1 Gel dosimetry with radio-fluorogenic coumarin 2

In radiotherapy, accurate deposition of energy to the targeted volume is vital to ensure 9 effective treatment. Gel dosimeters are attractive detection systems, as tissue substitutes with 10 potential to yield three-dimensional dose distributions. Radio-fluorogenesis is creation fluorescent 11 chemical products in response to energy deposition from high-energy radiation. This report shares 12 studies of a radio-fluorogenic gel dosimetry system, gelatin with coumarin-3-carboxlyic acid 13 (C3CA), for the quantification of imparted energy. Aqueous solutions exposed to ionizing radiation 14 result in the production of hydroxyl free radicals through water radiolysis. Interactions between 15 hydroxyl free radicals and coumarin-3-carboxylic acid produce a fluorescent product. 16 7-hydroxy-coumarin-3-carboxylic acid has a blue (445 nm) emission, following UV to near UV 17 (365–405 nm) excitation. Effects of C3CA concentration and pH buffers were investigated for this 18 system. The response of the system was explored with respect to strength, type, and exposure rate 19 of high-energy radiation. Results show a linear dose response relationship with a dose-rate 20 dependency and no energy or type dependencies. This report supports the utility of gelatin-C3CA 21 for phantom studies of radio-fluorogenic processes. 22


Introduction
Advancements in radiation therapy technology have supported study of tissue-equivalent gels containing active chemical sensors for the measurement of absorbed dose of radiation.Gel dosimeters have radiological properties similar to biological tissue and are suitable substitutes with the potential to resolve three-dimensional dose distributions.The development of gel dosimeters was dormant for many years, but has recently been developing at a rapid pace.The first reported use of a gel dosimeter was in 1950 with the colorimetric dye methylene blue [1].Other early investigators explored chloral hydrate and trichloroethylene in agar [2].Gelatin with ferricyanide, Fricke-type, gel dosimeters were first studied using colorimetric methods, and later magnetic resonance (MR) imaging [3][4][5].Further developments introduced polymer and leuco-dye systems [6][7].Recently, a radio-fluorogenic polymer system has been introduced [8].Each of the current gel dosimeters have their own limitations such as rapid diffusion of chemical products with Fricke-type, toxicicty of with polymer systems, intricate fabrication methods with leuco-dyes, and the water-insolubility of radio-fluorogenic polymers [9].The hunt for the ideal sensor element and gel substrate is ongoing.Two of the most common gel substrates are agarose and gelatin.Gelatin is derived from bovine or porcine collagen; primary element of skin, bone, and connective tissue.Agarose is a polysaccharide isolated from agar with highest gelling potential; agar is derived from seaweed.
Gelatin and agarose are both capable of creating hydrogels with low percentages of gelling agent.However, agarose is opaque and induces light scattering, while gelatin is relatively translucent.The opacity of agar makes it less than ideal for optical analysis. 32Clarity and transparency of gelatin is strongly dependent on raw material history, purity, and preparation.Commercial gelatin consists of tropocollagen rods in the order of 300 nm in length with 1.5 nm diameter [10].Raw material is processed with acid or base solutions yielding "Type A" (hydrogen chloride) or "Type B" (sodium hydroxide).Type A is denser than type B with a greater intrinsic viscosity [11].Gelation speed also affects rigidity with structure a function of formation temperature, slow gelatin yields increased organization and orientation of chain elements with greater lateral bonding, this results in the formation of fine well-ordered lattices [12].Additionally, gelation is not susceptible to ionic effects [10].Derived from biological tissue with well understood mechanisms of gelation, gelatin is an attractive substrate for exploration of optically active sensors.
Radio-fluorogenic sensors are chemical elements that allow for dosimetry, quantification of energy deposition from of ionizing radiation, through measurement of molecular fluorescence.
Fluorescent detection methods are particularly promising due to their ability to form selective high-resolution images.Initially reported by Day and Stein in 1949, fluorescence spectroscopy can be used to determine absorbed dose in aqueous solutions of aromatic compounds [13][14][15].Ionizing radiation initiates radiolysis of water, yielding hydroxyl free radicals that hydroxylate aromatic compounds via electrophilic substitution.Numerous aromatic compounds are recognized as radio-fluorogenic, with hydroxylation producing fluorescent products.The first fluorescent sensor investigated for radiation dosimetry was aqueous benzoic acid [15].Other potential sensors are terephthalic, trimesic, and pyromellitic acid [16][17][18].Each improved the yield of fluorescent products by restraining positions for substitution.However, each of those compounds possesses excitation wavelengths unsuitable for a gel substrate.Rayleigh scattering is wavelength dependent, proportional to 1/λ 4 , resulting in rapid reduction of transmission for shorter wavelengths of light.
Organic gels are naturally turbid due to their macromolecular nature, thus it is preferable to use longer excitation wavelengths with greater potential for penetration.Fluorescence of aromatic compounds is due to their conjugated system of alternating single and double-bonds; overlapping pi-orbitals allow for delocalization of electrons.Larger conjugated systems require less energy for excitation [19].Selection of a multi-cyclic radio-fluorogenic sensor would provide a more attractive fluorescent product; ideally with excitation from visible light.Multi-cyclic coumarin-3-carboxylic acid (C3CA) is a sensor candidate.
Aqueous C3CA has been identified as chemical dosimeter for application to radiotherapy with favorable traits including linear dose response, reproducibility, and long-term stability [20].The radio-fluorogenic mechanism of C3CA has been studied within aqueous solution [21].Positive features of C3CA include high solubility in aqueous solutions, simple organic composition, and favorable excitation and emission spectra.C3CA reacts with hydroxyl radicals to yield the fluorescent product, 7-hydroxycoumarin-3-carboxylic acid (7HOC3CA), Figure 1.respect to relative fluorescent yield.Ionizing radiation response was examined subject to dose, rate, energy, and type for megavoltage electron and photon energies.Instrumental analysis was conducted with a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.; Pal Alto, California).Excitation and emission slit widths were set to 5 nm, emission scans were performed and peak emission values recorded and plotted.The dose response curve was created by plotting intensity of 445nm emission versus nominal dose.

pH Response
The influence of pH buffers on fluorescent response was examined.Several solutions yielded various pH's; deionized water (pH 6.0), phosphate buffered saline (pH 7.4), and sodium hydroxide with sodium bicarbonate (pH 10).Results show positive correlation between pH and quantum yield.
A spectral shift of the excitation maxima was also demonstrated.Specifically, peak excitation shifted from 365nm in normal (pH 7) solution to 405 nm in basic (pH = 10) solution, Figure 2.

C3CA Concentration
With sodium hydroxide with sodium bicarbonate solutions, varying the concentration of C3CA revealed a stronger response with concentrations 5 mM and greater, Figure 3.The peak normalized response, fluorescent intensity divided by dose, produced a decreasing exponential curve, Figure 4.

Dose Response
Dose response was studied with ionizing radiation with respect to type, rate, and energy.
Relative response was measured with respect to 445nm emissions and plotted against nominal dose,

Discussion
The basic solutions (pH 10) were observed to double emission intensity and shift the peak excitation wavelength from 365 nm to 405 nm.Transition between excited and ground states, the energy gap, is known to be influenced by the micro-environment through molecular motion, collision, rotational and translational diffusion, and formation of complexes.Smaller quantum yields are observed with large energy gaps due to availability of alternative relaxation pathways.The observed increase in quantum yield is consistent with previous studies in aqueous solution; however, greater than previously observed (385 nm) [22].The increased spectral shift may be due to interactions with gelatin, additional study could clarify these effects.
The dose response was notably more pronounced for concentrations of C3CA above 5 mM.The normalized response curves of various concentrations of C3CA suggest saturation, diminishing population of radio-fluorogenic reactants in the dose range studied.Future work investigating absolute yields and a larger range of doses would be beneficial.
Normalized data demonstrate an independent linear response with respect to dose, energy, and type of ionizing radiation (electron and photon).With respect to type, an independent response is expected since photon dose deposition is predominately by delta rays, secondary electrons.A dose rate dependency was observed, consistent with other findings [20].Previously suggested to be due to metallic impurities in C3CA and alleviated by successive distillations.The MDA was estimated to be 1.5 Gy, this value should be determined rigorously, by study of an expanded dose range.

Conclusions
Optical imaging of biomarkers is an active area of study with C3CA a recognized radiation activated sensor for fluorescent imaging [18].Investigators have explored the use of coumarin attached to peptide ligands, designed for DNA binding, with potential for assessment of radiological response [23].Other work is currently studying the application of fluorescent labels for radiometric assay [24,25].Advances in the fabrication of gelatin based phantom materials with 3D printability make further study particularly attractive [26].Further study radio-fluorescent sensors in a gelatin matrix would help advance these prospective in vivo applications.
The potential of C3CA in gelatin for determination of spatial dose distributions has been demonstrated in a separate report [27].The use of planar laser induced fluorescence (PLIF) has been shown as a method to yield high-resolution three-dimensional images [28].This method of image collection and analysis has been recognized and is currently being explored with polymer based radio-fluorogenic gel [29].However, it is the author's belief that the greatest depth of penetration and finest imaging resolution will be obtained by applying methods of two-photon excitation microscopy.

Figure 5 .
Figure 5. Repeated measures, using four samples for each data point, demonstrated relative error less than 1%.A linear response was observed in the range investigated (R > 0.99), independent of type (photon or electron) and energy (9 MeV, 6 MV, and 23 MV), Figure 6.A strong negative correlation (R > 0.99) with dose rate was observed; the intensity of normalized fluorescent response decreased with increasing dose rate, Figure 7. Using a definition of three times the standard deviation of the background, the minimum detectable amount (MDA) was extrapolated from 9MeV electron data and estimated to be 1.5Gy, Figure 8.

Figure 7 .
Figure 7. Plot of 9 MeV dose response plotted with extrapolated MDA.
.preprints.org) | NOT PEER-REVIEWED | Posted: 11 May 2018 doi:10.20944/preprints201805.0176.v1Peer-reviewed version available at Bioengineering 2018, 5, 53; doi:10.3390/bioengineering5030053 were readied with water from EASYpure water purification system (Barnstead To allow for dispersion, gelatin was 'wet,' placed in a beaker to soak with half the total volume of water for 20 minutes.C3CA was brought into solution by boiling a small volume in a separate beaker.After sufficient 'wetting', aqueous C3CA solution was added with the remaining portion of water and temperature of gel solution raised to 35° C; care was taken to ensure the temperature remained below 40° C to prevent denaturation.Gel was maintained at 35° C for 90 minutes, or until optically clear and free of visible colloidal structures.The solution was then removed from heat and pipetted into poly-methyl-methacrylate (PMMA) cuvettes.Gels were left to cool overnight at ambient temperature.For initial pH buffer studies, 7% gelatin solutions were made with 7HOC3CA to mimic the radio-fluorogenic product.Sodium bicarbonate/hydroxide and phosphate buffered saline (PBS) solutions were prepared with 0.9 mM C3CA and 0.1 mM 7HOC3CA.For concentration and dose response studies, 1 mM, 5 mM, 10 mM, and 20 mM C3CA solutions were prepared with the sodium bicarbonate/hydroxide buffer.Irradiations were conducted with a C-series high-energy medical linear accelerator (linac) (Varian Medical Systems; Palo Alto, CA), with two megavoltage (MV) photon and five electron energies; 6 and 23 and 6, 9, 12, 15, and 20 MeV.Irradiations were conducted with a polystyrene phantom containing a void for 4 cuvettes; the phantom was designed expressly to provide geometry favorable for establishment of electronic equilibrium.A computed tomography (CT) scan was carried out on the phantom, images were imported into Eclipse treatment planning system (Varian Medical Systems; Palo Alto, CA), and nominal dose calculated.