 978 nm diode laser was used for excitation. The absorption spectra were measured using a Perkin Elmer Lambda 1050 UV/Vis/NIR spectrometer. Nitrogen adsorption/desorption analysis was measured using a Micromeritics ASAP 2020 M apparatus. The specific surface area was determined by the Brunauer–Emmett–Teller (BET) method. The pore volume was obtained from the T-plot method and the average pore size was calculated using Barrett–Joiner–Halenda (BJH) method. Confocal images. 100 μL of S. aureus ATCC25923 and E. coli ATCC25922 solutions (CFU = 1.5 x 107) were incubated with 10 μL of DAPI (5 μg/mL) for 10 min followed by mixing with 20 µL of UCNP (1-6) dispersions (2 mg/mL). The samples were placed in a confocal dish and the fluorescence images of DAPI (λexc = 400 nm, λem = 405 - 500 nm), SiPc (λexc = 633 nm, λem = 680 - 750 nm) and UCL from UCNP (λexc = 980 nm, λem = 520 - 600 nm) were collected using an inverted Zeiss LSM780 confocal microscope. Fluorescence Quantum Yield. The fluorescence quantum yield (ΦF) was determined with the relative method by comparison with SiPc in CH2Cl2 as a reference (ΦF = 0.47).23 The quantum yield was calculated using equation 1, where R and S refer to the reference and sample, respectively. I is the area under the fluorescence specrum, A is the solution absorption at the excitation wavelength, and (nS/nR)2 is the refractive index correction.

(1) Singlet Oxygen Quantum Yield. The singlet oxygen quantum yield (ΦΔ) was measured in water in order to address their use in aqueous media. Due to the low phosphorescence intensity of the spinforbidden radiative 1O2 → 3O2 transition, 1O2 photogeneration rates were derived using 9,10anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as a fluorescent monitor (λex = 380 nm) for photosensitized bleaching rates. A 660 nm LED irradiation source was used to carry out the experiments and methylene blue (ΦΔ =

0.52)

[22]

was used as reference. The singlet oxygen

photogeneration rates for the samples were calculated using equation 2, where r is the slope of the monitor’s bleaching over time (1O2 photogeneration rate), λ the irradiation wavelength, I0 the incident spectral photon flow, which can be cancelled from the equation (approximated to be a constant value), A the absorbance, and subscripts R and S refer to the reference and sample, respectively.

(2)

In vitro antibacterial therapy 660 nm irradiation. The used bacteria were a donation from FioCruz, Rio de Janeiro, Brazil. S. aureus ATCC25923 and E. coli ATCC25922 solutions (~ 108 CFU/ mL, 100 μL) were mixed with 100 μL of ACS Paragon Plus Environment

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functionalized UCNPs (2 mg/mL). After 1 h irradiation with a 660 nm LED device (a prototype developed by the Technological Support Laboratory, from São Carlos Institute of Physics, São CarlosSão Paulo, Brazil; 108 J/ cm²) under continuous stirring, 10 μL of each sample was plated on BHI brain heart infusion agar medium with appropriate dilution (serial dilution until 10-5) and cultured at 37 °C for 24 h followed by counting the colony forming units (CFU/mL). Bacterial colonies treated with the synthesized particles in the absence of irradiation and only with irradiation were counted following the aforementioned procedures. All experiments were realized in triplicate. 976 nm irradiation. S. aureus ATCC25923 and E. coli ATCC25922 solutions (~ 108 CFU/ mL, 100 μL) were mixed with 100 μL of UCNP2 (2 mg/mL) and irradiated using a 976 nm laser with a power density of 400 mW/cm² during 30 min. 20 μL aliquots were taken every 10 min and plated on solid medium with appropriate dilution and cultured at 37 °C for 24 h followed by counting of the bacterial colonies. The bacterial colonies treated only with light were counted the same way. All experiments were realized in triplicate.

Associated Content Supporting Information. N2 adsorption-desorption isotherms of UCNP and UCNP@mSiO2, zeta potentials in water of UCNP 1 - 6, dynamic light scattering size distribution by intensity in water of UCNP 1 - 6, excitation and emission spectra of functionalized particles UCNP 2 - 6 in aqueous dispersions, fluorescence intensity against fraction of absorbed light for SiPc in CH2Cl2 and UCNP 2 in water, FT-IR spectra of UCNP 1 - 6, Raman spectra of UCNP 1 - 6 between 500 - 4000 cm-1 , Raman spectra of UCNP 1 - 6 between 500 – 1700 cm-1.

Acknowledgements This work was supported by the Center for Research, Technology and Education in Vitreous Materials (CeRTEV), Project 2013/07793-6, funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), which also granted the post-doctorate fellowship to M.C.G. (Grant Number 2015/24118-6). We would like to thank Benjamin J. A. Moulton for assisting with the Raman measurements.

References [1]

Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J. The Epidemic of Antibiotic-Resistant Infections: A Call to Action for the Medical Community from the Infectious Diseases Society of America. Clin. Infect. Dis. 2008, 46, 155164.

[2]

Fair, R. J.; Tor, Y. Antibiotics and Bacterial Resistance in the 21st Century. Perspect. Medicin. Chem. 2014, 6, 25–64. ACS Paragon Plus Environment


[3]

Pogue, J. M.; Kaye, K. S.; Cohen, D. A.; Marchaim, D. Appropriate Antimicrobial Therapy in the Era of Multidrug-Resistant Human Pathogens. Clin. Microbiol. Infect. 2015, 21, 302–312.

[4]

Castano, A. P.; Demidova, T. N.; Hamblin, M. R. Mechanisms in Photodynamic Therapy: Part One - Photosensitizers, Photochemistry and Cellular Localization. Photodiagnosis Photodyn. Ther. 2004, 1, 279-293.

[5]

DeRosa, M.; Crutchley, R. J.; Photosensitized Singlet Oxygen and Its Applications. Coord. Chem. Rev. 2002, 233-234, 351-371.

[6]

Skovsen, E.; Snyder, J. W.; Lambert, J. D. C.; Ogilby, P. R. Lifetime and Diffusion of Singlet Oxygen in a Cell. J. Phys. Chem. B 2005, 109, 8570-8573.

[7]

Maisch, T. Anti-Microbial Photodynamic Therapy: Useful in the Future? Lasers Med Sci. 2007, 22, 83-91.

[8]

Wang, F.; Banerjee, D.; Liu, Y.; Chen, X.; Liu, X. Upconversion Nanoparticles in Biological Labeling, Imaging, and Therapy. Analyst, 2010, 135, 1839-1854.

[9]

Tree-Udom, T.; Thamyongkit, P.; Wiratkasem, N.; Chanchao, C.; Palaga, T.; Insin, N.; Wanichwecharungruang, S. Harmonization of Upconverting Nanocrystals and Photosensitizer for Antimicrobial Application. RSC Adv. 2015, 5, 102416-102423.

[10]

Ye, Y.; Li, Y.; Fang, F. Upconversion Nanoparticles Conjugated with Curcumin as a Photosensitizer to Inhibit Methicillin-Resistant Staphylococcus aureus in Lung under Near Infrared Light. Int. J. Nanomed. 2014, 9, 5157-5165.

[11]

Joshi, P.; Ahmadov, T. O.; Wang, P.; Zhang, P. Singlet Oxygen Generation under NIR Light and Visible Light Excitations of Photosensitizers on Upconversion Nanoparticle Surface. RSC Adv. 2015, 5, 67892-67895.

[12]

Yin, M.; Li, Z.; Ju, E.; Wang, Z.; Dong, K.; Ren, J.; Qu, X. Multifunctional Upconverting Nanoparticles for Near-Infrared Triggered and Synergistic Antibacterial Resistance Therapy. Chem. Commun. 2014, 50, 10488-10490.

[13]

Wei, W.; Bing, W.; Ren, J.; Qu, X. Near Infrared-Caged D-Amino Acids Multifunctional Assembly for Simultaneously Eradicating Biofilms and Bacteria. Chem. Commun. 2015, 51, 12677-12679.

[14]

Dong, K.; Ju, E.; Gao, N.; Wang, Z.; Ren, J.; Qu, X. Synergistic Eradication of Antibiotic-Resistant Bacteria Based Biofilms In Vivo Using a NIR-Sensitive Nanoplatform. Chem. Commun. 2016, 52, 5312-5315. ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30 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


[15]

Watkins, Z.; Taylor, J.; D’Souza, S.; Britton, J.; Nyokong, T. Fluorescence Behaviour and Singlet Oxygen Production of Aluminium Phthalocyanine in the Presence of Upconversion Nanoparticles. J. Fluoresc. 2015, 25, 1417-1429.

[16]

Li, S.; Cui, S.; Yin, D.; Zhu, Q.; Ma, Y.; Qian, Z.; Gu, Y. Dual Antibacterial Activities of a ChitosanModified Upconversion Photodynamic Therapy System Against Drug-Resistant Bacteria in Deep Tissue. Nanoscale 2017, 9, 3912-3924.

[17]

Grüner, M.; Tuchscherr, L.; Löffler, B.; Gonnissen, D.; Riehemann, K.; Staniford, M. C.; Kynast, U.; Strassert, C. A. Selective Inactivation of Resistant Gram-Positive Pathogens with a LightDriven Hybrid Nanomaterial. ACS Appl. Mater. Interfaces 2015, 7, 20965.

[18]

Barroso, Á.; Grüner, M.; Forbes, T.; Denz, C.; Strassert, C. A. Spatiotemporally Resolved Tracking of Bacterial Responses to ROS-Mediated Damage at the Single-Cell Level with Quantitative Functional Microscopy. ACS Appl. Mater. Interfaces 2016, 8, 15046.

[19]

Renero-Lecuna, C.; Martín-Rodríguez, R.; Valiente, R.; González, J.; Rodríguez, F.; Kramer, K. W.; Gudel, H. U. Origin of the High Upconversion Green Luminescence Efficiency in β-NaYF4: 2% Er3+, 20% Yb3+. Chem. Mater. 2011, 23, 3442-3448.

[20]

Sedlmeier, A.; Gorris, H. H. Surface Modification and Characterization of Photon-Upconverting Nanoparticles for Bioanalytical Applications. Chem. Soc. Rev. 2015, 44, 1526-1560.

[21]

Kostiv, U.; Janoušková, O.; Šlouf, M.; Kotov, N.; Engstová, H.; Smolková, K.; Ježek, P.; Horák, D. Silica-Modified Monodisperse Hexagonal Lanthanide Nanocrystals: Synthesis and Biological Properties, Nanoscale 2015, 7, 18096-18104.

[22]

Fernández, D. A.; Awruch, J.; Dicelio, L. E. Photophysical and Aggregation Studies of t-ButylSubstituted Zn Phthalocyanines. Photochem. Photobiol. 1996, 63, 784-792.

[23]

Eisfeld, A.; Briggs, J. S. The J- and H-Bands of Organic Dye Aggregates. Chem. Phys. 2006, 324, 376-384.

[24]

Muhr, V.; Würth, C.; Kraft, M.; Buchner, M.; Baeumner, A. J.; Resch-Genger, U.; Hirsch, T. Particle-Size-Dependent Förster Resonance Energy Transfer from Upconversion Nanoparticles to Organic Dyes. Anal. Chem. 2017, 89, 4868-4874.

[25]

Wilkinson, F.; Helman, W. P.; Ross, A. B. Quantum Yields for the Photosensitized Formation of the Lowest Electronically Excited Singlet State of Molecular Oxygen in Solution. J. Phys. Chem. Ref. Data 1993, 22, 113-262.

[26]

Grüner, M.; Siozios, V.; Hagenhoff, B.; Breitenstein, D.; Strassert, C. A. Structural and ACS Paragon Plus Environment


Photosensitizing Features of Phthalocyanine—Zeolite Hybrid Nanomaterials. Photochem. Photobiol. 2013, 89, 1406-1412. [27]

Galstyan, A.; Block, D.; Niemann, S.; Grüner, M. C.; Abbruzzetti, S.; Oneto, M.; Daniliuc, C. G.; Hermann, S.; Viappiani, C.; Schäfers, M.; Löffler, B.; Strassert, C. A.; Faust, A. Labeling and Selective Inactivation of Gram-Positive Bacteria Employing Bimodal Photoprobes with Dual Readouts. Chem. - Eur. J. 2016, 22, 5243-5252.

[28]

Grüner, M. C.; Niemann, S.; Faust, A.; Strassert, C. A. Axially Decorated SiIV-phthalocyanines Bearing Mannose- or Ammonium-conjugated Siloxanes: Comparative Bacterial Labeling and Photodynamic Inactivation. Photochem. Photobiol. 2018, DOI: 10.1111/php.12881.

[29]

Minnock, A.; Vernon, D. I.; Schofield, J.; Griffiths, J.; Parish, J. H.; Brown, S. B. Photoinactivation of Bacteria. Use of a Cationic Water-Soluble Zinc Phthalocyanine to Photoinactivate Both Gram-Negative and Gram-Positive Bacteria. J. Photochem. Photobiol. B 1996, 32, 159-164.

[30]

Lopez, A.; Reins, R. Y.; McDermott, A. M.; Trautner, B. W.; Cai, C. Antibacterial Activity and Cytotoxicity of PEGylated Poly(Amidoamine) Dendrimers, Mol. BioSyst. 2009, 10, 1148-1156.

[31]

Li, Z.; Zhang, Y. Monodisperse Silica-Coated Polyvinylpyrrolidone/NaYF4 Nanocrystals with Multicolor Upconversion Fluorescence Emission. Angew. Chem. Int. Ed. 2006, 45, 7732-7735.

[32]

Li, C.; Liu, J.; Alonso, S.; Li, F.; Zhang, Y. Upconversion Nanoparticles for Sensitive and In-Depth Detection of Cu2+ ions. Nanoscale 2012, 4, 6065-6071.


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Figure 6. Confocal fluorescence images of samples incubated with A) E. coli and B) S. aureus. Bacterial dispersions (CFU = 1.5 x 107) were incubated with DAPI (c = 5 µg/mL) for 10 min followed by mixing with 20 µL of UCNP sample dispersions (2 mg/mL). Lane 1: DAPI (λexc = 400 nm, λem = 405 - 500 nm); Lane 2: SiPc (λexc = 633 nm, λem = 680 - 750 nm); Lane 3: UCL (λexc = 980 nm, λem = 520 - 600 nm); Lane 4: merged images; Lane 5: magnification of indicated part of merged image. The scale bar represents 20 µm and in the magnification 5 µm. 163x379mm (150 x 150 DPI)


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Figure S1. N2 adsorption–desorption isotherms of A) core UCNP and B) UCNP@mSiO2. C) Mesopore size distribution of the as-synthesized UCNPs@mSiO2.



Figure S2. Zeta potentials measured in water for A) UCNP1; B) UCNP2; C) UCNP3; D) UCNP 4; E) UCNP 5; F) UCNP6.


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Figure S3. Dynamic light scattering size distribution by intensity in water of A) UCNP1; B) UCNP2; C) UCNP3; D) UCNP4; E) UCNP5; F) UCNP6.



Figure S4. A-E) Excitation and emission spectra of the functionalized particles 2 - 6 in aqueous dispersions (c = 100 µg/mL, λexc = 350 nm, λem = 730nm).


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Figure S5. A) Plot of fluorescence intensity against the fraction of absorbed light for SiPc in CH2Cl2 (black squares) and for UCNP 2 in H2O for three different concentrations (1, 2 and 3). B) Absorbance spectra of three different concentrations of SiPc in CH¬2Cl2; C) Fluorescence emission spectra of SiPc for three different concentrations; D) Absorbance spectra of three different concentrations of UCNP 2 in H2O; E) Fluorescence emission spectra of SiPc for three different concentrations (λexc = 615 nm).



Figure S6. FT-IR spectra of α-NaYF4:Yb3+/Er3+ of the prepared samples UCNP1-UCNP6. UNCP1 is stabilized by PVP, UCNP2-UCNP6 are silica coated with their different functional groups respectively. 118x84mm (96 x 96 DPI)


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Figure S7. Raman spectra of samples of A) UCNP1; B) UCNP2; C) UCNP3; D) UCNP4; E) UCNP5; F) UCNP6. Characteristic shifts are marked, and the blue and red boxes can be assigned to the loaded photosensitizer SiPc. 270x138mm (96 x 96 DPI)



Figure S8. Raman spectra magnified between 500 – 1700 cm-1 for samples UCNP1-UCNP6 (A-F) with colorcoded typical shifts for different chemical bonds. 254x130mm (96 x 96 DPI)


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Functionalizing the Mesoporous Silica Shell of Upconversion

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