1. Introduction

To improve medical diagnostics and therapies, researchers have begun to turn to x-ray radiation as the primary excitation source over traditional ultraviolet (UV) and visible light as x-ray radiation has the ability to pass through tissue at much greater penetration depths than UV or visible light [1, 2]. While traditional x-ray radiography and computed tomography (CT), where CT requires the use of heavy metal contrast agents to interact with the bombarding x-ray radiation to generate an image, have been used for decades clinically [3, 4], more sensitive techniques are needed for early detection, treatment, and prevention of diseases. Currently, x-ray luminescence arising from molecules that can absorb ionizing radiation are being used as positive contrast agents in x-ray luminescence computed tomography (XRCT) to enhance images and generate an elemental map of the sample upon optical detection [5–9]. While x-rays penetrate tissue much more effectively than UV or visible light, optical detection of the signal generated by the x-ray probe may remain a limiting factor. While enhanced imaging based on x-ray radiation is promising, current contrast agents contain heavy metals (due to their high density and ability to stop and absorb x-ray radiation), which can be toxic and require chelating agents or passivation layers to separate the probe from the body [10,11]. Molecules containing gadolinium, such as gadolinium oxide [12], gadolinium oxysulfide with various dopants [13], sodium gadolinium tungstate [14], and alkali metal gadolinium fluorides [15] are especially promising though there are studies that have shown successful imaging of lanthanum oxysulfide doped with europium or terbium [13], and doped alkali metal yttrium fluorides [16,17], but their use is limited due to the potential for toxicity.

However, fully organic, potentially less toxic [18,19], x-ray imaging probes that incorporate organic scintillators have been less studied [20]. The current approach focuses on a versatile system with stilbene as the organic scintillator encapsulated inside polystyrene-co-poly(propargyl acrylate) (PS-pPA) nanoparticles that exhibit a significant surface charge. The long-range electrostatic interactions result in a significant interparticle repulsion which yields in the adoption of a minimum energy crystalline colloidal array (CCA) with either body-centered cubic (bcc) or face-centered cubic (fcc) symmetry and spatial periodicities that range from ca. 10²– 10³ nm, resulting in the appearance of optical band gap effects such as a rejection wavelength. These CCAs can be photopolymerized into stable, monolithic crystalline hydrogels, which are more biologically applicable and mechanically robust than when in their initial liquid state [21]. The hydrogels could then be post-processed through copper(I) catalyzed azide/alkyne cycloaddition (CuCAAC) reactions in the hydrogel with a variety of azide modified fluorophores to achieve versatile hydrogels with various emission characteristics [22].

2. Results & discussion

Stilbene, an organic scintillator, was encapsulated inside polystyrene-co-poly(propargyl acrylate) (PS-pPA) colloidal particles of size 131.1 nm ± 9.90 nm through a doped emulsion co-polymerization. The polymerized stilbene/PS-pPA particles were electrostatically self-assembled into a liquid crystalline colloidal array (CCA) with deionized (DI) water as the dispersion media (cf. Fig. 1(a), stilbene/PS-pPA). The stilbene/PS-pPA liquid CCA can be photopolymerized into a hydrogel; upon x-ray irradiation, the hydrogel will emit blue light due to the presence of stilbene (cf. Fig. 1(b) emitter series n°1). As the nanoparticles have free alkynes on their surface (due to propargyl acrylate and its higher solubility in water when compared to styrene) the hydrogel can be infiltrated with an azide modified fluorophore(s), such as 2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (AzNap), and appropriate catalyst and reducing agent, such as copper(II) sulfate and sodium ascorbate, which yields a hydrogel with dye covalently attached to the surface of the nanoparticles through a copper(I) catalyzed azide/alkyne cycloaddition (CuCAAC) reaction. The AzNap fluorophore forms a Förster Resonance Energy Transfer (FRET) pair with stilbene, so the hydrogel as a whole emits green light upon x-ray irradiation (cf. Fig. 1(c) emitter series n°2). Furthermore, covalently attaching both AzNap and N-(9-(2-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)carbonyl)phenyl)-6-(diethylamino)9,9a-dihydro-3H-xanthen-3-ylidene-N-ethylethanaminium (AzRhod) through CuCAAC reactions shifts the x-ray radioluminescence to orange as AzRhod is a FRET pair with AzNap (cf. Fig. 1(d) emitter series n°3).

 

Fig. 1 Crystalline colloidal array (CCA) composed of electrostatically assembled polystyrene-co-poly(propargyl acrylate) with stilbene encapsulated inside the particle in (a) liquid form and (b) encapsulated in a methacrylate based hydrogel that emits blue light upon x-ray stimulation (emitter series n°1). (c) Methacrylate based hydrogel composed of polystyrene-co-poly(propargyl acrylate) with stilbene encapsulated inside the particle with 2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (AzNap) covalently attached to the particles through a copper(I) catalyzed azide/alkyne cycloaddition (CuAAC) performed in the hydrogel (emitter series n°2). Emitter series n°2 emits green light upon x-ray irradiation. (d) Methacrylate based hydrogel composed of polystyrene-co-poly(propargyl acrylate) with stilbene encapsulated inside the particle with AzNap and N-(9-(2-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)carbonyl)phenyl)-6-(diethylamino)9,9a-dihydro-3H-xanthen-3-ylidene-N-ethylethanaminium (AzRhod) covalently attached to the particles through copper(I) catalyzed azide/alkyne cycloaddition (CuAAC) reactions performed in the hydrogel; emitter series n°3 emits orange light upon x-ray stimulation.

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Stilbene in its polycrystalline form has two main peaks and two observable shoulders in its x-ray radioluminescence spectra: the main peak occurs at ca. 461 nm with the secondary peak occurring at ca. 406 nm and two shoulders appearing at ca. 484 nm and 502 nm (cf. Fig. 2(a)). However, the luminescence of the stilbene/PS-pPA system could be manipulated by moving the rejection wavelength by adding DI-water to the liquid array. Presented in Fig. 2(b) are optical photographs of the liquid stilbene/PS-pPA CCA under white light (left) and x-ray irradiation (right). The x-ray emission from stilbene gives the droplet a blue color with the CCA appearing white under white light. However, when approximately half of the CCA was diluted and photographed under white light and x-ray irradiation as presented in Fig. 2(c), the opalescent edge of the diluted CCA can clearly be seen under white light (left), while the CCA under x-ray stimulation (right) appears the same color blue to the left of the opalescent edge (i.e. where the additional deionized (DI) water did not infiltrate the original CCA), while to the right of the opalescent edge of the CCA (i.e. where the added DI-water changed the spacing between the nanoparticles) the resulting color is darker due to the decrease in luminescence because the rejection wavelength has been red shifted such that it inhibits a portion of the x-ray radioluminescence. As the rejection wavelength moves through the x-ray radioluminescence of the stilbene, there is a dramatic decrease in the emission (cf. Figs. 2(d.1) and 2(d.4)) at the corresponding pseudo-band gap, which can be observed spectroscopically [23–25]. At a rejection wavelength of 423 nm, the x-ray luminescence of stilbene exhibits a main peak occurring at 388 nm with a broad peak ranging from ca. 430 nm to 517 nm resulting from the decrease of the emission around the 423 nm rejection wavelength (cf. Fig. 2(d.1)). The dominant wavelength, calculated using the 1931 International Commission on Illumination (CIE) chromaticity diagram, was 487 nm, which corresponded to a light blue color. The primary colors (red, green, and blue) define the 1931 CIE chromaticity space accessible by the additive color approach. The dominant wavelength for any given color is defined as the wavelength corresponding to a line drawn through the color point (CIE (x,y) color coordinates), which connects both the color point and the white point to the edge of the chromaticity diagram. Upon further dilution to yield a rejection wavelength at 470 nm, the x-ray radioluminescence energy profile of the stilbene/PS-pPA CCA shifts to have a maximum emission peak at 395 nm with a broad peak centered at ca. 502 nm (cf. Fig. 2(d.2)); a similar x-ray radioluminescence spectrum was obtained with a rejection wavelength of 480 nm with a 10 nm shift where the decrease of the emission occurs (cf. Fig. 2(d.3)). However, once the stop band has moved through the main emission of stilbene, the majority of the x-ray emission profile of stilbene could be recovered though the emission is still suppressed in the pseudo-band gap at 530 nm resulting in a non-Gaussian peak with a peak maximum at 397 nm (cf. Fig. 2(d.4)) [26, 27]. The dominant wavelength of the x-ray radioluminescence spectrum with rejection wavelength at 530 nm, is 454 nm, which corresponds to a dark blue color. The varying shapes of the x-ray radioluminescence spectra exhibiting varying pseudo-stop bands and the corresponding dominant wavelengths confirm the observations from the optical photographs in Figs. 2(b) and 2(c).

 

Fig. 2 (a) X-ray radioluminescence spectrum of stilbene in its crystalline powder form. (b) Optical photographs of stilbene/PS-pPA liquid CCA illuminated under white light (left) and under x-ray irradiation (right). Under x-ray excitation, the droplet appears a bright blue color while the droplet appears white under white light illumination. (c) Optical photographs of stilbene/PS-pPA liquid CCA illuminated under white light (left) and under x-ray stimulation (right). The droplet has been diluted on the right side, and an opalescent line can clearly be seen in the white light image (left), while the diluted portion of the CCA results in a dark blue color, and the undiluted portion results in a bright blue color under x-ray irradiation (right). X-ray radioluminescence (blue) and reflection (red) spectra of stilbene/PS-pPA liquid CCA with rejection wavelength at (d.1) 423 nm, (d.2) 470 nm, (d.3) 480 nm, and (d.4) 530 nm. X-ray irradiation performed with an AmpTek Mini-X x-ray unit equipped with a tungsten target operating at 50 kV and 70 μA.

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While the pseudo-stop band, and thus the x-ray radioluminescence spectrum, could easily be manipulated in liquid form, the liquid CCA is not mechanically stable and the crystal is easily destroyed without the ability to recover. These sensitivities can be assuaged by polymerizing the liquid CCA into a hydrogel. The liquid stilbene/PS-pPA CCAs were mixed with poly(ethylene glycol) methacrylate (PEGMA, monomer), poly(ethylene glycol) dimethacrylate (PEGDMA, crosslinker), and 2,2-diethoxyacetophenone (DEAP, photo-initiator) and were photopolymerized using ultraviolet light [21]. This resulted in stable hydrogels, emitter series n°1, where the pseudo-stop band could be tuned through a reversible dehydration/hydration process. Optical photographs revealed a pale, translucent blue hydrogel under white light (cf. Fig. 3(a)), while the hydrogel appears bright blue under x-ray irradiation (cf. Fig. 3(b)). In the x-ray radioluminescence spectra of emitter series n°1, there is a large shoulder around 410 nm with a broad peak centered ca. 505 nm, which is reminiscent of the secondary peak in the liquid stilbene/PS-pPA CCAs. The expected peaks of stilbene are present in the radioluminescence spectra; however, it appears that the peak at 505 nm dominates and the 410 nm peak becomes the secondary peak. At a rejection wavelength of 583 nm, which is inside the emission of stilbene, the magnitude of x-ray radioluminescence of emitter series n°1 is at its minimum (cf. Fig. 3(c)). At an intermediate rejection wavelength of 510 nm, the magnitude of the x-ray luminescence of the hydrogel is greater than that associated with the 483 nm rejection wavelength but less than that associated with the pseudo-band gap at 530 nm (cf. Fig. 3(d)), while the maximum in x-ray radioluminescence for emitter series n°1 is observed at a rejection wavelength of 530 nm, which is outside the majority of the emission from stilbene (cf. Fig. 3(e)). The coupling of the rejection wavelength to the maximum x-ray induced emission of the stilbene nanoparticle results in a decrease in the overall x-ray radioluminescence but does not result in a divot in the spectrum like the liquid CCA because multiple crystals that are slightly offset from each other are being sampled in the hydrogel while a single crystal is being sampled in the liquid CCA. While the dominant wavelength varies from only 491 to 495 nm, the color purity ranges from 0.394 to 0.451 to 0.554 as the rejection wavelength passes through the x-ray radioluminescence of stilbene [28]. Color purity, under the 1931 CIE chromaticity space, is defined as the ratio of the distance between (1) the white point (1/3, 1/3) and the color point (i.e. CIE (x,y) color coordinates) and (2) the dominant wavelength and white point. Colors with high color purities (i.e. the value approaches 1) appear saturated and monochromatic, while colors with low color purities (i.e. the value approaches 0) appear pale.

 

Fig. 3 Optical photographs of stilbene/PS-pPA methacrylate-based hydrogels (emitter series n°1) under (a) white light and (b) x-ray irradiation. X-ray radioluminescence (blue) and reflection (red) spectra of emitter series n°1 corresponding to reflection wavelength at (c) 483 nm, (d) 510 nm, and (e) 530 nm. X-ray irradiation performed with an AmpTek Mini-X x-ray unit equipped with a tungsten target operating at 50 kV and 70 μA.

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While polymerizing the stilbene/PS-pPA methacrylate-based CCAs mechanically stabilizes the CCA, the luminescence of emitter series n°1 is poor. Many organic scintillators emit blue light poorly, but this potential downfall can be utilized in a FRET system where the chosen fluorophore(s) is a bright emitter, to not only increase the light output, but to also change the color emitted. To increase the overall luminescence and manipulate the emitted color, emitter series n°1 was utilized in CuCAAC reactions. Emitter series n°1 was swollen with AzNap, copper(II) sulfate, and sodium ascorbate solutions. The CuCAAC reaction occurred in the swollen hydrogel between the free alkyne functional groups on the surface of the PS-pPA nanoparticles and the azide group on AzNap [22]. After reaction completion, the catalysts and unreacted fluorophores were removed, and emitter series n°2 appeared a bright yellow color (cf. Fig. 4(a)); once emitter series n°2 was bombarded with x-ray radiation, the hydrogels emit green light (cf. Fig. 4(b)). Considering the maximum integral of the x-ray radioluminescence spectra, which is proportional to power, the luminescence of emitter series n°2 increased by 92% by adding the AzNap and exploiting FRET when compared to emitter series n°1. By employing CuCAAC reactions and FRET, the x-ray excited optical luminescence of a poor emitting hydrogel can be manipulated to become a much brighter system overall by coupling a poor emitting x-ray excited probe to an x-ray inactive fluorophore.

 

Fig. 4 Optical photographs of stilbene/PS-pPA methacrylate-based hydrogels functionalized with AzNap (emitter series n°2) through a CuAAC reaction in the hydrogel under (a) white light and (b) x-ray irradiation. X-ray radioluminescence (blue) and reflection (red) spectra of emitter series n°2 at rejection wavelength corresponding to (c) 478 nm and (d) 541 nm. X-ray irradiation performed with an AmpTek Mini-X x-ray unit equipped with a tungsten target operating at 50 kV and 70 μA.

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Additionally, the maximum emission peak of emitter series n°2 has shifted to 534 nm and 540 nm when the pseudo-stop band is at 478 nm and 541 nm, respectively (cf. Figs. 4(c) and 4(d)). As the photoluminescence of AzNap occurs at 520 nm (monomeric peak) and at ca. 535 nm (J-aggregate peak), the maximum emission peak for emitter series n°2 is attributed to the AzNap [29–31]. FRET, a dipole-dipole energy transfer mechanism, occurs between the stilbene and AzNap since the emission of stilbene overlaps well with the absorbance of AzNap and the two molecules are in close proximity. The Förster distance, i.e. the distance at which FRET is 50% efficient, is ca. 2.30 nm. When the rejection wavelength overlaps with the maximum emission of the AzNap (i.e. 530 nm), the x-ray luminescence decreased by 22% as calculated by comparing the integral of the luminescence curve at each rejection wavelength. [32] It should be noted that AzNap shows far less emission when excited through x-rays than stilbene, so all x-ray luminescence is attributed to FRET between stilbene and AzNap. Clearly, the rejection wavelength plays a crucial role in the amount of light allowed to escape the hydrogel. To obviate the possibility of inter-particle FRET, the inter-planar spacing (d_hkl) and nearest neighbor spacing (a) was calculated using Bragg diffraction theory: λ=2d_hkln_csinθ where λ is rejection wavelength, θ is diffraction angle, which is equal to 90° since the particles arrange in a face centered cubic (FCC) crystal lattice and rejection wavelength was sampled normal to the CCA, the plane of interest is (111), and n_c, the refractive index of the composite, is 1.363 [21]. Given those parameters, the d₁₁₁ spacing ranges from 174.7 nm to 193.7 nm, while the nearest neighbor spacing ($\text{a}=\sqrt{3/2}{\text{d}}_{hkl}$) ranges from 214.0 nm to 237.2 nm, which is clearly more than the Förster distance (2.30 nm).

To further manipulate the color emitted, emitter series n°1 was modified with both AzNap and AzRhod through CuCAAC reactions in the hydrogel (emitter series n°3). Due to the broadness of the x-ray radioluminescence spectrum of stilbene, AzRhod does form a FRET pair with stilbene, but the Förster distance is small, 2.07 nm, which indicates that AzRhod and stilbene do not form as strong of a FRET pair as AzRhod and AzNap as the Förster distance between these fluorophores is 3.62 nm, which indicates that most of the FRET arises from the AzNap/AzRhod transition. Similarly to AzNap, AzRhod also shows almost no x-ray induced luminescence. In this way, all emission is due to the x-ray stimulation of stilbene which transfers a small amount of energy directly to AzRhod with most of its energy being transferred to AzNap, which, in turn, transfers its energy to AzRhod (cf. Fig. 1(d), emitter series n°3). While emitter series n°3 is red under white light (cf. Fig. 5(a)), the hydrogel emits orange light upon x-ray irradiation (cf. Fig. 5(b)). As observed with both emitter series n°1 and n°2, as the rejection wavelength approaches the emission of the main emitter, the x-ray radioluminescence is significantly decreased. At a pseudo-stop band of 490 nm, the total emission is 74% greater than when the rejection wavelength is at 532 nm [32]. At a rejection wavelength of 490 nm only a small portion of the x-ray emission of stilbene is suppressed; therefore most of the energy of stilbene is still transferred to the AzNap and the AzRhod (cf. Fig. 5(c)), yielding a maximum peak of 605 nm, which is indicative of the aggregate peak of AzRhod, while the monomeric peak occurs at 585 nm [33–35]. While the 532 nm rejection wavelength is mostly outside of the emission of AzRhod, the pseudo-band gap covers the majority of the emission of AzNap. By suppressing the emission of AzNap, the energy transferred to the AzNap by the stilbene is quenched due to the smaller local density of optical states; thus, the emission from AzRhod is not fully activated (cf. Fig. 5(d)), resulting in an overall decrease in emitted light [36] though the exact mechanism of multiple FRETs in a photonic crystal remains a highly debated question [37–41]. Emitter series n°3 emits orange with dominant wavelengths at 606 and 601 nm for rejection wavelengths of 532 nm and 490 nm, respectively with a high color purity of 0.97 or greater as calculated using the 1931 CIE color space. As observed with emitter series n°2, the possibility of inter-particle FRET does not exist for emitter series n°3 as the d₁₁₁ spacing ranges from 179.1 nm to 194.4 nm, and the nearest neighbor spacing ranges from 219.3 nm to 238.1 nm. Nonetheless, when comparing the integrals of the x-ray radioluminescence spectra at their maximum, emitter series n°3 is 90% more luminescent than emitter series n°1, which demonstrates that the x-ray radioluminescence can be greatly increased from emitter series n°1 even with multiple FRETs. By utilizing stilbene as the x-ray active “pump” source and coupling it to a variety of fluorophore combinations, CCA hydrogels could be made to emit blue light (emitter series n°1), green light (emitter series n°2), and orange light (emitter series n°3).

 

Fig. 5 Optical photographs of stilbene/PS-pPA methacrylate-based hydrogels functionalized with AzNap and AzRhod (emitter series n°3) through CuAAC reactions in the hydrogel under (a) white light and (b) x-ray irradiation. X-ray radioluminescence (blue) and reflection (red) spectra of emitter series n°3 corresponding to rejection wavelength at (c) 490 nm and (d) 532 nm. X-ray irradiation performed with an AmpTek Mini-X x-ray unit equipped with a tungsten target operating at 50 kV and 70 μA.

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3. Conclusion

X-ray imaging is becoming increasingly used as a diagnostic and treatment tool for a variety of diseases as x-ray radiation more easily penetrates tissues when compared to visible or ultraviolet light. The current effort presents fully organic, emission tunable hydrogels composed of water dispersed electrostatically self-assembled polystyrene-co-poly(propargyl acrylate) nanoparticles in which stilbene, an organic scintillator, has been encapsulated. Due to the alkyne groups on the surface of the nanoparticle, the methacrylate based hydrogels can be swollen with a single or multiple azide modified fluorophores and appropriate CuCAAC catalyst and reducing agent to covalently attach the fluorophore(s) to the nanoparticle while already assembled into a hydrogel. Stilbene serves to absorb x-ray radiation, which it converts to visible blue light. However, the amount of light and the color of light can be manipulated through fluorophore(s) that FRET with one another. In this way, the emission of the hydrogel can be heavily tuned and used in x-ray based imaging technologies without the need for traditional toxic, heavy metal based imaging agents.

4. Experimental

4.1. Reagents and solvents

All reagents were purchased from commercial suppliers, such as Aldrich or Alfa Aesar. All solvents used for reactions were distilled under nitrogen after drying over an appropriate drying reagent. All other commercial reagents were used without further purification unless otherwise stated. Deionized water was obtained from a Nanopure System and exhibited a resistivity of ca. 10¹⁸ Ohm⁻¹cm⁻¹. Analytical thin-layer chromatography was performed on glass plates coated with 0.25-mm 230–400 mesh silica gel containing a fluorescent indicator. Column chromatography was performed using silica gel (spherical neutral, particle size 63–210 μm). All monomers used in the synthesis of hydrogels were mixed with mixed bed ion exchange resin (IER, BioRad Laboratories, AG 501-X8 Resin, 20–50 mesh) for at least 24 hours prior to use.

4.2. Preparation of glass cells

Glass slides (Corning, 3 in x 1 in x 0.039 in) were cut in half, washed with soap and DI-water, and dried under nitrogen. The dried, cut glass was cleaned using freshly made Piranha solution (concentrated sulfuric acid:hydrogen peroxide, 3:1 by volume) for 24 hours. Then the glass slides were washed with DI-water and dried under nitrogen and set aside. A solution of octadecyltrichlorosilane (OTS) in toluene (30% by volume) was prepared in the nitrogen glove box. The solution was taken from the glove box and the glass slides were coated with it to change the surface of the glass from hydrophilic to hydrophobic. The coated slides were allowed to completely dry. The white film that coated the slides was removed by friction. All glass slides were exposed to nitrogen prior to use. Spacers were made by cutting a 2 × 1 cm section, using a die, from 2 adhered sheets of Parafilm for a total film thickness of 250 μm. Spacers were stored in DI-water with excess IER until needed. Glass cells were assembled by pressing the spacer onto one glass slide. The CCA (60 μL) mixed with all monomers and initiator was placed in the center of the glass slide. Another glass slide was slowly placed on top of the glass slide with spacer.

4.3. Chemical characterization methods

¹H NMR spectra were recorded on a JEOL ECX-300 spectrometer. Chemical shifts for protons are reported in parts per million downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent (CDCl₃: δ 7.26 ppm).

4.4. Syntheses

Synthesis of 6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1)

6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione was synthesized according to a previously reported method [30].

Synthesis of 1-azido-3-iodopropane (2)

1-azido-3-iodopropane was synthesized according to an established method [42].

2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (AzNap)

6-(Piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1) (0.3 g, 1.07 mmol), potassium carbonate (0.3 g, 2.17 mmol), and 1-azido-3-iodopropane (2) (0.249 g, 1.18 mmol) were mixed with dry DMF (5 ml). The obtained mixture was stirred at 80°C for 4 hours. After cooling, the mixture was quenched with deionized water and was left overnight for crystallization. The solid was decanted and extracted with dichloromethane. The solution was dried with Na₂SO₄ and filtered. The filtrate was evaporated under reduced pressure, and the residue was re-crystallized from hexane (cf. Fig. 6). Yield 0.25 g, 64%, m.p = 87 °C. ¹H (CDCl₃): δ 1.73 (m, 2H, J = 5.5 Hz), 1.88 (m, 4H, J = 5.5 Hz), 2.04 (m, 2H, J = 6.9 Hz), 3.23 (t, 4H, J = 5.5 Hz), 3.42 (t, 2H, J = 6.9 Hz), 4.27 (t, 2H, J = 6.9 Hz), 7.17 (d, 1H, J = 8.3 Hz), 7.67 (m, 1H, J = 7.2 Hz), 8.39 (m, 1H, J = 7.2 Hz, J = 1.4 Hz), 8.49 (d, 1H, J = 8.3 Hz), 8.56 (m, 1H, J = 7.2 Hz, J = 1.4 Hz).

 

Fig. 6 Synthetic scheme to yield 2-(3-azidopropyl)-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (AzNap).

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Synthesis of N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)-9,9a-dihydro-3H-xanthen-3-ylidene)-N-ethylethanaminium (3)

N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)-9,9a-dihydro-3H-xanthen-3-ylidene)-N-ethylethanaminium was synthesized according to a previously reported method [43].

Synthesis of 2-[2-(2-Azidoethoxy)ethoxy]ethanol) (4)

2-[2-(2-Azidoethoxy)ethoxy]ethanol) was synthesized by the method described in literature [44].

Synthesis of N-(9-(2-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)carbonyl) phenyl)-6-(diethyl amino)9,9a-dihydro-3H-xanthen-3-ylidene-N-ethylethanaminium (AzRhod)

Compound (3) (0.41g, 0.82 mmol) was dissolved in dry dichloromethane (10ml). Then compound 4 (0.173g, 0.99 mmol) was added into solution. The obtained solution was stirred and cooled to 0 °C. Triethylamine (0.166g, 1.64 mmol) was added dropwise into solution. The mixture was stirred at room temperature for 24 hours. After 24 hours, the reaction was quenched with water. The organic layer was separated, dried with Na₂SO₄, filtered, and evaporated under reduced pressure. The residue was dissolved in dichloromethane (0.5ml), and the crude product was precipitated by the addition of diethyl ether (10ml). The mixture was then separated by centrifugation. The product obtained was a deep red oil and was purified by column chromatography on silica with an eluent of dichloromethane:acetone (5:1) to wash out impurities followed by an eluent of dichloromethane:methanol (10:1) to recover the desired product (cf. Fig. 7). R_f =0.2. Yield 0.34g (69%), dark-red oil. ¹H NMR (CDCl₃) δ 1.32 (t, 12H, J=7.2 Hz), 3.35 (m, 2H, J=4.8 Hz), 3.55–3.70 (m, 16H), 4.17 (m, 2H, J=4.5 Hz), 6.82 (d, 2H, J=2.4 Hz), 6.90 (d.d, 2H, J=9.3 Hz, J=2.4 Hz), 7.06 (d, 2H, J=9.3 Hz), 7.28 (m, 1H, J=7.6 Hz), 7.70–7.84 (m, 2H, J=7.6 Hz, J=1.4 Hz), 8.33 (m, 1H, J=7.6 Hz, J=1.4 Hz). ¹³C NMR (CDCl₃) δ 12.74, 46.22, 50.75, 64.77, 68.86, 70.16, 70.57, 70.65, 96.41, 113.64, 114.27, 129.76, 130.28, 130.49, 131.43, 131.65, 133.29, 133.78, 155.62, 157.87, 165.03. ESI+ Mass (m/z): calc. for C₃₄H₄₂N₅O₅, 600.3; found, 600.5.

 

Fig. 7 Synthetic scheme to yield N-(9-(2-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)carbonyl)phenyl)-6-(diethylamino)9,9a-dihydro-3H-xanthen-3-ylidene-N-ethylethanaminium (AzRhod).

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Synthesis of stilbene/polystyrene-co-poly(propargyl) acrylate nanoparticles

Polystyrene-co-poly(propargyl acrylate) with stilbene doped inside the nanoparticles were synthesized using a standard free radical emulsion polymerization [45]. Styrene monomer was purified from the inhibitor by extracting with an aqueous KOH solution (10% by volume, 3×100 mL) followed by washing with deionized water (DI-water, 3×100 mL). The styrene was dried over Na₂SO₄ and distilled under vacuum. Stilbene (1.5 g, 8.32 mmol) was dissolved in purified styrene (30 mL, 261.83 mmol) with heat and sonication. DI-water (120 mL) was added to a 4-neck round bottom flask equipped with a mechanical stirrer and thermocouple. A nitrogen purge was started with the purge gas below the solution level. Purified styrene (10 mL, 87.28 mmol) was injected into the reaction flask while stirring the DI-water at 370 rpm. Propargyl acrylate (0.91 mL, 8.24 mmol), purified by passing through basic alumina, was injected into the flask. After 5 minutes, sodium lauryl sulfate (0.35 mL, 29% by mass in DI-water) was injected dropwise into the reaction vessel and was allowed to mix for 5 minutes. Then divinylbenzene (0.25 mL, 1.76 mmol) was added to the flask dropwise. The reaction vessel was heated to 65°C. Once the vessel reached temperature, a solution of sodium hydrogen phosphate (0.35 g, 2.47 mmol, dissolved in 2 mL DI-water) and a solution of potassium persulfate (0.35 g, 1.29 mmol, dissolved in 8 mL of DI-water) were added dropwise to the reaction flask. The purge gas was moved above the solution level to keep a blanket of nitrogen over the reaction. The temperature of the reaction was increased to 70°C. Purified propargyl acrylate (2.75 mL, 24.9 mmol) was added to the stilbene/polystyrene solution. Once the reaction reached temperature, the stilbene/propargyl acrylate/styrene solution (16.375 mL) was added to the reaction vessel dropwise. Next, divinylbenzene (0.6 mL, 4.212 mmol) was injected into the vessel dropwise. The remaining stilbene/propargyl acrylate/styrene solution (16.375 mL) was added to the 4-neck flask. The reaction was allowed to proceed for 2.5 hours. After 2.5 hours, the reaction was allowed to cool to ca. 37°C, and the product was gravity filtered. The filtrate was placed into dialysis bags (50,000 MWCO) and was dialyzed against DI-water until the conductivity of the bath remained constant. The dialyzed product was stored in Nalgene containers with IER. After two days of storage with IER, the zeta potential, as measured with a ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp.), of the colloid was −74.22 mV. The particle size was 131.1 nm ± 9.90 nm as measured by dynamic light scattering (DLS, Coulter N4Plus dynamic light scatter). After dialysis, the particles were subjected to a Soxhlet extraction in order to estimate how much, if any, stilbene escaped the particles. In boiling benzene, no stilbene was detected in the washing solution up to 24 hours with absorbance spectroscopy. It should be noted that benzene is a good solvent for stilbene and will swell the polystyrene particle; therefore, at 24 hours, the stilbene detected in the the benzene washing solution is likely from stilbene inside the particle diffusing out of the swollen particle.

Synthesis of emitter series n°1

To increase the solids concentration of stilbene/PS-pPA colloids, the colloids (1.5 mL) were centrifuged at 21,100 g for 20 min three times. After each centrifuge cycle, a small layer of the supernatant (ca. 200 μL) that was no longer luminescent was removed. The colloids (150μL) were mixed with poly(ethylene glycol) methacrylate (MW= 360 g/mol, 60 μL), poly(ethylene glycol) dimethacrylate (MW= 550 g/mol, 6 μL), and IER; the mix was placed on a shaker table at ca. 116 motions/min for approximately 24 hours. After 24 hours, the photo-initiator, 2,2-diethoxyacetophenone (4 μL), was added to the mix, and the mix was shaken for an additional 15 min. Glass cells were made as described above. After assembly, the glass cell was photopolymerized using an UV oven (ELC-500 Light Exposure System, Electro-Lite Corporation) for 1 min on each side of the cell, alternating sides, for a total of 6 minutes. After photopolymerization, the glass cell was submerged in DI-water, and the top glass slide was removed along with the spacer. The polymerized hydrogel (emitter series n°1) was removed and stored in DI-water. The total number of particles in the polymerized hydrogel is approximately 5.4×10¹³.

Synthesis of emitter series n°2

Emitter series n°2 was synthesized using CuAAC. Emitter series n°1 was swollen with solutions of AzNap (12 mM), sodium ascorbate (58 mM), and copper(II) sulfate (23.4 mM) in DI-water:tetrahydrofuran (2:1). The swollen gel in excess solution was shaken at 49 motions/min using a shaker table for 2 hours. After 2 hours, the hydrogel was removed and placed in a fresh solution of DI-water with 4% tetrahydrofuran (THF). The solution was changed until no AzNap was in the supernatant as detected by UV/vis. At this point, the hydrogel was considered cleaned of any non-covalently attached fluorophore.

Synthesis of emitter series n°3

Emitter series n°3 was synthesized using CuAAC. Emitter series n°1 was swollen with solutions of AzNap (4.67 mM), AzRhod (2.83 mM), sodium ascorbate (36 mM), and copper(II) sulfate (14 mM) in DI-water:tetrahydrofuran (2:1). The swollen gel in excess solution was shaken at 49 motions/min using a shaker table for 2 hours. After 2 hours, the hydrogel was removed and placed in a fresh solution of DI-water with 4% tetrahydrofuran (THF). The solution was changed until no AzNap and AzRhod was in the supernatant as detected by UV/vis. At this point, the hydrogel was considered cleaned of any non-covalently attached fluorophore.

4.5. Optical and x-ray radioluminescence characterization methods

Absorbance spectra were obtained using a Perkin Elmer Lambda 950 spectrophotometer. X-ray radioluminescence spectra were collected by irradiating the sample with a mini X-ray tube (Amptek Inc., MA, USA), equipped with a tungsten target operating at a tube voltage of 50 kV and a tube current of 79 μA. The radioluminescence was collected with a fiber bundle (Oriel) coupled to a MicroHR (Horiba Jobin Yvon) monochromator and a cooled CCD detector (Synapse, Horiba Jobin Yvon). The signal was collected on a grating with 600 line mm⁻¹ and a blaze of 500 nm. The spectra was analyzed with SynerJY (Horiba Jobin Yvon) software. The exposure time varied from 20–60 sec based on the luminescence of the liquid CCA or hydrogel. None of the spectra were corrected for the emission of the donor.