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Radiation Responsive Esculin-derived Molecular Gels as Signal Enhancers for Optical Imaging Julian R. Silverman, Qize Zhang, Nabendu B Pramanik, Malick Samateh, Travis M Shaffer, Sai Sateesh Sagiri, Jan Grimm, and George John ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15548 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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ACS Applied Materials & Interfaces

Radiation Responsive Esculin-derived Molecular Gels as Signal Enhancers for Optical Imaging 1,2

Julian R. Silverman , Qize Zhang2,4,7‡, Nabendu B. Pramanik§1‡, Malick Samateh1,2, Travis M. Shaffer2,3,4,7, Sai Sateesh Sagiri1, Jan Grimm3,4,5,6* and George John1,2* 1

Department of Chemistry and Biochemistry, the City College of New York, 160 Convent Avenue, New York, NY 10031 2

Doctoral Program in Chemistry, The City University of New York Graduate Center, 365 5th

Avenue, New York, NY 10016 3

Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 4

Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New

York, NY 10065 5

Pharmacology Program, Weill Cornell Medical College, 1300 York Avenue, New York, NY

10065 6

Department of Radiology, Weill Cornell Medical College, 1300 York Avenue, New York, NY

10065 7

Department of Chemistry, Hunter College, 695 Park Avenue, New York, NY 10065

*Corresponding Authors: [email protected], Tel: 212-650-8353, Fax: 212-650-6107 [email protected], Tel: 646-888-3095, Fax: 646-888-3095 ‡

Authors contributed equally

ACS Paragon Plus Environment

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ABSTRACT Recent interest in detecting visible photons that emanate from interactions of ionizing radiation with matter have spurred the development of multifunctional materials that amplify this optical signal from radiotracers. Tailored stimuli-responsive systems may be paired with diagnostic radionuclides to improve surgical guidance and aid in detecting therapeutic radionuclides otherwise difficult to image with conventional nuclear medicine approaches. Since light emanating from these interactions is typically low in intensity and blue-weighted (i.e. greatly scattered and absorbed in vivo), it is imperative to increase or shift the photon flux for improved detection. To address this challenge, a gel that is both scintillating and fluorescent is used to enhance the optical photon output in image mapping for cancer imaging. Tailoring biobased materials to synthesize thixotropic thermoreversible hydrogels (MGC 0.12 wt.%) offers imageaiding systems which are not merely functional, but also potentially economical, safe and environmentally friendly. These robust gels (0.66 wt.%, ~900 Pa) respond predictably to different types of ionizing radiation including β- and γ-emitters, resulting in up to a doubling of the detectable photon flux from these emitters. The synthesis and formulation of such a gel is explored with a focus on its physicochemical and mechanical properties, before being utilized to enhance the visible photon flux from a panel of radionuclides as detected. The possibility of developing a topical cream of this gel makes this system an attractive potential alternative to current techniques, and the multifunctionality of the gelator may serve to inspire future nextgeneration materials.

KEYWORDS: Esculin; biobased; molecular gels; self-assembly; cancer imaging; Cerenkov imaging; scintillation


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1. INTRODUCTION Ionizing radiation (IR) has been used for non-invasive imaging since the discovery of X-rays over a century ago. As IR by its nature results in the ionization of surrounding matter, it is also used for therapies including beam, immuno-, and brachytherapy. IR emanates from radionuclides in routine clinical use and includes positrons for positron emission tomography (PET) or highenergy photon emitters for single photon emission computed tomography (SPECT).1 While these methods have repeatedly demonstrated their value in the clinic, the resolution and imaging times of these modalities are not ideal, and certain therapeutic radionuclides, such as yttrium-90 (90Y) and actinium-225 (225Ac), cannot easily be imaged; therefore, alternative imaging techniques have been developed to address these limitations.2,3 Rather than detecting IR itself, it is possible to image visible photons produced from the interactions between IR and matter. One mechanism, scintillation, produces visible light that occurs via excitation of the medium by high energy electrons, positrons, or gammas followed by radiative relaxation. Another mechanism, Cerenkov luminescence, occurs when a charged particle (such as an electron or positron) travels faster than the speed of light in a certain medium.4,5 Cerenkov luminescence imaging (CLI) typically uses charged-coupled devices (CCDs) to detect the photons. However, the well-known limitations of this imaging modality are that the light is blue-weighted (a region where CCDs typically have low quantum efficiency and tissue absorbs and scatters photons) and low photon flux (approximately a billion time less than ambient light). To address these challenges, fluorescent nanoparticles or dyes can be combined with CL emitters, resulting in a technique known as secondary Cerenkov induced fluorescence imaging, or SCIFI.6 However, this technique often utilizes quantum dots composed of rare earth elements, and the possible toxicity precludes their widespread use in the clinical application.7


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Another method is combining a scintillating material with ionizing radiation, which results in blue-weighted visible photon emission and is referred to as radioluminescence imaging.8 To address several limitations of current CL systems, we investigated biocompatible agents that follow the principles of green chemistry. Esculin, a naturally occurring coumarin 6O-β-D-glucoside, is a highly versatile molecule used in bacterial identification, for its antiapoptotic effect, and as an additive in liquid scintillation cocktails due to its β-glucosidase specificity, antioxidant activity and scintillating fluorescence respectively.9-14 Esculin’s chemical versatility and functional structure make it a choice candidate for the development of utile sugar fatty acid esters, a class of molecules known for their ability to self-assemble in solution to afford molecular gels.15,16 Molecular gelators represent a class of low molecular weight chemicals capable of 17,18

structuring liquids for a variety of food, fuel, personal care and medical applications. Capitalizing on the variety of distinctive non-covalent forces, these systems self-assemble in various media to afford higher order structures, which can grow to encompass the volume of a liquid to structure and immobilize the solution into a viscoelastic gelatinous phase.19,20 Molecular gels are easily transitioned between the solid-like gel and liquid-like sol states, which may be leveraged to create versatile medical and materials solutions.18 The dispersed selfassembled fibrillar network, which results from the small-scale ordering and packing of the gelator may allow for an increased signal response compared to bulk or condensed systems.18 The prime advantage of using an esculin-based system for optical image enhancement is the emission spectrum in high quantum efficiency region of most CCDs and biocompatibility.21 Previous work has focused on using organics22, solid scintillators23 or fluorescent nanoparticles24 for increasing the optical output detected by the CCD. However, these systems are typically


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comprised of a ‘cocktail’, often a mixture of an aromatic solvent, surfactant, and scintillant placed into a glass container and kept adjacent to a photomultiplier tube capable of capturing the visible light and counting the response.25 These systems typically work through two mechanisms. The first is scintillation, where the agent is excited by ionizing radiation (such as a high-energy photon). The second is excitation via the blue-weighted Cerenkov light, and re-emit photons in areas where a CCD has higher quantum efficiency excite the agent. Here we aim to utilize an aqueous-based gelator that has both fluorescent and scintillating properties, to enhance visible photon output while avoiding organic solvents. Molecular gels, which may consist of unidirectional hierarchically assembled amphiphilic molecules, are prime candidates for such unique applications; some systems even demonstrate an aggregation enhanced fluorescence emission due the higher-order fiber-like structures.26 Herein, we describe the formulation and first reported characterization of a naturally derived small molecule hydrogel capable of increasing the detected photon flux emanating from medical radionuclides as detected by an IVIS Spectrum small animal optical imaging system. Following a sustainable synthetic procedure and formulation affords a multifunctional material capable of responding smartly to different forms of radiation and shear, making this system highly customizable for application. Imaging studies are performed to best understand potential clinical uses for these systems. This work may help inspire the development of practical biocompatible solutions from de novo design to meet the unique challenges found with medical technologies.

2. EXPERIMENTAL SECTION 2.1. Materials and Methods


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Materials: Esculin hydrate and vinyl caprylate were purchased from Acros Organics (New York, New York). Lipase acrylic resin from Candida Antarctica (≥ 5,000 U/g), recombinant, expressed in Aspergillus Niger was provided by Novozymes North America as Novozyme 435 (Franklinton, North Carolina). Silica Gel (200-300 MESH), hexanes, ethyl acetate, methanol and acetone were purchased from Fisher Scientific (IL). Acetone used for reactions was distilled over calcium chloride before use.

2.2. Synthesis of Esculin Caprylate (E8) In a 500 mL screw-cap Erlenmeyer flask solid Novozyme 435 lipase catalyst (0.3 g/mmol glucoside) was added to mixture of esculin hydrate (2.0 mmol, 0.680 g), caprylic acid vinyl ester, or caprylic acid methyl ester (3:1 mmol acyl donor/glucoside ratio) in 50 mL dried acetone. The reaction proceeded in an orbital shaker at 250 rpm heated to 50 ºC. The reaction was monitored by thin layer chromatography (TLC) with 95% ethyl acetate 5% methanol eluent and visualized using 5% sulfuric acid and gentle heating. After 24 hours the bottom glucoside spot (Rf = 0) faded and a product spot (Rf = 0.4) appeared indicating the completion of the reaction. Before the solution is allowed to cool to room temperature the enzymes are filtered out and rinsed with acetone until the washings show no spots on TLC before they are air-dried and stored for recycling. Acetone is evaporated leaving behind a crude solid mixture of glucoside/ester product (84% yield from vinyl ester, 24% yield from methyl ester). The solid mixture is triturated thrice with 50 mL hexanes at 50 ºC to remove the excess fatty acid or derivatives from the opaque light green bulk solid. To remove trace elements of unreacted sugar, ester or acids the product (0.85 g) is dissolved in 25 mL methanol, and coated by evaporation onto 5.0 grams of silica gel or Celite before being spread onto a short silica plug (40.0 g). The column is eluted with 200 mL 1:1 ethyl


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acetate-hexanes once, and twice with 200 mL 95% ethyl acetate, 5% methanol and the solvent evaporated from the second fraction to afford the pure fatty acid glucoside ester product. Pure glucoside-ester was dissolved in deuterated solvent (20 mg in 1 mL deuterated dimethyl sulfoxide, o r d e u t e r a t e d c h l o r o f o r m ) and the solution is filtered through glass wool before recording a spectrum on a 300 MHz Bruker 1H NMR Spectrometer.

2.3. Preparation and Characterization of Molecular Gels Gel samples were prepared by adding the solid glassy glucoside solid (8.25, 16.5 and 33 mg) to deionized water (5.0 mL). The mixture is sealed and heated to disperse the gelator. The sol was then cooled to room temperature to allow for self-assembly and after a length of time (between 5 to 15 minutes) the samples are inverted to confirm the presence of the gel state. The efficacy of gel formation was examined by determining the minimum gelation concentration (MGC) and gel-to-sol transition temperature of gelation (Tgel). Gel samples were diluted with solvent until after setting and vial inversion a gel, partial gel, or sol was formed. The gel transition temperature was determined by submersing a gel sample in an oil bath, and increasing the temperature until the gel flowed like a liquid sample, indicating the disassembly of the gelator structure. Minimum gelation concentration was determined beginning from a 1.0 wt. % sample or below the concentration of precipitation for shorter chain samples, and diluting the samples until a sol is formed.

2.4. Characterization FT-IR Spectroscopy: Gel and Gelator Characterization


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The infrared spectra (FT-IR) of the bulk gelators and gel samples were measured using a Thermo Scientific Nicolet iS 10 FT-IR Spectrometer with an attenuated total reflectance configuration sampling between 4000-600 cm-1.

Rheological Characterization of Gels Dynamic rheology measurements were performed on a stress-controlled rheometer (AR 2000 ex) with a cone and plate geometry (1° cone angle and 40 mm diameter with a truncation gap of 45 µm). About 1 mL of gel was loaded onto the plate, and the cone was lowered to minimize the truncation gap. Precautions were taken to minimize shear-induced disruption of the gel network: before experiments samples were equilibrated within the geometry for 10 minutes. Excess gel was trimmed away from the cone to ensure optimal filling. Linear viscoelastic region (LVR) and critical strain were examined for hydrogels (0.16, 0.33 and 0.66% by weight) by performing oscillatory strain sweep measurements from 0.01 to 100% deformation at a fixed frequency of 1 Hz. Oscillatory frequency sweep measurements were performed at different temperatures in the frequency domain of 0.01 to 10 Hz, with a constant strain of 0.1%, which is within the linear viscoelastic regime of the hydrogels. Temperature for the frequency step were maintained from 5 °C to 45 °C at an interval of 10 °C for each frequency sweep. Thixotropic studies were performed by oscillatory strain sweep in series with 60 seconds intervals between sweeps. Viscometric studies were also conducted by strain ramp in a cyclic fashion to investigate thixotropic properties.

Gelator Interactions with Ionizing Radiation Emanating from Radionuclides 10 µCi (0.37 MBq) of the radionuclide of interest was added to 100 µL of gel samples in a 96 well black-wall plate, with Millipore water added for a final volume of 200 µL. H2O with


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radioactivity added was used as a control. Images were taken over 120 seconds and regions of interest (ROIs) were drawn over each well to determine the radiance, with the background subtracted from each ROI to account for gamma interactions with the CCD. All trials were conducted in triplicate (Figure S8).

3. RESULTS AND DISCUSSIONS The synthesis of the coumarin glucoside molecular gelator (E8) was achieved by coupling esculin with an acyl donor,27 either methyl or vinyl caprylate by a heterogeneous lipase catalyzed esterification reaction (Figure 1 and Figure S1).20 While methyl esters represent a sustainable source of acyl donor as they may be derived from plant oils, byproducts and waste materials.28

Esculin Caprylate (E8)

Figure 1. Esculin Caprylate (E8) Biocatalytic Synthesis Gelator characterization was performed in an effort to probe the noncovalent hydrogen bonding and van der Waals forces, which serve to assemble the gelator in solution (Figure S1-3).15 In probing the ultraviolet-visible absorbance and fluorescence emission it is evident that by acylating esculin’s primary hydroxyl group, there is no significant change in the coumarin’s absorbance and emission. (Figure S4). The retention of electronic character is important in the development of a functional scintillator, as modifying the electronic nature of the coumarin fluorophore might diminish its sensing capability.11,13


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Esculin caprylate (E8) gels were prepared by dispersion of the gelator in water by heating and mixing followed by cooling at ambient conditions (Figure S5). E8 gelator (0.01-2 mg) in water (0.1-1 mL) was heated until the solid was completely dissolved. The resulting solution was allowed to cool to room temperature and gelation was observed after the solution transitions from clear to translucent and back to clear. Gel formation was confirmed when the solution exhibited no gravitational flow upon inversion of the vial.18 Gels demonstrated thermoreversibility in a sealed vial; the gels dissolved by heating the mixture, but could be returned to the gel state upon cooling. E8 demonstrated gelation in water at concentrations as low as 0.12 wt. %, by the vial inversion method and can be classified a supergelator with an MGC less than 1.0 %.18 In examining the temperature of gelation in open vials (Tgel) the boiling point of water was reached before gels transitioned from gel to sol in both open and closed systems. The gelation of the system in water is caused by the self-assembly of E8 molecules into an entangled fibrous network structure (evident from SEM images, Figure S6).

3.1. Rheological Studies of the E8 Gels As the fiber’s microstructure affects the viscoelastic properties of the molecular gels different rheological studies were performed on the stable hydrogels.19,29 In the first series of experiments, strain sweep (from 0.01 % to 100 %) at a low deformation frequency (1 Hz) was conducted on three hydrogels containing E8 concentrations at 0.16, 0.33 and 0.66 wt.%. Figure 2A shows the dynamic moduli (G´, storage/elastic modulus and G´´, loss/viscous modulus) as a function of percent strain. The dynamic moduli of all the three hydrogels are independent of strain up to a critical strain. Beyond this critical strain, the viscoelastic behavior of the hydrogels is not linear as the dynamic moduli decline and approaching each other. The linear viscoelastic region (LVR)


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of the hydrogels is dependent on the concentration gelator and solvent. The dynamic moduli of the hydrogel with 0.16 wt.% E8 deviate from linearity at critical strain of ~ 1.0 %, while hydrogels with 0.33 wt.% and 0.66 wt.% E8 concentration have shown the critical strain at ~ 3.0 % and ~ 10.0 %, respectively. In general, the presence of a LVR implies the existence of entangled gelator network in liquid sol and the onset critical strain or non-linear viscoelastic regime refers to the occurrence of structural changes in the gels. The result of finding different critical strains suggests that the disruption of gelator network occurs at different strain amplitudes, depending on the gelator concentration in the hydrogels. The increase in critical strain with the increase in gelator concentration suggests that the entanglements of the fibrous network are increased with the gelator concentration. In addition to the network stability, the increase in gelator concentration has also enhanced the stiffness of the hydrogels progressively; which is evident from the increase in G´ value of the LVR of the hydrogels (Figure 2A). For all the samples, G´ is greater than G´´ at low strain, indicating the solid-like nature of the hydrogels, but at higher amplitudes, definite crossover of G´ and G´´ is seen; suggesting the yielding of gelator network (yield strain: ~ 15%). The frequency (rate of deformation) dependence of elastic and viscous moduli of the hydrogels was further tested by sweeping the frequency (0.1 Hz to 10 Hz) at a constant strain within the limits of the LVR of a 0.66 wt.% E8 hydrogel. To determine the effect of temperature on the stability of the hydrogels, frequency sweep studies were performed at different temperatures that may be clinically relevant.16,30 As seen from Figure 2B, the G´ curves at all the temperatures are higher over the G´´ curves, indicating that the hydrogels have retained the solidlike nature or elastic nature during the entire frequency sweep. The G´ curves have shown slightly positive slopes at all temperatures, indicating that the prepared hydrogels possess the


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characteristics of weak gels. Below the critical strain, the G´ is often expected to be nearly independent of frequency for a structured solid-like material. Figure 2B shows similar elastic modulus profile but with slightly positive slopes and this behavior is consistent at all the test temperatures. Retention of elastic nature at higher temperature is crucial for photo-responsive gels. Since photo irradiation may result in a raise in temperature; this feature enables the usage of these gels in photo irradiation applications. As most of the gels tend to lose their elastic strength and start to flow with the increase in temperature, esculin hydrogels may find potential utility in topical applications.


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Figure 2. Rheology of thixotropic gel formulations: (A) Strain sweep showing the LVR of the three hydrogels, (B) Temperature effect on the frequency sweep, (C) Thixotropic, and (D) Viscometric studies demonstrating a thixotropy, or time dependence to gel reformation. In general, during clinical or diagnostic applications, hydrogels are applied to the site (e.g. the tumor bed after surgery) and then diagnostic studies are conducted. These conditions call for hydrogels, which possess pseudoplastic properties (shear thinning and thixotropy) for better performance. Since the developed E8 hydrogels are intended for clinical applications, the pseudoplastic properties of the gels were tested by viscometric studies in addition to the rheological studies. The thixotropic response of the hydrogels were first probed by running the strain sweep from 0.01 % to 100 % at 1 Hz for three consecutive times in series at 25 °C. A recovery time of 60s was maintained after each sweep during the study. Figure 2C shows that the dynamic moduli of the gel containing 0.66 wt.% of E8, remained the same during each strain sweep. There is no appreciable change in the viscoelastic behavior of all the hydrogels after three strain sweeps (Figure S7). This indicates that the developed hydrogels possess the ability to regain their structural integrity after being mechanically sheared or disrupted. To distinguish non-time dependent shear-thinning from time-dependence thixotropy viscometric studies were performed as well. Figure 2D shows the change in apparent viscosity of 0.66 wt.% gel when shear rate was first increased and then decreased. The change in apparent viscosity suggests that the developed hydrogel has non-Newtonian behavior with pseudoplastic nature. The decrease in viscosity with the corresponding increase in shear rate clearly shows the shear thinning behavior of the hydrogels. The hydrogel reformulates to the initial viscosity with a hysteretic loop when the applied shear rate was reduced, this indicates that the developed hydrogels are thixotropic in nature.


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3.2. Luminescence Enhancement Next, E8 gels were paired with radionuclides to assess the effect on visible photon output. The PET tracer zirconium-89 (89Zr) was chosen due to widespread clinical use, while other PET radionuclides (18F,

64

Cu) were also tested (Figure S8). PET radionuclides (18F,

64

Cu,

89

Zr)

solutions emit visible photons via the Cerenkov mechanism. The radionuclides were combined with various concentrations of E8 gels and optically imaged on an IVIS spectrum.31 Figure 3A shows the visible photon output of 89Zr combined with various concentrations of E8 gels.

Figure 3A: Photon flux enhancement of

89

Zr through combination with E8 gels at various

concentrations. Figure 3B shows the photon counts of a constant amount of

89

Zr (10 µCi) with various

concentrations of E8 gel (100 µL volume). As minimum gelation concentration (MGC) of E8 is


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0.12 %. Concentrations of the gelator above and below the MGC were used to determine the effect of gelation on enhancement. For partial gels (concentrations MGC), the enhancement in photon flux was also minimal. Interestingly, at gelator concentrations between 0.1 – 0.2 %, the highest enhancement of ~3-fold photon flux was seen. The dilution effect on photon flux behavior of the gel samples were also studied and shown in Figure S9.

150000

Avg Radiance [p/s/cm2/sr]

100000

50000

1%

0. 8%

0. 6%

0. 35 %

0. 25 %

0. 15 %

0. 12 %

0. 07 %

0. 04 %

0

co nt ro l 0. 01 %

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


Concentrations of E8 gel (% w/v) Figure 3B: Photon counts of E8 gels having different E8 gelator concentrations in presence of 89

Zr radionuclide.

The photon flux enhancement of radionuclides by the E8 gels were due to the presence of fluorescent coumarin moieties and layer-by-layer stacking of E8 gelator molecules. To demonstrate that the enhancement was a result of these interactions rather than scattering or other effects, esculin copolymers and small molecular sugar-derived gelators were examined.


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Poly(esculin methacrylate) (PEMA) polymers were tested at various concentrations (0.14% 0.52%), with no change in photon flux (Figure 4). Esculin methacrylate (EMA) was prepared via the enzymatic reaction of esculin and vinyl methacrylate in acetone (Figure S10) and radically polymerized to prepare its polymer poly(esculin methacrylate) (PEMA) (Figure S11). Copolymers of EMA and poly(ethylene glycol) methyl ether methacrylate (PEGMEA), poly(EMA-co-PEGMEA) (PEP) were also tested, having EMA amounts about 40 mol %. Two PEP polymers were used for the photon flux study, PEP1 where the EMA amount was 30 mol % and PEP2 where the EMA amount was 40 mol %. The synthetic procedure of copolymers of EMA and PEGMEA, PEP is shown in Figure S12. For each of the aforementioned samples, no enhancement in photon flux was seen when combined with 89Zr (Figure 4), in contrast to the E8 gel, demonstrating that layer by layer stacking of E8 gels results in an increase in the visible photon flux in contrast to covalently bonded polymeric chains. To better demonstrate that the enhancement seen with E8 gels is due to the presence of coumarin moiety, non-emitting gels of an Amygdalin-18, poly(ethylene glycol) (PEG) and gelatin were also tested. Amygdalin-18 gels at 0.11 wt. % and amygdalin 0.35 wt. % were prepared and tested (Figure S13). The amygdalin gels, gelatin, and polyethylene glycol (PEG) gels all showed no enhancement compared to H2O though the gels also form via layer-by-layer stacking of gelators (Figure 4).


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150000

Avg Radiance [p/s/cm2/sr]

100000

50000

2O

co nt r Es ol 8 0. 1 Es 2 8 A 0 .1 M 5 Y G A M 0.11 Y G PE 0 . 3 5 M A PE 0 . 1 4 M A 0. 52 PE P1 PE G el P2 at in 0. G 5 el at in 1 PE 0 G ge l

0

H

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


Concentration (%w/v)

Figure 4: Comparative photon-count study of different molecular gels, polymers and commercial gels combined with 89Zr.

While others have postulated that increases in the photon flux when using an open filter on the IVIS demonstrate that scintillation is occurring, the CCD in the IVIS system is most efficient between 450-850 nm, whereas it drops to less than 20% quantum efficiency below 300 nm.21 Therefore, when using a fluorescent agent, an increase in photon flux may be observed due to either scintillation or the absorption of photons at a wavelength where the CCD quantum efficiency is low (450 nm). As the gelator displays fluorescent properties and precursor is a scintillator, it is likely that a combination of the two mechanisms results in the visible photon enhancement.14

3.3. In vivo Lymph Node Imaging


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To demonstrate the potential medical application of the E8 gel silica nanoparticles were radiolabeled with 1.0 mCi

89

Zr using a chelator free approach and injected these into the right

footpad of male athymic nude mice for a lymph node imaging with an IVIS system32. Figure 5A shows the CL image of the two proximal lymph nodes (position 1 and position 2, blue and red arrows) after 24 h. Afterwards, we applied E8 gel (0.12 wt. % gelator concentration) at position 1 and position 2, then imaged immediately afterwards. ROI on position 1 and 2 allowed quantification of the average radiances (Figure 5C and 5D). There is only a small enhancement on position 1, and stronger enhancement on position 2 for both average radiances (1.43-fold) and imaging area (total radiances 3-fold) (Figure 5B, C and D). The right lower lymph node (position 2) is more evident to see than figure 5A after E8 gel is applied. Overall, the enhancement in mice is not as robust as observed in the well plate due to complicated in vivo environments such as tissue absorption and scattering.33,34 Since the blue Cerenkov light does not penetrate well through the tissue of the mice; most of the blue-weighted light is absorbed prior to reaching the gel applied to the skin. Therefore, enhancement of the E8 gel is limited. However, the E8 gel still resulted in a statistically higher photon flux in lymph node mapping.


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Figure 5. E8 gel lymph node imaging application: A) 24 h post injection lymph node imaging without apply of E8 gel. B) 24 h post injection lymph node imaging after applied E8 gel (0.12 % gelator concentration) at position 1 and 2. C) Average radiance of position 1 and 2 before and after E8 gel applied. D) Total radiance comparison only at position 2 before and after E8 gel applied.

4. CONCLUSIONS


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Multifunctional stimuli responsive molecular gelators and their gels can be fashioned from renewable materials and applied to enhancing the detected photon output by up to 1.8-fold for positron and gamma emitters. The application of these materials towards a wide array of imaging strategies due to their ability to sense various types particulate and electromagnetic radiation makes them a strong candidate to be components of scintillation counting or radionuclide imaging. While small gelation concentration ranges, short wavelength absorption and emission windows, and metastable gels are limitations of these first generation systems, by vetting the wide array of functional molecules, especially glucosides, there is the possibility to create systems specific to a wide variety of desired applications. In the future, a molecular gelator composed of green scintillation molecules for topical application could be developed that avoids the pitfalls of solid and liquid scintillation systems presently used for in vivo imaging. It is hoped that this work will help inspire the next generation of scintillators to be renewably sourced to provide dually sustainable and functional systems.

Funding Sources: This work was in part funded by the following grants to G.J: CBET-1512458 from the National Science Foundation, and GRANT11890945, NIFA, United States Department of Agriculture and J.G. acknowledges the grant support from NIH R01EB014944 and R01CA183953 and the MSKCC NIH Core Grant (P30-CA008748) a grant from the Center for Molecular Imaging and Nanotechnology. J.S. was partly supported by NSF grant CBET1512458 and T.M.S. was supported by NSF INGERT grant DGS 0965983. ACKNOWLEDGEMENT J.R.S. would like to thank Dr. J. Morales for his help with electron microscopy, Dr. A. Bykov for support with the x-ray instrument, and Dr. P. Pradhan for aid with the NMR studies.


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ABBREVIATIONS MGC, minimum gelation concentration, PET, positron emission spectroscopy, SPECT, single photon emission computed tomography, IR, infrared radiation, CLI, Cerenkov luminescence imaging, CCD, charge coupled device, SCIFI, secondary Cerenkov induced fluorescence imaging, IVIS, in vivo imaging system, E8, esculin caprylate, G’, elastic modulus, G’ viscous modulus, Tgel, temperature of gelation, FT/ATR, Fourier Transform/Attenuated Total Reflectance, ROI, Region of Interest, NMR, Nuclear Magnetic Resonance

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Table of Contents Graphic:


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Radiation-Responsive Esculin-Derived Molecular ... - ACS Publications

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