PolyRad – Protection Against Free Radical Damage

The effects of elevated levels of radiation contribute to the instability of pharmaceutical formulations in space compared to those on earth. Existing technologies are ineffective at maintaining the therapeutic efficacies of drugs in space. Thus, there is an urgent need to develop novel space-hardy formulations for preserving the stability and efficacy of drug formulations. This work aims to develop a novel approach for the protection of space pharmaceutical drug molecules from the radiation-induced damage to help extend or at least preserve their structural integrity and potency. To achieve this, free radical scavenging antioxidant, Trolox was conjugated on the surface of poly-lactic-co-glycolic acid (PLGA) nanoparticles for the protection of a candidate drug, melatonin that is used as a sleep aid medication in International Space Station (ISS). Melatonin-PLGA-PLL-Trolox nanoparticle as named as PolyRad was synthesized employing single oil in water (o/w) emulsion solvent evaporation method. PolyRad is spherical in shape and has an average diameter of ~600 nm with a low polydispersity index of 0.2. PolyRad and free melatonin (control) were irradiated by UV light after being exposed to a strong oxidant, hydrogen peroxide (H2O2). Bare melatonin lost ~80% of the active structure of the drug following irradiation with UV light or treatment with H2O2. In contrast, PolyRad protected >80% of the active structure of melatonin. The ability of PolyRad to protect melatonin structure was also carried out using 0, 1, 5 and 10 Gy gamma radiation. Gamma irradiation showed >98% active structures of melatonin encapsulated in PolyRads. Drug release and effectiveness of melatonin using PolyRad were evaluated on human umbilical vein endothelial cells (HUVEC) in vitro. Non-irradiated PolyRad demonstrated maximum drug release of ~70% after 72 h, while UV-irradiated and H2O2-treated PolyRad showed a maximum drug release of ~85%. Cytotoxicity of melatonin was carried out using both live/dead and MTT assays. Melatonin, non-radiated PolyRad and irradiated PolyRad inhibited the viability of HUVEC in a dose-dependent manner. Cell viability of melatonin, PolyRad alone without melatonin (PolyRad carrier control), non-radiated PolyRad, and irradiated PolyRad were ~98, 87, 75 and 70%, respectively at a concentration ~\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \sim $$\end{document} 0.01 mg/ml\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${mg}/{ml}$$\end{document} (10μg/ml\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$10\mu g/{ml}$$\end{document}). Taken together, PolyRad nanoparticle provides an attractive formulation platform for preventing damage to pharmaceutical drugs in potential space mission applications.


Materials and Methods
Materials. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
PolyRad characterization: size, surface charge, and melatonin encapsulation efficiency. The size and surface charge of particles before and after PLL-Trolox conjugation on melatonin encapsulated PLGA nanoparticles were measured using dynamic light scattering (DLS) and zeta potential measurements, respectively using a NanoSeries Zetasizer ZS 90 (Malvern). Samples were prepared in deionized (DI) water. Encapsulation of melatonin in PolyRad nanoparticles was assessed by measuring the absorbance at 300 nm using a microplate reader (BioTek Synergy 2) and a melatonin standard curve (S.I. Figure 1). The percentage of melatonin encapsulation capacity was calculated using the following Eq. 1:

Characterization of PLGA-PLL-Trolox formulation using Fourier Transform Infrared (FT-IR)
Spectroscopy. FTIR spectroscopy was used to confirm the structural changes that occurred after PLL-Trolox conjugation with PLGA nanoparticles. Samples for FT-IR spectroscopy were prepared by mixing and grinding freeze-dried nanoparticles with FT-IR grade potassium bromide (KBr; Alpha Aesar) (w:w ratio of 1:100) to make pulverized pellets. FT-IR absorption spectra were obtained using a Thermo Nicolet NEXUS 470 FTIR instrument in the wavelength range from 4000 to 400 cm −1 at a resolution of 4 cm −1 using 32 scans per sample. The effect of background noise from the ambient air without a sample in place was subtracted from the sample spectra. All spectra were analyzed using EZ OMNIC E.S.P v.5.1 software (Thermo Scientific). The resulting FTIR spectra of PLGA-PLL-Trolox were compared to individual spectra of PLGA, PLL, and Trolox to characterize the conjugation reaction based on the intensity or shift of the vibrational bands.
Determination of the composition of melatonin-PLGA-PLL-Trolox (PolyRad) nanoparticles using 1 H-NMR spectroscopy. For 1 H-NMR spectroscopy, 10 mg of PolyRad nanoparticles, melatonin alone, PLGA alone, PLL alone, and Trolox alone were dissolved in 750 μl of DMSO-d6 except for PLGA, which was dissolved in d-Chloroform. Each solvent was used as the internal reference to determine chemical shifts (δ) in ppm. 1 H-NMR spectra were then recorded using Bruker advanced III 400 MHz Liquid-State NMR instrument at R.T.
Fluorometric Hydroxyl Radical Scavenging Capacity (HOSC) assay. A fluorometric HOSC assay was applied to determine the mechanisms of highly reactive free radical absorption by PLGA-PLL-Trolox composite and Trolox alone was used as a standard 50 . OH• radicals were generated using Fe (III)/H 2 O 2 Fenton-like reaction system in a pH 7.4 sodium phosphate buffer 51 . Assay reactions were carried out in Costar black 96-well polystyrene plates and analyzed using a microplate reader (BioTek Synergy 2) with an excitation wavelength of 485 nm, emission wavelength of 530 nm, and 0.1 s read time for each well with each plate read once per minute for 3 h. The reaction mixture contained 170 μl of 9. Photochemical degradation of melatonin. To examine the resistance to photochemical degradation of melatonin, 2 mg of PolyRads containing 2.5 μg melatonin were dissolved in 1 ml 40% acetonitrile-60% water (v/v) solvent and spiked with 0.75 mM hydrogen peroxide (H 2 O 2 ) followed by UV radiation at 254 nm and 39 W spectral output for 0, 1, 2, 4, 6 and 8 h. H 2 O 2 is known to be decomposed into hydroxyl radical (OH•) and superoxide ions (O 2 . − ) when exposed to UV radiation. Pure melatonin powder was used as a control and were treated with 0.75 mM H 2 O 2 followed by UV irradiation at every 20 min for up to 2 h. The reaction progress was monitored by determination of melatonin concentration and its two degradant products (N 1 -acetyl-N 2 -formyl-5-methoxykynuramine, AFMK and 6-hydroxymelatonin) using Agilent 1260 Liquid Chromatography (LC) High-Performance Liquid Chromatography (HPLC) apparatus equipped with Waters C18 column of 4.6 × 250 mm (particle size 5 μm). The system consisted of a vacuum degasser, a binary pump unit, an autosampler for sample injections, a thermostat column compartment, and a photodiode array detector (DAD) for peak analysis at 304 nm. The mobile phase used was acetonitrile −25 mM ammonium acetate (40:60, v/v). The system was run at ambient temperature (~22 °C) with a flow rate of 1.0 ml/min. Sample injections of 30 μl were taken and (2020) 10:8335 | https://doi.org/10.1038/s41598-020-65247-y www.nature.com/scientificreports www.nature.com/scientificreports/ observed for characteristic peaks over 8 min intervals between injections. Quantification of melatonin and degradation products were carried out using calibration curves obtained by repeated injections of standard solutions of known concentration.
Gamma irradiation of PolyRad. The effects of gamma irradiation on the chemical structure of melatonin were carried out using radioactive cobalt 60 ( 60 Co) isotope of activity 200 mCi. Two sample groups of melatonin alone and PolyRad were prepared in 1 ml of 10 mg/ml aqueous solutions and irradiated at 1, 5 and 10 Gray (Gy) doses. Non-radiated (0 Gy) melatonin and nanoparticles were used as controls. The samples were lyophilized and dissolved in DMSO-d6. The effects of gamma irradiation on the structure of melatonin alone and melatonin in PolyRad nanoparticles were investigated using 1 H-NMR spectroscopy (Bruker instrument 400 MHz). The spectra were calibrated and normalized relative to DMSO-d6 at 25 °C (δ = 0 ppm). After 1 H-NMR measurements, the relative area under the curves were calculated with respect to non-radiated melatonin standards and plotted as a function of radiation doses.
In vitro drug release kinetics. In vitro release of melatonin from non-irradiated and irradiated PolyRad was carried out in PBS at pH 7.4. Each 10 mg of PolyRad and melatonin powder were put in separate 1.5 ml centrifuge tubes and exposed to 0.75 mM H 2 O 2 followed by UV irradiation for 2 h. PolyRad was suspended in 10 ml PBS and kept in a 37 °C water bath. PBS solution of 500 μl was withdrawn at 0, 5, 10, 15 and 30 min and then at 1, 2, 4, 8, 12, 24, 48 and 72 h. The solution was centrifuged at 1000 rcf for 1 min. The supernatant (150 μl) was put in a 96 well assay plate in triplicates. PBS was used as a blank. Melatonin concentrations were measured using absorbance at 300 nm using the microplate reader and melatonin standard curve. The drug release kinetics data were plotted as log cumulative drug release versus log time (Eq. 2).
∞ is the fractional drug release at time t, τ is the lag time, b is the fractional drug burst release, k is a kinetic constant that measures the drug release rate characteristic of the drug/polymer system, and n is the release exponent which characterizes the drug release mechanism 52-58 .

Melatonin cytotoxicity.
To determine and compare the cytotoxicity of melatonin on endothelial cells, human umbilical vein endothelial cells (HUVEC) were treated with free melatonin, melatonin-loaded PolyRad (carrier control), PolyRad, PolyRad treated with 10 Gy of gamma radiation, and PolyRad treated with 0.75 mM of H 2 O 2 followed by 1 h of UV radiation. HUVEC were seeded in Costar 96 well assay plate (sterile, black, clear-bottom with lid) at a cell density of 10,000 cells/well. The cells were incubated in PromoCell medium with 10% FBS and 1% penicillin-streptomycin (10,000 units/ml) at a 37 °C and 5% CO 2 incubator. Cells were allowed to grow overnight. The cells were incubated for 72 h with melatonin solution control and PolyRad at melatonin concentrations of 0.01, 0.1, 1 and 10 mg/ml. PBS was used as a negative control. Untreated viable (live) HUVEC were used as a positive control. After 72 h, cells were rinsed with PBS and cellular viability were measured using MTT assay (Invitrogen). The reduction of MTT to formazan by viable HUVEC was quantified measuring absorbance at 570 nm. Absorbance of treated cells were measured and calculated by subtracting the mean background level of wells containing the medium. The % cell viability was calculated as absorbance ratio of treated cells to live cells as shown in Eq. To visualize cells under fluorescence microscope, live and dead cells were stained using 2 μM calcein AM and 4 μM of EthD-1, respectively following the manufacturer's protocol. Images were acquired using a 10X Plan Fluor (NA = 0.3) objective equipped with a Carl Zeiss Axio Observer Z1 microscope at excitation/emissions of 470 ± 40/525 ± 50 and 545 ± 25/605 ± 70, respectively.

Characterization of PolyRad.
We synthesized melatonin-encapsulated PLGA nanoparticles with an average diameter of 325 ± 21 nm (Fig. 2a), polydispersity index (pDI) of 0.07 ± 0.05, and a zeta potential of about −23 ± 8.5 mV (Fig. 2b) as confirmed by the DLS analysis. The PLL-Trolox shell attributed to the increase in particle size due to the deposition of its complex onto the surface of melatonin-PLGA nanoparticles. The average diameter of PLL-Trolox conjugated PolyRad nanoparticles were approximately 90% larger (622 ± 114 nm; Fig. 2c) than melatonin-PLGA control nanoparticles without PLL-Trolox shell as seen by the shift in the average peak intensity to the right-hand side. The pDI and zeta potential increased to 0.23 ± 0.12 and 29 ± 10 mV (Fig. 2d), respectively, indicating the successful conjugation of positively charged PLL on the surface of nanoparticles. The encapsulation efficiency of melatonin inside nanoparticles was determined as 44 ± 12% (Table 1).
Confirmation of the composition of PolyRad nanoparticles using 1 H NMR Assay. Formation of melatonin-PLGA-PLL-Trolox nanoparticles was evaluated using 1 H-NMR (Fig. 4). The NMR results depicted a successful conjugation of PLL-Trolox on melatonin encapsulated PLGA nanoparticles to form PolyRad nanoparticles compared to their independent control NMR peaks (melatonin, PLGA, PLL, and Trolox alone). In Fig. 4, proton peaks at 2.5 and 7.25 ppm represent solvent background signals from DMSO-d6 and d-Chloroform, while the broad peak at 3.25 ppm represents residue water proton peak. Proton peaks from PLL, denoted by a, b, c and d which correspond to -CH-, -NH-, -CH 2 -, and -NH 2 groups occurred at 1.7, 3.5, 4.75 and 5.2 ppm, respectively. Also, melatonin distinctive peaks denoted by letters 'e-n' correspond to the -CH 3 -, the two -CH 2 -, -CH 3 O-, the three phenyl -CH-, the cyclic -CH-, and the two -NH-respectively. These peaks were recorded at 1.8, 2.75, 3.25, 3.75, 6.75-7.25, 7.25, 7.8 and 11.6 ppm, respectively. For Trolox, distinctive peaks were lettered 'o-v' . Trolox 1 H-NMR peaks showed propionic acid -CH 3 -(o) and -OH-(p) protons at 1.5 and 1.75 ppm respectively, cyclic -CH 2 -(q and r) at 1.9-2.1 ppm, phenyl methane protons (s, t and u) at 2.0-2.2 and 2.6 ppm, and the phenyl -OH-(v) protons at 7.25 ppm. Proton peaks for PLGA were labeled from 'w-z' and then '#' . PLGA proton peaks displayed lactide -CH 3 -(w), -CH-(y), and glycolide -CH 2 -(x) protons at 1.5, 6.2 and 5.5 ppm respectively. However, the lactide end and the glycolide end -OH-protons (z and # respectively) in PLGA were missing probably by being lost to neighboring protons. Comparing with the 1 H-NMR of the individual components, 1 H-NMR of nanoparticles displayed virtually all the peaks that were shown in the individual components as shown in Fig. 4. However, the chemical shift of protons (b) in PLL formed an amide bond with the lactide end -OH-protons (z) of PLGA, hence we had that this amide bond occurred around 3-3.5 ppm in the NPs' 1 H-NMR. Likewise, proton peak at 5.25 ppm in the NPs' 1 H-NMR corresponds to the -NH-group of the corresponding amide bond (CONH) between primary amino group of PLL (d) and the carboxyl group of Trolox (p).    www.nature.com/scientificreports www.nature.com/scientificreports/ Scavenging capacity of Trolox using fluorometric HOSC assay. Trolox serves as a potent scavenger of OH• radicals, efficiently blocks the effects of highly reactive free radicals even at low concentration and therefore, is tested to prevent the photochemical degradation of melatonin in PolyRad nanoparticulate form. Fluorometric Hydroxyl Radical Scavenging Capacity (HOSC) was performed to analyze the OH• scavenging capacity of Trolox in its free solution. Results showed that an increase in Trolox concentration in the HOSC medium increased the free radical scavenging capacity of Trolox. Figure 5 shows the fluorescence decay curve of fluorescein with time in the presence of different Trolox concentrations. In the absence of Trolox (Blank), the fluorescence intensity of fluorescein was quenched rapidly with more than 50% reduction in the relative fluorescence intensity within 10 min, indicating the reaction of fluorescein with OH• radicals caused by the Fenton like Fe 3+ /H 2 O 2 system. Trolox worked as OH• radical scavenging agent that delayed the decay in fluorescence intensity of fluorescein by diminishing the quenching rate significantly in a dose-dependent manner. As the Trolox concentration was increased from 20 to 100 μM, the capacity for scavenging OH• radicals increased. The ~100% fluorescence intensity retention time were 10, 50, 80, 100 and 120 min using 20, 40, 60, 80 and 100 μM Trolox, respectively.

Effects of photochemical degradation on PolyRad.
To study the effect of photochemical oxidation on the structure of melatonin in PolyRad formulation, irradiation experiments were performed on PolyRad suspensions using H 2 O 2 in 40% acetonitrile-60% water solvent followed by UV light exposure. Bare melatonin was used as a control. This process relies on the production of OH• through the irradiation of H 2 O 2 with UV radiation: that degrades melatonin 12,15 . The influence of H 2 O 2 and UV on melatonin degradation was analyzed with time using HPLC (Fig. 6a). The melatonin peak did not change by more than ~20% by UV treatment only for 2 h (Fig. 6a; hatched column). UV irradiation in the presence of H 2 O 2 influenced the disappearance of the melatonin peak by ~80% within 2 h (Fig. 6a; filled column). Pure melatonin control showed only one characteristic peak with a retention time of 5.67 min eluted from the HPLC column (Fig. 6b). The photochemical degradation of melatonin (N-acetyl-5-methoxytryptamine) formed two major degradant peaks (B and C) at 4.7-and 1.98-min retention times for N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK) and 6-hydroxymelatonin, respectively 4,59-61 . The measured changes of AFMK and 6-hydroxymelatonin concentrations increased as a function of different UV exposure time in the presence of H 2 O 2 (SI Figs. 2-4). Also, a peak at ~ 3.95 min, likely due to the melatonin degradation product, N-acetyl-5-methoxykynuramine (AMK) is seen. The overall reaction followed a first-order reaction:  (SI Fig. 5a). In contrast to bare melatonin (Fig. 6c,  hatched column), a remarkable reduction of melatonin degradation was observed using PolyRad nanoparticles (Fig. 6c, filled column). PolyRad protected ~75, 65, 55 and 15% of the active structure of melatonin after UV irradiation only, and 2 h of UV followed by 0.75, 1, and 5 mM of H 2 O 2 exposure, respectively indicating that nanoparticles scavenged H 2 O 2 in a concentration-dependent manner. Encapsulation of melatonin using PolyRad nanoparticles reduced the formation of AFMK by ~50% (SI Fig. 6).

H-NMR spectrometry studies on molecular structure of melatonin after gamma radiation.
Based on the 1 H NMR spectrum of melatonin as presented in Fig. 7a and Table 2, major melatonin peaks are observed at 1.9 (NHCOCH 3 ), 2.9 (CH 2 ), 3.5 (CH 2 NH), 3.9 (OCH 3 ) and 6.9-7.4 ppm (benzene aromatic rings and www.nature.com/scientificreports www.nature.com/scientificreports/ NHCHO). Free melatonin is degraded into other compounds that appear at 3.2, 3.6 and 3.78 ppm and predominantly at 10.6 (OH•) and 10.9 (NHCO) ppm. Melatonin in PolyRad nanoparticles did not show any significant differences between the non-radiated control and gamma irradiated PolyRad samples. The percentage of active melatonin remaining was calculated using the integral ratio of peaks of groups I through V (Table 2) based on the non-radiated melatonin control (Fig. 7b). The structural integrity of melatonin alone was completely degraded to 25.7, 10.8 and 5.5% at 1, 5 and 10 Gy, respectively compared to non-radiated free melatonin. The nanoparticle plays a critical role in the protection of melatonin by >98% most likely by scavenging of free radicals generated from gamma radiation 62 .  The areas under the curve of 1 H NMR peaks were integrated to determine the percentage melatonin degradation. Experiments were performed at least in triplicate to estimate the mean and standard deviation of each peak area.  www.nature.com/scientificreports www.nature.com/scientificreports/ In vitro drug release kinetics. The melatonin release kinetics from PolyRad is shown in Fig. 8a before and after radiation. A total of 70% melatonin was released from non-radiated nanoparticles after 72 h at pH 7.4. Notably, more than 50% of the drug was released after only 2 h, suggesting a spontaneous rapid degradation of solid nanoparticles under physiological conditions. Exposure to radiation elicited higher drug release, reaching >90% drug release in 72 h. The drug release profile follows a biphasic pattern, indicating a controlled drug release mechanism. The data from in vitro experiments were fitted to a power-law model to investigate the kinetics of melatonin release (Fig. 8b and Table 3). The cumulative release is linear with respect to time, t ( > . Cytotoxicity assay. The effects of melatonin solution, PolyRad without melatonin and PolyRad (with melatonin) on the viability of HUVEC were analyzed using MTT assay and live/dead assay imaging (Fig. 9). As the concentration of melatonin in PolyRad (♦) increases, cell viability decreases from 75% to ~60% at melatonin concentrations ranging from 0.01 to 10 mg/ml. Free melatonin (■) showed ~90% cell viability at 0.01 mg/ml which dropped to ~65% at 10 mg/ml. Independent radiation treatments using gamma (×) and UV irradiation (+) after  Table 3. Kinetic modeling of melatonin release from PolyRad nanoparticles. www.nature.com/scientificreports www.nature.com/scientificreports/ H 2 O 2 exposure showed similar effects on cell viability as non-radiated PolyRad, indicating radiation did not affect the release of melatonin from the nanoparticles. Melatonin free PolyRad (•) had no significant toxicity at 0.01 and 0.1 mg/ml, the cell viability dropped to ~65% at 10 mg/ml. Results show an increased melatonin cytotoxicity in the order of free melatonin, non-irradiated PolyRad and irradiated PolyRad. We further studied if melatonin and PolyRad interfered with the cell viability of HUVEC. As shown in Fig. 9b, live cells after treated with 0.01 mg/ ml each of melatonin and PolyRad fluoresce green as stained with calcein AM. Cells show a slight cytotoxicity as assessed by dead cell staining (red fluorescence) using EthD-1.

Discussion
Melatonin is an endogenous neurohormone that regulates many biological functions such as sleep, circadian rhythm, immune responses and cardiac functions, among others [63][64][65][66] . Changes of environment in spaceflight compared to that on Earth disrupt sleep and circadian clock in astronauts' physiology decreasing the performance 1,67,68 . Sleep medications such as melatonin are used to improve astronauts' circadian rhythms and sleep 4 . Space medication is a challenge because of the instability of active pharmaceutical ingredients (API) in the presence of harsh spaceflight environmental conditions. Altered physical and chemical stability in pharmaceuticals can result in reduced potency which can result in reduced efficacy. It has been tested that the API of melatonin degrades in the adverse environments of space station 4,60 . The traditional approach to overcome the limitation is to replace medicines from the International Space Station (ISS) before their expiration. However, this is not a practical solution for longer duration missions to planets (e.g., Mars). Hence, it is desired that medicines maintain their shelf-life throughout the duration of the space mission. Contrary to standard dispensing practices on Earth, pharmaceuticals are packed and dispensed in special flight-certified containers and stored inside compactly packed kits. The packaging could affect the active pharmaceutical ingredients and therapeutic efficacy of medications in space 60,69 . The shielding material should meet the requirement of application in radiotherapy, should be light-weight, flexible, environmental friendly and easily recyclable. According to these requirements, there is a need to provide effective shielding against space medications with materials of appropriate designs that can maintain the stability of space medicines.
Our results show that antioxidant conjugated polymeric nanoparticles (PLGA-PLL-Trolox) can act as an effective and protective shield for pharmaceuticals that are being exposed to high energy radiations in space. Trolox antioxidant effectively acts as a radiation-induced highly reactive OH• scavenger that protects the pharmaceuticals prolongating the shelf-life and thus the potency. Trolox can scavenge free radicals through a variety of reaction mechanisms, as it is the case for many other scavengers 62 . Four reaction mechanisms have been reported to significantly contribute to the OH• scavenging capacity of Trolox in aqueous solution: hydrogen transfer (HT), radical adduct formation (RAF), single electron transfer (SET) and sequential proton loss electron transfer (SPLET) 62 . The free radical scavenging capacity of Trolox has been shown to reduce oxidative stress in aqueous systems as well as epithelial cells, hepatic injury and red blood cells in vitro and in vivo [70][71][72][73][74] . Fluorescein, organic compound used in this assay, has characteristic of high photostability, stability at physiological pH and is highly sensitive to OH• attacks. Accordingly, Trolox protected fluorescein from OH• attacks by scavenging the radicals, prolonging the relative fluorescein intensity. Figure 5 shows that conjugation of Trolox to the surface of drug-loaded PolyRad leads to successful shielding of encapsulated pharmaceuticals from the damaging effects of photochemical degradation. When PolyRads were photochemically degraded using UV light and H 2 O 2 , part of their covalent bonds dissociated which in turn protected the active structure of melatonin (Fig. 6c). AFMK and 6-hydroxymelatonin appear to be the two main degradants of pure melatonin which is blocked in PolyRad formulations. The antiradical activity of PolyRad was further detected using gamma radiation and analyzed via 1 H NMR showing>98% protection of active melatonin structure in nanoparticles (Fig. 7a,b).
In vitro drug release follows a biphasic kinetics pattern. The in vitro release for non-irradiated PolyRad showed a burst release of ~20% of the encapsulated melatonin (Fig. 8a). The initial burst release decreased after photochemical degradation. The polymer coating began to disintegrate, releasing ~65% and 75% of total melatonin from non-radiated and irradiated PolyRad, respectively after 24 h and ~70% and 85% from non-radiated and irradiated PolyRad, respectively after 72 h. The exponent, n, characteristic of the overall mechanism of drug release increased after photochemical degradation (Fig. 8b). The values of the exponent of time, n, indicating that the release of melatonin (high diffusivity) is at least partially controlled by the scission of the polymer. The in vitro data provide guidance for understanding the fundamentals of melatonin drug release. In vitro cell culture studies indicate that melatonin and PolyRad induce cytotoxicity to HUVEC in a dose-dependent manner (Fig. 9a) 75,76 . Generation of reactive oxygen species has been reported to promote apoptosis in HUVEC while maintaining normal redox homeostasis 77,78 . It is possible that PolyRad increases ROS production suggesting higher cell death at concentrations above 0.01 mg/ml (10 μg/ml). The ability of PolyRad to release melatonin was not affected after irradiation. PolyRad is as potent as free melatonin in eliciting therapeutic effects in HUVEC growth (Fig. 9b).
conclusion Here, we have described a new technology for protection of pharmaceuticals against radiation induced damages (i.e., free radical exposure), which is likely to occur during space missions. We synthesized PolyRad nanoparticles, 325 ± 21 nm in hydrodynamic diameter, using the emulsion solvent evaporation method by encapsulating melatonin within the nanoparticles to protect APIs from radiation damage. Conjugation of Trolox to the surface of PLGA nanoparticles significantly inhibited the photochemical degradation of active melatonin. Nonetheless, the current results are promising and continue to shed light on the inherent benefits of Trolox as an antioxidant that acts as a free radical scavenger to shield spaceflight pharmaceuticals from the deleterious effects of incident radiation and helps maintain their shelf-life and potency. PolyRad is demonstrated to be particularly useful for shielding space pharmaceuticals from the toxic effects of space radiation as induced by free radicals with the conjugation of antioxidants on the surface of the nanoparticles. Results from this research can be further extended to