Fluorescent Sulfonate-Based Inorganic–Organic Hybrid Nanoparticles

Jul 13, 2018 - Compounds showing efficient staining and/or fluorescence are a most relevant tags for application in polymer science as well as biology...
1 downloads 0 Views 3MB Size
Subscriber access provided by The University of British Columbia Library

Article

Fluorescent Sulfonate-Based Inorganic-Organic Hybrid Nanoparticles for Staining and Imaging Marieke Poß, Eva Zittel, Anna Meschkov, Ute Schepers, and Claus Feldmann Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00423 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 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

Bioconjugate Chemistry

Fluorescent Sulfonate-Based Inorganic-Organic Hybrid Nanoparticles for Staining and Imaging

Marieke Poßa, Eva Zittelb, Anna Meschkovb, Ute Schepersb,c, Claus Feldmanna*

a

Institut für Anorganische Chemie, Karlsruhe Institute of Technology (KIT), Engesserstrasse 15, D-76131 Karlsruhe (Germany)

b

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-vonHelmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany

c

Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber Weg 6, 76131 Karlsruhe, Germany

KEYWORDS Nanoparticles, inorganic-organic hybrids, sulfonate, staining, fluorescence, imaging

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 2 of 35

ABSTRACT: Sulfonate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general saline composition [Gd(OH)]2+n/2[Rdye(SO3)n]n– showing optical absorption and emission in the blue to red spectral regime are presented for the first time. All IOH-NPs are prepared via straightforward aqueous synthesis and instantaneously result in colloidally highly stable suspensions with mean particle diameters of 40-50 nm and high zeta potentials (–20 to –40 mV at pH 7.0). Specifically, the IOH-NPs

comprise

[Gd(OH)]2+2[CSB]4–,

[Gd(OH)]2+2[DB71]4–,

[Gd(OH)]2+[NFR]2–,

[Gd(OH)]2+[AR97]2–, and [Gd(OH)]2+2[EB]4– showing blue, orange, red and infrared absorption and emission ([CSB]: Chicago Sky Blue; [DB71]: Direct Blue 71; [NFR]: Nuclear Fast Red; [AR97]: Acid Red 97; [EB]: Evans Blue). The novel IOH-NPs are characterized by electron microscopy, dynamic light scattering, infrared spectroscopy, energy-dispersive X-ray analysis, thermogravimetry, elemental analysis, and fluorescence spectroscopy. In vitro studies based on HeLa and HUVEC cells were exemplarily performed with [Gd(OH)]2+2[EB]4– IOH-NPs and show intense fluorescence and only moderate toxicity at concentrations of 1 to 10 µg/mL. Based on aqueous synthesis, good colloidal stability, absence of severely toxic metals (e.g. Cd2+, Pb2+), use of molecular dyes that are already known for staining in cell biology and histology, extremely high dye load per nanoparticle (70-80 wt-%), and blue to red absorption and fluorescence, the sulfonate-based IOH-NPs can be highly interesting for staining, fluorescence microscopy and optical imaging.

Graphical Abstract / TOC

2 ACS Paragon Plus Environment

Page 3 of 35 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

Bioconjugate Chemistry

INTRODUCTION

Compounds showing efficient staining and/or fluorescence are most relevant tags for application in polymer science as well as biology and medicine.1-4 Thus, compounds with strong absorption or emission in the visible spectral regime are widely used as keycomponents for labelling of cells, tissue as well as whole organs in vitro and in vivo. This includes staining agents in cell biology and histology as well as contrast agents for optical imaging.2-10 Moreover, fluorescent nanoparticles are used for labelling and advertising in/on various types of polymers.11-13 For all these purposes, molecular organic dyes – such as coumarins, rhodamines, oxazines, cyanines, etc. – are conventionally used.14 However, they often suffer from limited emission intensity, low photostability and low contrast (i.e., the difference between absorptive/emissive dye molecule and non-absorbing/non-emitting background).1-14 To evade the weaknesses of molecular organic dyes, nanoparticles have turned out as promising alternatives many times. Due to the quasi-infinite number of absorptive/emissive centers per nanoparticle the optical contrast is greater in magnitudes order. By the same token photostability and chemical stability are greater as well. Aiming at fluorescence, semiconductor-type quantum dots (Q-dots, e.g. CdSe@ZnS) represent the most prominent class of nanoparticles that is well-known for unrivalled brightness and excellent photostability.15,16 On the other hand, Q-dots suffer from disadvantages such as high demands on chemical synthesis, elaborate core-shell structures, and harmful elements. As an alternative, absorptive and/or fluorescent molecular organic dyes were encapsulated in inorganic matrices (e.g., silica, phosphates),17-22 organic polymers (e.g., polyglycolic acid/PGA, polylactic acid/PLA, poly(lactic-co-glycolic acid)/PLGA, polycaprolactone/PCL, chitosan, polydopamine/PDA, polyethylene glycol/PEG),23-26 or liposomes27-31 to overcome the intrinsic weaknesses of insufficient emission intensity and low photostability. These measures, on the one hand, complicate the material synthesis, and on the other hand, increase the material complexity. Furthermore, the concentration of dye molecules in relation to the amount of the inert matrix becomes unfavorable. Typically, the dye concentration is only in a range of 5 mol-% and below.17-31 Taken together, alternative concepts are eligible for the use of standard, less harmful fluorescent dyes, but at increased dye concentration. Aiming at high dye concentration and low photobleaching in combination with low material complexity and straightforward synthesis, we here suggest the novel sulfonate-based inorganicorganic

hybrid

nanoparticles

(IOH-NPs)

having

a

general

saline

composition

[Gd(OH)]2+n/2[Rdye(SO3)n]n–. Specifically, the dye anions comprise Chicago Sky Blue ([CSB]4–), Direct Blue 71 ([DB71]4–), Nuclear Fast Red ([NFR]2–), Acid Red 97 ([AR97]2–), and Evans Blue 3 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 4 of 35

([EB]4–). These molecular dyes are commercially available, and they have been widely used in solution for staining in cell biology and histology already.14,32-48 Especially, this holds for Chicago Sky Blue,9 Nuclear Fast Red,38-42 and Evans Blue.45-48 Here, these molecular dyes are made insoluble upon addition of Gd3+ as a cation to obtain well-dispersible sulfonate-based nanoparticles with unprecedentedly high dye load (70-80 wt-% per nanoparticle) as aqueous suspensions. The aqueous [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NP suspensions show strong absorption and emission ranging from blue to orange, red and infrared light.

RESULTS AND DISCUSSION

Synthesis of sulfonate-based IOH-NPs. The material concept and chemical composition of the IOH-NPs – according to the Latin origin “hybrid = crossbreed” – combines two parts that are intermixed on the molecular level. Thus, an inorganic cation (e.g. [GdO]+, [Gd(OH)]2+) is combined with a fluorescent organic anion [Rdye(SO3)n]n– to form saline IOHNPs that are insoluble in water. Based on the stoichiometric ratio of inorganic cation and fluorescent organic anion, unprecedented high loads of the fluorescent organic anion are reached with 70-80 wt-% per nanoparticle. We have already proven the feasibility and multifunctionality of such IOH-NPs in regard of multimodal imaging with [GdO]+[ICG]– (ICG: indocyanine green) as the first example of sulfonate-based IOH-NPs.49-52 [GdO]+[ICG]– allows multimodal imaging including OI, photoacoustic imaging (PAI) and magnetic resonance imaging (MRI) (Figure 1).51 In vitro and in vivo studies, moreover, demonstrated high biocompatibility and biodegradability of [GdO]+[ICG]– (in suspension) with longer detectability and greater emission intensity than for ICG solutions. Moreover, a reduced toxicity and a comparable T1-relaxivity in comparison to the clinically used MRI contrast agent GdDTPA (DTPA: diethylenetriamine pentaacetate) were evidenced.51

4 ACS Paragon Plus Environment

Page 5 of 35 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

Bioconjugate Chemistry

Figure 1. Multifunctional imaging based on [GdO]+[ICG]– IOH-NPs (ICG: indocyanine green) including: a) Composition with inorganic [GdO]+ cation and fluorescent organic [ICG]– anion; b) Aqueous suspension with green color and red-light emission; c) Magnetic resonance imaging (MRI, mouse model); d) Photoacoustic imaging (PAI, dead mouse phantom); e) Optical imaging (OI, mouse model) (summary of data published in [51]). Based on the promising synthesis and the multimodal properties of [GdO]+[ICG]–,51 we here intend to broaden the material platform of sulfonate-based IOH-NPs by introducing further sulfonate-based fluorescent organic anions. This could allow establishing IOH-NPs with specific absorption and emission suitable for different requirements of staining and/or imaging. In this regard, we have chosen different fluorescent anions that are already used as molecular species in solution, including Chicago Sky Blue (CSB), Direct Blue 71 (DB71), Nuclear Fast Red (NFR), Acid Red 97 (AR97), and Evans Blue (EB) (Figure 2). All these fluorescent organic anions indeed can be transferred into insoluble, saline compounds upon addition of Gd3+ via straightforward aqueous synthesis, resulting in the IOH-NPs [Gd(OH)]2+2[CSB]4–, [Gd(OH)]2+2[DB71]4–, [Gd(OH)]2+[NFR]2–, [Gd(OH)]2+[AR97]2–, and [Gd(OH)]2+2[EB]4– that show blue, orange, red and infrared absorption and emission (Figure 2).

5 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 6 of 35

Figure 2. Scheme illustrating the saline composition of the sulfonate-based inorganicorganic hybrid nanoparticles (IOH-NPs) [Gd(OH)]2+n/2[Rdye(SO3)n]n–. The IOH-NPs are composed of stoichiometric amounts of the inorganic cation [Gd(OH)]2+ and a molecular organic dye anion [Rdye(SO3)n]n– containing sulfonate groups. Specifically, the synthesis of the [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs was performed via uncomplex precipitation in water, which is exemplarily illustrated for [Gd(OH)]2+[NFR]2– (Figure 3a). To this concern, an aqueous solution of the sodium salt of the respective molecular dye anion was prepared, in which an aqueous solution of GdCl3×6H2O was injected under intense stirring. For obtaining nanoparticles and colloidally stable suspensions, general aspects of controlling particle nucleation and particle growth have to be considered following the LaMer-Dinegar model.53 Hence, rapid mixing was performed in order to establish a high oversaturation. Moreover, the fluorescent organic anion was applied with 5 mol% excess to guarantee an anion termination of the as-nucleated nanoparticles. Subsequent to the synthesis, the IOH-NPs were centrifuged/redispersed three times from/in water to remove all remaining salts and starting materials. All sulfonate-based IOH-NPs can be easily dispersed in polar solvents such as water or biological buffers such as HEPES or aqueous dextran.

6 ACS Paragon Plus Environment

Page 7 of 35 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

Bioconjugate Chemistry

Figure 3. Scheme illustrating the aqueous synthesis of [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs with [Gd(OH)]2+[NFR]2– as specific example: a) Molecular structure of [NFR]2– anion; b) SEM image; c) Zeta potential in water; d) Aqueous suspension of [Gd(OH)]2+[NFR]2– IOHNPs; e) Size and size distribution according to SEM and DLS (see SI: Figure S1 for Zeta potential of all other sulfonate-based IOH-NPs). It is to be noted that the [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs are readily available as colloidally stable aqueous suspensions after synthesis. Neither advanced multistep procedures nor core-shell structures or specific demands regarding materials crystallinity and surface conditioning for dispersion in water have to addressed, which is a major difference to Qdots,15,16 and which facilitates the synthesis enormously. In fact, all sulfonate-based IOHNPs are amorphous and do not show any specific Bragg peak. The concept of sulfonatebased IOH-NPs, moreover, comprises several advantages over simple solutions of the fluorescent anions or matrix-encapsulated dye molecules:14,17-31 i) The availablility of colloidally stable aqueous suspensions instantaneously after synthesis; ii) The unprecedented high dye load (70-80 wt%) with a great number of absorptive/emissive centers per nanoparticle; iii) The different uptake mechanisms of nanoparticles (i.e. phagocytosis, pinocytosis) resulting in very efficient internalization; iv) Optional multimodal detection via absorption (color), emission (fluorescence) and magnetism (paramagnetism of Gd3+).54,55 Based on the waterbased synthesis strategy, the sulfonate-based IOH-NPs are accessible in large quantities (e.g. 1 g of IOH-NPs made in 100 mL of water) and concentrated suspensions (up to 10 mg/mL).

Particles

size

and

[Gd(OH)]2+n/2[Rdye(SO3)n]n–

chemical

composition.

The

particle

diameter

of

the

IOH-NPs was investigated via scanning electron microscopy 7 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 8 of 35

(SEM). Accordingly, average particle diameters of 40-50 nm were determined at narrow size distribution by statistical evaluation of at least 100 nanoparticles from SEM overview images (Table 1). Representative SEM images of all sulfonate-based IOH-NPs are shown in Figure 3b and Figure 4. In view of the very comparable composition of all IOH-NPs and in view of similar conditions of synthesis, in fact, the observed coincidence of particle size and shape is not a surprise. Table 1. Average particle size of the sulfonate-based IOH-NPs [Gd(OH)]2+n/2[Rdye(SO3)n]n– as obtained from SEM and DLS as well as Zeta potential (in water at pH 7.0) and total dye load (for Zeta potential analysis see SI: Figure S1). Compound [Gd(OH)]2+2[CSB]4–

SEM*

DLS**

Zeta potential

(nm)

(nm)

(mV, at pH 7.0) (wt-%)

38(5)

91(11)

–26

74

43(7)

–42

73

44(9)

91(11)

–28

70

47(6)

50(8)

–19

79

42(10)

114(13) –42

77

[Gd(OH)]2+2[DB71]4– 37(7) 2+

2–

[Gd(OH)] [NFR] 2+

[Gd(OH)] [AR97] [Gd(OH)]2+2[EB]4–

2–

Dye load

(*Statistical average of >100 nanoparticles; **hydrodynamic diameter)

Besides electron microscopy, the particle diameter of the as-prepared sulfonate-based IOH-NPs was also validated via dynamic light scattering (DLS) in water, resulting in mean hydrodynamic diameters of 43(7) to 114(13) nm (Table 1, Figure 3e, Figure 4). Diameter and size distribution of all IOH-NPs are again very comparable (Table 1). Here, it needs to be noted that the IOH-NPs are dispersed in water without any specific surface-active agent or surface functionalization, such as dextran, polyethylene glycol, oleate, oleylamine, etc.). Due to the interaction of the extended shell of absorbed water molecules (caused by the high dielectricity as well as by extensive hydrogen bridges),56 the hydrodynamic diameter is considerably larger than the average particle diameter obtained from SEM (Table 1, Figure 3e, Figure 4). Therefore, particle clustering driven by merged solvent shells is to be expected. Besides the absolute values, however, the particle size distribution obtained from SEM and DLS is very comparable, excluding any considerable agglomeration and indicating the stability of the aqueous suspensions. In fact, the aqueous suspensions show excellent colloidal stability without any visible agglomeration and/or sedimentation on a timescale of several weeks (Figure 3d, Figure 4). 8 ACS Paragon Plus Environment

Page 9 of 35 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

Bioconjugate Chemistry

Figure 4. Size and size distribution of the as-prepared, sulfonate-based IOH-NPs [Gd3+(OH)]2+2[EB]4–,

[Gd3+(OH)]2+2[DB71]4–,

[Gd3+(OH)]2+[AR97]2–

and

[Gd3+(OH)]2+2[CSB]4– including SEM images and size distribution analysis based on SEM (statistical evaluation of at least 100 nanoparticles) and DLS (hydrodynamic diameter of aqueous suspensions).

The excellent colloidal stability of the sulfonate-based IOH-NPs can be directly correlated to their Zeta potential in water. Thus, all [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs exhibit negative charging of –19 to –42 mV in water within the physiologically relevant pH range of 6.5 to 7.5 (Figure 3c; SI: Figure S1). [Gd(OH)]2+[NFR]2– as our representative example shows a Zeta potential of –28 mV at neutral pH (Figure 3c). In fact, a Zeta potential of –20 to –30 mV is considered as optimal for both sufficient charge stabilization and optimal uptake, for instance, in tumor cells.57 9 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 10 of 35

The negative surface charge and the low solubility of the sulfonate-based IOH-NPs, finally, account for the option of preparing nanosized and colloidally stabilized particles in water. To increase the membrane permeability and cell uptake in biomedical applications, the as-prepared sulfonate-based IOH-NPs can be also coated by dextran or protamine,58 which was already shown elsewhere.49-52 To validate the chemical composition of the sulfonate-based IOH-NPs, different analytical methods were involved. First of all, proving the presence of the fluorescent organic anion and of the inorganic Gd3+ cation are of course most essential. In this regard, Fourier-transform infrared (FT-IR) spectroscopy qualitatively evidences the presence of the fluorescent organic anion (Figure 5a; SI:

Figure S2). All IOH-NPs show the characteristic vibrations of the respective pure molecular dye. In detail, vibrations can become broader, and they can show less fine-structure and modulated intensities due to the non-crystallinity of the IOH-NPs and due to the high dye concentration in the IOH-NPs. [Gd(OH)]2+[NFR]2–, which was again selected as representative example, shows vibrations related to ν(O–H) (3500-3000 cm–1) as well as the most characteristic vibrations ν(C=O): 1700-1500 cm–1, ν(S=O) (1250-100 cm–1) and the fingerprint area (1250-500 cm–1). As expected these absorption bands are very comparable to pure nuclear fast red as a reference (Figure 5a). Similar

findings

were

also

observed

for

[Gd3+(OH)]2+2[EB]4–,

[Gd3+(OH)]2+2[DB71]4–,

[Gd3+(OH)]2+[AR97]2– and [Gd3+(OH)]2+2[CSB]4– (SI: Figures S2). Moreover, presence and function of the respective fluorescent organic anion are proven by their characteristic absorption and emission (Figures 6,7a).

Figure 5. Exemplary FT-IR spectra and TG of [Gd(OH)]2+[NFR]2– IOH-NPs: a) FT-IR (with pure NFR as a reference); b) TG (see SI: Figures S2,S3 for FT-IR and TG of all other sulfonate-based IOH-NPs).

Beside the fluorescent organic anion, the presence of gadolinium and sulfur was qualitatively proven by energy dispersive X-ray analysis (EDX) (Table 2). To quantify the chemical composition 10 ACS Paragon Plus Environment

Page 11 of 35 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

Bioconjugate Chemistry

and the cation-to-anion ratio in the sulfonate-based IOH-NPs, thermogravimetry (TG), and elemental analysis (EA) were performed. Prior to analysis, the as-prepared IOH-NPs were dried in vacuum at room temperature for 8 hours to remove all adsorbed solvents and water. Thereafter, TG shows a weight loss of 62-83% in a temperature range of 30-1000 °C due to total combustion of the organic content, i.e, the thermal combustion of the fluorescent organic anion (Figure 5b; SI:

Figure S3). For all sulfonate-based IOH-NPs, the thermal remnant was identified via X-ray powder diffraction (XRD) as Gd2O2(SO4). For the specific example of [Gd(OH)]2+[NFR]2–, the thermal combustion can be rationalized based on the following reaction: 2[Gd(OH)]2+[C14H7SNO7]2– + 29O2 → Gd2O2(SO4) + 28CO2 + 8H2O + N2 + 2SO2 Table 2. Chemical composition of sulfonate-based IOH-NPs [Gd(OH)]2+n/2[Rdye(SO3)n]n– as obtained by EA (C,H,N,S content) and TG (total combustion weight loss). Compound

[Gd(OH))2[CSB] [(Gd(OH)]2[DB71] [Gd(OH)][NFR]

[Gd(OH)][AR97]

[Gd(OH)]2[EB]

C content

H content

N content

S content

Weight loss

(%-wt)

(%-wt)

(%-wt)

(%-wt)

(%-wt)

(calcd)

(calcd)

(calcd)

(calcd)

(calcd)

29.8

3.6

6.2

8.9

68.0

(32.7)

(2.1)

(6.7)

(10.3)

(63.5)

25.6

3.2

5.3

6.8

58.6

(37.2)

(2.3)

(7.6)

(9.9)

(66.2)

35.0

2.6

3.0

6.7

62.4

(33.1)

(1.8)

(2.8)

(6.3)

(56.4)

46.1

3.6

6.4

7.7

75.1

(47.5)

(2.5)

(6.9)

(7.9)

(73.1)

30.8

3.7

6.4

9.2

67.2

(33.4)

(2.1)

(6.9)

(10.5)

(63.6)

Based on the above reaction, a weight loss due to total combustion of 56.4% is calculated for [Gd(OH)]2+[NFR]2–, which fits well with the experimental observation of 62.4 wt-% (Table 2, Figure 5b). A similar coincidence between experimental observation and calculated values was also found for all other sulfonate-based IOH-NPs (Table 2; SI: Figure S3). Due to the high weight of the Gd-based cation as well as of the fluorescent organic anion, the cation-to-anion ratio of the sulfonate-based IOH-NPs can be deduced with good accuracy. The obtained 1:1 or 2:1 ratios fit well with the charges of the respective cation and anion. Last but not least, the chemical composition was validated by EA (Table 2). Here, the observed carbon, hydrogen, and nitrogen contents are also well in agreement with the calculated values within the significance of the analysis. 11 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 12 of 35

All in all, the chemical composition and the cation-to-anion ratio of the novel sulfonate-based IOHNPs [Gd(OH)]2+2[CSB]4–, [Gd(OH)]2+2[DB71]4–, [Gd(OH)]2+[NFR]2–, [Gd(OH)]2+[AR97]2–, and [Gd(OH)]2+2[EB]4– are reliably determined based on different analytical methods. It is to be noted that the negative charge of the fluorescent organic anions is directly correlated to the number of available SO3 groups. Only in the case of [NFR]2– an additional negative charge stems from the acidic OH group (Figure 4a).38-42 Optical properties. The presented [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs show intense absorption over the whole visible regime resulting in bright absorptive colors that are easy to detect (Table 3, Figure 7a). The absorption of the organic dye anions results in the blue color of [Gd(OH)]2+2[CSB]4– and [Gd(OH)]2+2[EB]4– as well as the orange to red color of [Gd(OH)]2+[NFR]2–, [Gd(OH)]2+[AR97]2– and [Gd(OH)]2+2[DB71]4– (Figure 6). The absorptive color of the IOH-NPs – as expected – is identical to the pure organic dyes. However, the absorption is much stronger due to the great number of dye anions in each single IOH-NP. Therefore, suspensions of the IOH-NPs can be highly interesting for staining in cell biology and histology in alternative to the molecular dyes in solution. In contrast to the molecular dyes in solution, the [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs not only show strong absorption, they also show intense emission upon excitation with visible light (i.e. blue-light light-emitting diode/blue-light LED) (Table 3). Specifically, the emission occurs for [Gd(OH)]2+2[CSB]4– and [Gd(OH)]2+2[DB71]4– in the blue spectral range (Table 3, Figures 6,7b). [Gd(OH)]2+[NFR]2– and [Gd(OH)]2+2[AR97]4– show emission of yellow and red light (Table 3, Figures 6,7b). [Gd(OH)]2+[EB]2–, finally, emits in the deep red to infrared spectral range (Table 3, Figures 6,7b). Such emission is extremely interesting for optical imaging in cell biology and histology but also for in vitro and in vivo studies.1-10 Most interestingly, emissive properties were yet only reported for NFR, CSB, and EB in the case of the molecular dyes,60-63 whereas a fluorescence of DB71 and AR97 was not reported before. The extremely high dye load per IOH-NP (70-80 wt-%) and the resulting great number of fluorescent centers per nanoparticle volume, on the one hand, guarantee for intense light absorption, and on the other hand, result in an emission that is intense enough to be visible for the human eye or to be detected via spectroscopy and microscopy (Figures 6,7b). Another advantage of the IOH-NPs and the quasi-infinite number of fluorescence centers per nanoparticle is related to low bleaching. Even after partial bleaching, the reservoir of intact fluorescent organic anions in the IOH-NPs is high enough to guarantee intense absorption and emission. This latter aspect was validated in detail for [GdO]+[ICG]–,51 Gd3+[AMA]– (AMA: amaranth red),52 or [ZrO]2+[MFP]2– (MFP: methylfluorescein phosphate).50 12 ACS Paragon Plus Environment

Page 13 of 35 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

Bioconjugate Chemistry

Table 3. Excitation and emission of the sulfonate-based IOH-NPs. IOH-NP composition

Excitation range

Emission range

Emission

(nm)

(nm)

λmax (nm)

[Gd(OH)]2+2[CSB]4–

240–460

400–550

437

[Gd(OH)]2+2[DB71]4–

320–440

400–550

444

[Gd(OH)]2+[NFR]2–

400–580

520–740

578

[Gd(OH)]2+[AR97]2–

550–730

550–640

592

[Gd(OH)]2+2[EB]4–

350–640

700–880

782

Figure 6. Aqueous suspensions of the as-prepared, sulfonate-based [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs with the fluorescent organic anion and the respective absorptive color and visible emission ([Gd(OH)]2+2[CSB]4–, [Gd(OH)]2+2[DB71]4–, [Gd(OH)]2+[AR97]2– excited via UV-LED; [Gd(OH)]2+[NFR]2– excited via halogen lamp with green glass filter; [Gd(OH)]2+2[EB]4– excited via white light halogen lamp). In vitro evaluation. All sulfonate-based [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs show intense absorption and emission ranging over the complete visible regime (Figure 7), so that they can be interesting for staining and optical imaging. [Gd(OH)]2+2[EB]4–, however, seems most relevant for biomedical application due to its deep red to infrared emission (Table 3, Figure 7), which is most suitable for deep tissue penetration. As a molecular dye in solution, moreover, Evans Blue is already widely used in viability assays as well as to assess the permeability of the blood-brain barrier.45-48,64,65 Therefore, we exemplarily address uptake, fluorescence imaging and toxicity of [Gd(OH)]2+2[EB]4– IOH-NPs in vitro via incubation with

13 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 14 of 35

human cervix carcinoma cells (HeLa cells) and human umbilical vein endothelial cells (HUVEC).

Figure 7. Optical properties of sulfonate-based [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs: a) Excitation spectra, b) Emission spectra (normalized on maximum intensity for direct comparison).

Figure 8. Uptake and emission of [Gd(OH)]2+2[EB]4– IOH-NPs at 1 and 10 µg/mL after incubation (24 h) with HeLa and HUVEC cells (untreated cell as control). Cells were treated with 2 µg/mL Hoechst 33342 for nuclear staining. Fluorescence confocal microscopy images were taken by excitation/emission with 405 nm/460-480 nm (for nuclei) and 635 nm/640740 nm (for Evans Blue). Depicted are merged images of both fluorescent emissions (scale bar: 20 µm).

14 ACS Paragon Plus Environment

Page 15 of 35 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

Bioconjugate Chemistry

The cellular uptake in HeLa and HUVEC cells is clearly demonstrated after incubation (up to 24 h at 37 °C) with [Gd(OH)]2+2[EB]4– suspensions (1 and 10 µg/mL). Thus, fluorescence microscopy clearly indicates the expected red emission of the IOH-NPs starting at concentrations of 1 µg/mL (Figure 8). Untreated cells (control) in comparison show only the blue emission of Hoechst 33342-stained nuclei. The IOH-NPs are mainly accumulating in the perinuclear endosomal compartments (Figure 8). Whereas cultivation of HUVEC cells with 1 µg/mL [Gd(OH)]2+2[EB]4– IOH-NPs already results in certain uptake and weak emission, uptake into HeLa cells at this level of concentration is weak. Cultivation with 10 µg/mL for both cell lines is sufficient and results in a massive uptake and an intense emission (Figure 8).

Figure 9. In vitro cytotoxicity of [Gd(OH)]2+2[EB]4– IOH-NPs assessed by MTT assays after 72 h of incubation with HeLa and HUVEC cells (statistical error bars calculated from n = 6). The cytotoxicity potential of the [Gd(OH)]2+2[EB]4– IOH-NPs was evaluated by MTT toxicity assays (Figure 9). By mitochondrial enzyme reactivity, metabolically active cells reduce the yellow tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) to a purple formazan, which can be easily quantified photometrically. The amount of formazan measured is directly proportional to the ratio of viable cells present in each well. Cells were incubated with the indicated concentration of [Gd(OH)]2+2[EB]4– IOH-NPs for 72 hours. As a result, the cytotoxicity of [Gd(OH)]2+2[EB]4– turned out as low in the concentration range needed for staining and fluorescence imaging. Thus, the viability is at 60-100% after incubation with 110 µg/mL (Figure 9). Especially, HUVEC cells show good viability at low concentrations of [Gd(OH)]2+2[EB]4–. Only at high concentrations (>20 µg/mL), the [Gd(OH)]2+2[EB]4– IOH-NPs become cytotoxic. Based on the MTT assays (Figure 9), finally, the median lethal dose (LD50) can 15 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 16 of 35

be deduced to 20 µg/mL for HUVEC cells and to about 100 ug/mL for HeLa cells. With the intense fluorescence and the moderate toxicity, all in all, the sulfonate-based [Gd(OH)]2+n/2[Rdye(SO3)n]n– IOH-NPs can become a useful addition for staining and fluorescence imaging.

CONCLUSION AND OUTLOOK In conclusion, five novel saline sulfonate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general composition [Gd(OH)]2+n/2[Rdye(SO3)n]n– are presented, specifically

including

[Gd(OH)]2+2[CSB]4–,

[Gd(OH)]2+2[DB71]4–,

[Gd(OH)]2+[NFR]2–,

[Gd(OH)]2+[AR97]2–, and [Gd(OH)]2+2[EB]4–. These compounds show visible absorption and emission in the blue, orange, red and infrared regime, respectively. Synthesis is performed by straightforward forced hydrolysis in water and results in colloidally highly stable suspensions with mean particle diameters of 40-50 nm. Colloidal stability and nucleation of nanoparticles are supported by the high zeta potential of –25 to –40 mV at neutral pH. Based on an aqueous synthesis, the low costs of constituents, the high colloidal stability, the absence of harmful metals (e.g. Cd2+, Pb2+), the high dye load (up to 80 wt-%), the use of conventional organic dyes molecules as anions, and the visible absorption and emission, the sulfonate-based IOH-NPs can become a highly interesting class of nanoparticles. Due to the quasiinfinite number of dye anions, the IOH-NPs offer strong absorption and emission as well as low photobleaching. In vitro studies based on HeLa and HUVEC cells that were exemplarily performed with [Gd(OH)]2+2[EB]4– show intense fluorescence and moderate toxicity (at 1-10 µg/mL) indicating the feasibility of the sulfonate-based IOH-NPs. Potential areas of application can relate to all kinds of staining, histology, microscopy, optical imaging, as well as the absorptive or emissive labelling of polymers. Aiming at biomedical application, moreover multimodality is optional including optical imaging and magnetic resonance imaging.

EXPERIMENTAL SECTION Analytical techniques

Dynamic light scattering (DLS) was used to determine the hydrodynamic diameter of the sulfonate-based IOH-NPs and their size distribution in suspension. Studies were conducted in polystyrene cuvettes applying a Nanosizer ZS (Malvern Instruments, United Kingdom).

Zeta potential measurements were conducted using an automatic titrator MPT-2 attached to the Nanosizer ZS (Malvern Instruments, United Kingdom). For a typical measurement, 1 mL of a suspension containing 5 mg/mL of the sulfonate-based IOH-NPs was diluted with 9 mL of 16 ACS Paragon Plus Environment

Page 17 of 35 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

Bioconjugate Chemistry

demineralized water. Titration was performed using 0.01 M and 0.1 M HCl as well as 0.01 M and 0.1 M NaOH.

Scanning electron microscopy (SEM) was carried out with a Zeiss Supra 40 VP (Zeiss, Germany), equipped with a field emission gun (acceleration voltage 1 kV, working distance 3 mm). Samples were prepared by placing a droplet of diluted aqueous suspensions of the sulfonate-based IOH-NPs on a silica wafer that was left for drying overnight.

Energy-dispersive X-ray (EDX) analysis was performed with an Ametek EDAX (Ametek, United States), device mounted on the above described Zeiss SEM Supra 40 VP scanning electron microscope. For this purpose, the sulfonate-based IOH-NPs were pressed to dense pellets in order to guarantee for a smooth surface and a quasi-infinite layer thickness. These pellets were fixed with conductive carbon pads on aluminum sample holders. EDX was only used to validate the presence of gadolinium and sulfur in the IOH-NPs. A quantification of Gd/S or determination of the lighter elements C/N/O/H was not performed via this method due to limited significance.

X-ray powder diffraction (XRD) measurements were conducted with a Stadi-P diffractometer (Stoe, Germany) with Ge-monochromatized Cu-Kα radiation. The dried sulfonate-based IOH-NP samples were fixed between Scotch tape and acetate paper and measured between –69 ° and +69 °of two-theta.

Fourier-transformed infrared (FT-IR) spectra were recorded with a Bruker Vertex 70 FT-IR spectrometer (Bruker, Germany) in the range 4000−370 cm–1 with a resolution of 4 cm–1. To this concern, 1 mg of dried sulfonate-based IOH-NP sample was pestled with 300 mg of KBr and pressed to a pellet.

Differential thermal analysis/thermogravimetry (DTA/TG) was performed with a STA409C device (Netzsch, Germany). The measurements were performed in air. The vacuum dried sulfonatebased IOH-NP samples (20 mg in corundum crucibles) were heated to 1000 °C with a rate of 1 K/min.

Elemental analysis (C/H/N/S analysis) was performed via thermal combustion with an Elementar Vario Microcube device (Elementar, Germany) at a temperature of about 1100 °C.

Photoluminescence (PL) was recorded with a Horiba Jobin Yvon Spex Fluorolog 3 (Horiba Jobin Yvon, France) equipped with a 450 W Xe-lamp and double grating excitation and emission monochromator.

Synthesis of sulfonate-based IOH-NPs For preparing [Gd(OH)]2+n/2[RdyeSO3]n– IOH-NPs, the following starting materials were used: Acid Red 97/sodium salt (Na2[AR97], 40%, Sigma Aldrich), Nuclear Fast Red/sodium salt (Na2[NFR], Sigma Aldrich), Direct Blue 71/sodium salt (Na4[DB71], 50%, Sigma Aldrich), 17 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 18 of 35

Chicago Sky Blue 6B/sodium salt (Na4[CSB], Sigma Aldrich), Evans Blue/sodium salt (Na4[EB], Alfa Aesar), and GdCl3×6H2O (99%, Sigma Aldrich).

[Gd(OH)] 2+2[EB]4– IOH-NPs were prepared by injecting an aqueous solution of 62 mg GdCl3×6H2O in 1 mL of H2O to a solution of 32 mg Na4[EB] in 30 mL of H2O at room temperature. Admixing was performed under vigorous stirring. After 2 min of intense stirring, the IOH-NPs were separated via centrifugation (25,000 RPM, 15 min). To remove all remaining salts, the nanoparticles were resuspended in and centrifuged from H2O three times.

[Gd(OH)] 2+2[DB71]4– IOH-NPs were prepared by injecting an aqueous solution of 62 mg GdCl3×6H2O in 1 mL of H2O to a solution of 32 mg Na4[DB71] in 30 mL of H2O at room temperature. Admixing was performed under vigorous stirring. After 2 min of intense stirring, the IOH-NPs were separated via centrifugation (25,000 RPM, 15 min). To remove all remaining salts, the nanoparticles were resuspended in and centrifuged from H2O three times.

[Gd(OH)] 2+[AR97]2– IOH-NPs were prepared by injecting an aqueous solution of 37 mg GdCl3×6H2O in 1 mL of H2O, to a solution of 69 mg Na2[AR97] in 30 mL of H2O at room temperature. Admixing was performed under vigorous stirring. After 2 min of intense stirring, the IOH-NPs were separated via centrifugation (25,000 RPM, 15 min). To remove all remaining salts, the nanoparticles were resuspended in and centrifuged from H2O three times.

[Gd(OH)] 2+[NFR]2– IOH-NPs were prepared by injecting an aqueous solution of 37 mg GdCl3×6H2O in 1 mL of H2O, to a solution of 35 mg Na2[NFR] in 30 mL of H2O at room temperature. Admixing was performed under vigorous stirring. After 2 min of intense stirring, the IOH-NPs were separated via centrifugation (25,000 RPM, 15 min). To remove all remaining salts, the nanoparticles were resuspended in and centrifuged from H2O three times.

[Gd(OH)] 2+2[CSB] 4– IOH-NPs were prepared by injecting an aqueous solution of 62 mg GdCl3×6H2O in 1 mL of H2O, to a solution of 33 mg Na4[CSB] in 30 mL of H2O at room temperature. Admixing was performed under vigorous stirring. After 2 min of intense stirring, the IOH-NPs were separated via centrifugation (25,000 RPM, 15 min). To remove all remaining salts, the nanoparticles were resuspended in and centrifuged from H2O three times.

Material composition of IOH-NPs Detailed information regarding the material characterization of [Gd(OH)]2+2[CSB]4–, [Gd(OH)]2+2[DB71]4–, [Gd(OH)]2+[AR97]2–, and [Gd(OH)]2+2[EB]4– including electron microscopy, dynamic light scattering, Zeta potential measurement, FT-IR spectroscopy, and thermogravimetry can be found in the Supporting Information.

18 ACS Paragon Plus Environment

Page 19 of 35 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

Bioconjugate Chemistry

Biological studies

Cell culture. Cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptavidin (Gibco) (in the case of HeLa cells) or EGM-2 (Lonza) (in the case of HUVEC cells) at 37 °C, 5% CO2, and humid atmosphere. For all in vitro experiments, cells were trypsinized (0.05% trypsin-EDTA, Gibco) and seeded onto 96-well-plates (toxicity assay), or ibidi 8-well-µslides (confocal microscopy) at the required densities. Incubation was performed with the culture conditions as described.

Cell viability assay. To determine the cytotoxicity potential (LD50 values), MTT toxicity assays were performed. By mitochondrial enzyme reactivity, metabolically active cells reduce the yellow tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) to a purple formazan, which can be quantified photometrically. The measured amount of formazan is directly proportional to the ratio of viable cells present in each well. Cells were incubated with the indicated concentrations of [Gd(OH)]2+2[EB]4– IOH-NPs for 3 days. For the evaluation, all wells were washed with DPBS and controls were treated with Triton X-100 before finally adding 15 µl MTT solution (Promega) to each well. After 1.5 h of incubation, the reaction was stopped by adding 100 µL Lysis Buffer (Promega), and the resulting absorbance of the converted dye was determined the next day at 595 nm (ultra microplate reader ELx808, BioTEK instruments). All results were obtained 6-folds and set into correlation with positive and negative controls, error bars indicate the standard deviation.

Confocal microscopy (using a Leica SPE Scanning confocal inverted microscope) was performed in order to investigate the cellular uptake and the distribution of the [Gd(OH)]2+2[EB]4– IOH-NPs. After incubation, cells were stained with 2 µg/mL Hoechst 33342 (nuclei stain) and three times washed with DPBS. Hoechst 33342 was excited at 405 nm (UV laser); the [Gd(OH)]2+2[EB]4– IOH-NPs were excited at 635 nm (HeNe laser). The emission was measured at 460-480 nm (nuclei) and 640-740 nm (IOH-NPs). Images were taken as sequential scans at 400 Hz and with a resolution of 1024×1024 pixels. Finally, the images were combined as overlays.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via ACS Publications website http://pubs.acs.org. This includes details regarding Zeta potential measurements, FT-IR spectroscopy, and thermogravimetry.

19 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 20 of 35

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful to the Deutsche Forschungsgemeinschaft (DFG) for funding. The work was supported by the Deutsche Forschungsgemeinschaft (DFG), within the Research Training Group 2039 (E.Z., C.F., U.S.). Furthermore, M.P. acknowledges the support of the DFG graduate School Karlsruhe School of Optics and Photonics (KSOP) at the KIT.

REFERENCES (1)

Fujimoto, J. G., and Farkas D. L. (2009) Biomedical Optical Imaging, Oxford University Press (Review).

(2)

Marks, K. M., and Nolan G. P. (2006) Chemical labeling strategies for cell biology. Nature

Methods 3, 591-596. (3)

Moon, H. C., Kim, C. H., Lodge, T. P., and Frisbie, C. D. (2016) Multicolored, Low-Power, Flexible Electrochromic Devices Based on Ion Gels. ACS Appl. Mater. Interfaces 8, 62526260.

(4)

Fleischmann, C., Lievenbruck, M., and Ritter, H. (2015) Polymers and Dyes: Developments and Applications. Polymers 7, 717-746.

(5)

Butkevich, A. N., Mitronova, G. Y., Sidenstein, S. C., Klocke, J. L., Kamin, D., Meineke, D. N. H., D'Este, E., Kraemer, P. T., Danzl, J. G., and Belov, V. N. (2016) Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells. Angew. Chem. Int. Ed. 55, 3290-3294.

(6)

Huang, Y. L, Walker, A. S., and Miller, E. W. (2015) A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing. J. Am. Chem. Soc. 137, 10767-10776.

(7)

Iwata, M., Ohno, Y., and Otaki, Y. M. (2014) Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue. PLoS One 9, e89500/1-e89500/15.

(8)

Miao, J. T., Fan, C., Sun, R., Xu, Y. J., and Ge, J. F. (2014) Optical properties of hemicyanines with terminal amino groups and their applications in near-infrared fluorescent imaging of nucleoli. J. Mater. Chem. B 2, 7065-7072. 20 ACS Paragon Plus Environment

Page 21 of 35 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

Bioconjugate Chemistry

(9)

McLintock, A., Cunha-Matos, C. A., Zagnoni, M., Millington, O. R., and Wark, A. W. (2014) Universal Surface-Enhanced Raman Tags: Individual Nanorods for Measurements from the Visible to the Infrared (514–1064 nm). ACS Nano 8, 8600-8609.

(10) Lichtman, J. W., and Conchello, J. A. (2005) Fluorescence microscopy. Nature Methods 2, 910-919. (11) Reisch, A., and Klymchenko, A. S. (2016) Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging. Small 12, 1968-1992. (12) Lai So, V. L., He, L., Fei, B., Cheuk, K. K. L., and Xin, J. H. (2014) Bio-inspired colouration on various textile materials using a novel catechol colorant. RSC Adv. 4, 41081-41086. (13) Li, C., and Liu, S. (2012) Polymeric assemblies and nanoparticles with stimuli-responsive fluorescence emission characteristics. Chem. Commun. 48, 3262-3278. (14) Booth, G. (2000) Dyes, General Survey. Wiley-VCH, Weinheim. (15) Wegner, K. D., and Hildebrandt, N. (2015) Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors. Chem. Soc. Rev. 44, 4792-4834. (16) Chen, O., Zhao, J., Chauhan, V. P., Cui, J., Wong, C., Harris, K. D., Wei, H., Han, H.-S., Fukumura, D., Jain, R. K., and Bawendi, M. G. (2013) Compact high-quality CdSe-CdS coreshell nanocrystals with narrow emission linewidths and suppressed blinking. Nature Mater. 12, 445-451. (17) Zhang, N., Ding, E., Feng, X., Xu, Y., and Cai, H. (2012) Synthesis, characterizations of dyedoped silica nanoparticles and their application in labeling cells. Colloids Surf B Biointerfaces

89, 133-138. (18) Knopp, D., Tang, D., and Niessner, R. (2009) Review: Bioanalytical applications of biomolecule-functionalized nanometer-sized doped silica particles. Anal. Chim. Acta 647, 1430. (19) Muddana, H. S., Morgan, T. T., Adair, J. H., and Butler, P. J. (2009) Photophysics of Cy3Encapsulated Calcium Phosphate Nanoparticles. Nano Lett. 9, 1559-1566. (20) Fuller, J. E., Zugates, G. T., Ferreira, L. S., Ow, H. S., Nguyen, N. , Wiesner, U. B., and Langer, R. S. (2008) Intracellular delivery of core-shell fluorescent silica nanoparticles.

Biomater. 29, 1526-1532. (21) Burns, A. A., Vider, J., Ow, H., Herz, E., Penate-Medina, O., Baumgart, M., Larson, S. M., Wiesner, U., and Bradbury, M. (2009) Fluorescent Silica Nanoparticles with Efficient Urinary Excretion for Nanomedicine. Nano Lett. 9, 442-448. (22) Altinoglu, E. I. , Russin, T. J., Kaiser, J. M., Barth, B. M., Eklund, P. C., Kester, M., and Adair, J. H. (2008) Near-Infrared Emitting Fluorophore-Doped Calcium Phosphate Nanoparticles for In Vivo Imaging of Human Breast Cancer. ACS Nano 2, 2075-2084. 21 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 22 of 35

(23) Zheng, C., Zheng, M., Gong, P., Jia, D., Zhang, P., Shi, B., Sheng, Z., Ma, Y., and Cai, L. (2012) Indocyanine green-loaded biodegradable tumor targeting nanoprobes for in vitro and in vivo imaging. Biomater. 33, 5603-5609. (24) Hughes, G. A. (2005) Nanostructure-mediated drug delivery. Nanomedicine: Nanotechnology,

Biology and Medicine 1, 22-30. (25) Zhang, X.; Wang, K.; Lin, M.; Zhang, X.; Tao, L.; Chen, Y.; Wei, Y. (2015) Polymeric AIEbased nanoprobes for biomedical applications: recent advances and perspectives. Nanoscale 7, 11486-11508. (26) Lin, M.; Zeng, G.; Wang, K.; Wan, Q.; Tao, L.; Zhang, X.; Wei, Y. (2016) Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications. Nanoscale 8, 16819-16840. (27) Shen, Z., Loe, D. T., Awino, J. K., Kroger, M., Rouge, J. L., and Li, Y. (2016) Self-assembly of core-polyethylene glycol-lipid shell (CPLS) nanoparticles and their potential as drug delivery vehicles. Nanoscale 8, 14821-14835. (28) Goyard, D., Shiao, T. C., Fraleigh, N. L., Vu, H. Y., Lee, H., Diaz-Mitoma, F., Le, H. T., and Roy, R. (2016) Expedient synthesis of functional single-component glycoliposomes using thiol–yne chemistry. J. Mater. Chem. B 4, 4227-4233. (29) Ng, K. K., Takada, M., Harmatys, K., Chen, J., and Zheng, G. (2016) Chlorosome-Inspired Synthesis of Templated Metallochlorin-Lipid Nanoassemblies for Biomedical Applications.

ACS Nano 10, 4092-4101. (30) Hu, T., Cao, H., Yang, C., Zhang, L, Jiang, X., Gao, X., Yang, F., He, G., Song, X., and Tong, A. et al. (2016) LHD-Modified Mechanism-Based Liposome Coencapsulation of Mitoxantrone and Prednisolone Using Novel Lipid Bilayer Fusion for Tissue-Specific Colocalization and Synergistic Antitumor Effects. ACS Appl. Mater. Interfaces 8, 6586-6601. (31) Parveen, S., Misra, R., and Sahoo, S. K. (2012) Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging. Nanomedicine: Nanotechnology, Biology and Medicine

8, 147-166. (32) Cui, X., Nichols, S. M., Arteaga, O., Freudenthal, J., Paula, F., Shtukenberg, A. G., and Kahr, B. (2016) Dichroism in Helicoidal Crystals. J. Am. Chem. Soc. 138, 12211-12218. (33) Prakash, R., Prashanth, H. V., Ragunatha, S., Kapoor, M., Anitha, T. K., and Krishnamurthy, V. (2016) Comparative study of efficacy, rapidity of detection, and cost-effectiveness of potassium hydroxide, calcofluor white, and Chicago sky blue stains in the diagnosis of dermatophytoses. Internat. J. Dermatol. 55, e172-e175.

22 ACS Paragon Plus Environment

Page 23 of 35 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

Bioconjugate Chemistry

(34) Normand, A., Riviere, E., and Renodon-Corniere, A. (2014) Identification and characterization of human Rad51 inhibitors by screening of an existing drug library. Biochem.

Pharmacol. 91, 293-300. (35) Tambosis, E., and Lim, C. (2012) A comparison of the contrast stains, Chicago blue, chlorazole black, and Parker ink, for the rapid diagnosis of skin and nail infections. Internat. J.

Dermatol. 51, 935-938. (36) Zhu, W., Liu, Y., Cao, X., Zhang, S., Wang, C., and Lin, X. (2010) Recovering organic matters and ions from wastewater by genetically engineered Bacillus subtilis biomass. J. Environment.

Managem. 161, 402-407. (37) Pazarlioglu, N. K., Akkaya, A., Akdogan, H. A., and Gungor, B. (2010) Biodegradation of Direct Blue 15 by Free and Immobilized Trametes versicolor. Water Environment Res. 82, 579-585. (38) Li, Z., Liu, P., Yang, T., Sun, Y., You, Q., Li, J., Wang, Z., and Han, B. (2016) Composite poly(l-lactic-acid)/silk fibroin scaffold prepared by electrospinning promotes chondrogenesis for cartilage tissue engineering. J. Biomater. Appl. 30, 1552-1565. (39) Bemenderfer, T. B., Harris, J. S., Condon, K. W., and Kacena, M. A. (2014) Tips and Techniques for Processing and Sectioning Undecalcified Murine Bone Specimens. Meth. Mol.

Biol. 1130, 123-147. (40) Frank, M., Dapson, R. W., Wickersham, T. W., and Kiernan, J. A. (2007) Certification procedures for nuclear fast red (Kernechtrot), CI 60760. Biotech. Histochem. 82, 35-39. (41) Thomas, M. A., and Lemmer, B. (2005) HistoGreen: a new alternative to 3,3′diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry? Brain Res. Prot. 14, 107-118. (42) Chowdhury, A. G., Dasgupta, G., and Ray, H. N. (1955) ‘Kernechtrot’ or Nuclear Fast Red in the Histochemical Detection of Calcareous Corpuscles in Taenia saginata. Nature 176, 701702. (43) Hosoya, Y., Taguchi, T., and Tay, F. R. (200) Evaluation of a new caries detecting dye for primary and permanent carious dentin. J. Dentistry 35, 137-143. (44) Kass, L., Harrison, G. J., and Lindheimer, C. (2002) A new stain for identification of avian leukocytes. Biotech. Histochem. 77, 201-206. (45) Jang, W. Y., Lee, B. R., Jeong, J., Sung, Y., Choi, M., Song, P., Kim, H., Jang, S., Kim, H., and Joo, K. I. et al. (2017) Overexpression of serum amyloid a 1 induces depressive-like behavior in mice. Brain Res. 1654, 55-65.

23 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Page 24 of 35

(46) Xu, L., Cao, F., Xu, F., He, B., and Dong, Z. (2015) Bexarotene reduces blood-brain barrier permeability in cerebral ischemia-reperfusion injured rats. PLoS One 10, e0122744/1e0122744/14. (47) Valigurova, A., Paskerova, G. G., Diakin, A., Kovacikova, M., and Simdyanov, T. G. (2015) Protococcidian Eleutheroschizon duboscqi, an unusual apicomplexan interconnecting gregarines and cryptosporidia. PLoS One 10, e0125063/1-e0125063/27. (48) Sandor, N., Walter, F. R., Bocsik, A., Santha, P., Schilling-Toth, B., Lener, V., Varga, Z., Kahan, Z., Deli, M. A., and Safrany, G. et al. (2014) Low dose cranial irradiation-induced cerebrovascular damage is reversible in mice. PLoS One 9, e112397/1-e112397/13. (49) Montes-Cobos, E., Ring, S., Fischer, H., Heck, J. G., Schwaninger, M., Feldmann, C., Lühder, F., and Reichardt, H. M. (2017) Targeted delivery of glucocorticoids to macrophages in a mouse model of multiple sclerosis using inorganic-organic hybrid nanoparticles. J. Contr. Rel.

245, 157-169. (50) Heck, J. G., Napp, J., Simonato, S., Möllmer, J., Lange, M., Reichardt, H. M., Staudt, R., Alves, F., and Feldmann, C. (2015) Multifunctional Phosphate-Based Inorganic-Organic Hybrid Nanoparticles. J. Am. Chem. Soc. 137, 7329-7336. (51) Poß, M., Tower, R. J., Napp, J., Appold, L C., Lammers, T., Alves, F., Glüer, C.-C., Boretius, S., and Feldmann, C. (2017) Multimodal [GdO]+[ICG]− Nanoparticles for Optical, Photoacoustic, and Magnetic Resonance Imaging. Chem. Mater. 29, 3547-3554. (52) Poß, M.; Napp, J.; Niehaus, O.; Pöttgen, R.; Alves, F.; Feldmann, C. (2015) M3+[Amaranth Red]3- (M = La, Gd): A Novel Sulfonate-based Inorganic-Organic Hybrid Nanomaterial for Multimodal Imaging. J. Mater. Chem. C 3, 3860-3868. (53) LaMer, V. K., and Dinegar, R. H. J. (1950) Theory, Production and Mechanism of Formation of Monodispersed Hydrosols. J. Am. Chem. Soc. 72, 4847-4849. (54) Zhang, S., Gao, H., and Bao, G. (2015) Physical Principles of Nanoparticle Cellular Endocytosis. ACS Nano 9, 8655-8671. (55) Balmert, S. C., and Little, S. R. (2012) Biomimetic Delivery with Micro- and Nanoparticles.

Adv. Mater. 24, 3757-3778. (56) Brown, M. A., Abbas, Z., Kleibert, A., Green, R. G., Goel, A., May, S., and Squires, T. M. (2016) Determination of Surface Potential and Electrical Double-Layer Structure at the Aqueous Electrolyte-Nanoparticle Interface. Phys. Rev. X 6, 011007/1-011007/12. (57) Davis, M. E. (2012) Fighting cancer with nanoparticle medicines - The nanoscale matters.

MRS Bulletin 37, 828-835.

24 ACS Paragon Plus Environment

Page 25 of 35 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

Bioconjugate Chemistry

(58) Chao, Y., Karmali, P. P., Mukthavaram, R., Kesari, S., Kouznetsova, V. L., Tsigelny, I. F., and Simberg, D. (2013) Direct Recognition of Superparamagnetic Nanocrystals by Macrophage Scavenger Receptor SR-AI. ACS Nano 7, 4289. (59) Bogart, L. K., Taylor, A., Cesbron, Y., Murray, P., and Levy, R. (2012) Photothermal Microscopy of the Core of Dextran-Coated Iron Oxide Nanoparticles During Cell Uptake.

ACS Nano 6, 5961. (60) Roller, B. T., Munson, J. M., Brahma, B., Santangelo, P. J., Pai, S. B., and Bellamkonda, R. V. (2015) Evans blue nanocarriers visually demarcate margins of invasive gliomas. Drug Deliv.

Transl. Res. 5, 116-124. (61) Cui, Y., Yu, J., and Feng, S. (2014) Nuclear fast red as highly sensitive “off/on” fluorescent probe for detecting guanine. Talanta 130, 536-541. (62) Tabatabaei, S. N., Girouard, H., Carret, A.-S., and Martel, S. (2015) Remote control of the permeability of the blood-brain barrier by magnetic heating of nanoparticles: A proof of concept for brain drug delivery. J. Contr. Rel. 206, 49-57. (63) Isak, S. J., Eyring, E. M., Spikes, J. D., and Meekins, P. A. (2000) Direct blue dye solutions: photo properties. J. Photochem. Photobiol. A 134, 77-85. (64) Xu, T., Zhang, W-G., Sun, J., Zhang, Y., Lu, J-F., Zhou, C-M., Han, H-B., Yan, J-H. (2015) Protective effects of thrombomodulin on microvascular permeability after subarachnoid hemorrhage in mouse model. Neurosci. 299, 18-27. (65) Li, H., Lu, Z.-Q., Sun, J., Wang, F., Ding, G., Chen, W., Fang, R., Yao, Y., Pang, M., Liu, J., (2016) Sodium butyrate exerts neuroprotective effects by restoring the blood-brain barrier in traumatic brain injury mice. Brain Res. 1642, 70-78.

25 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Graphical Abstract 72x48mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 26 of 35

Page 27 of 35 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

Bioconjugate Chemistry

Figure 1 142x94mm (300 x 300 DPI)

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Figure 2 125x82mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 28 of 35

Page 29 of 35 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

Bioconjugate Chemistry

Figure 3 141x79mm (300 x 300 DPI)

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Figure 4 116x166mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 30 of 35

Page 31 of 35 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

Bioconjugate Chemistry

Figure 5 103x39mm (300 x 300 DPI)

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Figure 6 141x69mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 32 of 35

Page 33 of 35 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

Bioconjugate Chemistry

Figure 7 158x67mm (300 x 300 DPI)

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Figure 8 256x170mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 34 of 35

Page 35 of 35 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

Bioconjugate Chemistry

Figure 9 171x112mm (300 x 300 DPI)

ACS Paragon Plus Environment