Oral nanotherapeutics: Redox nanoparticles attenuate ultraviolet B radiation-induced skin inflammatory disorders in Kud:Hr- hairless mice

Accepted Manuscript Oral nanotherapeutics: Redox nanoparticles attenuate ultraviolet B radiation-induced skin inflammatory disorders in Kud:Hr- hairless mice Chitho P. Feliciano, Yukio Nagasaki PII:

S0142-9612(17)30468-4

DOI:

10.1016/j.biomaterials.2017.07.015

Reference:

JBMT 18178

To appear in:

Biomaterials

Received Date: 9 June 2017 Revised Date:

20 June 2017

Accepted Date: 9 July 2017

Please cite this article as: Feliciano CP, Nagasaki Y, Oral nanotherapeutics: Redox nanoparticles attenuate ultraviolet B radiation-induced skin inflammatory disorders in Kud:Hr- hairless mice, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Oral Nanotherapeutics: Redox Nanoparticles Attenuate Ultraviolet B RadiationInduced Skin Inflammatory Disorders in Kud:Hr- Hairless Mice

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Chitho P. Feliciano1, 2 and Yukio Nagasaki2, 3, 4, *

Department of Materials Science, Graduate School of Pure and Applied Sciences,

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University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8573, Japan; 2Radiation

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Biology Research Group, Biomedical Research Section, Atomic Research Division, Philippine Nuclear Research Institute, Department of Science and Technology (PNRIDOST), Commonwealth Avenue, Diliman, Quezon City, Philippines; 3Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, Tennoudai 1-1-

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1, Tsukuba, Ibaraki, 305-8573, Japan; 4Satellite Laboratory, International Center of Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science

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(NIMS), University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8573, Japan.

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*Correspondence: [email protected] (Y. Nagasaki)

Abstract

The active participation of an anti-inflammatory drug in the biological pathways

of inflammation is crucial for the achievement of beneficial and therapeutic effects. This study demonstrated the development of redox nanoparticles that can circulate in the blood at significantly high levels, thus increasing their efficacy as an oral treatment

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against the deleterious effects of reactive oxygen species (ROS) in an in vivo inflammatory skin model. To confirm the blood bioavailability of the nanoparticles, mice were injected with the nanoparticles suspension (RNPN) via oral gavage. Using

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electron spin resonance and radioactive labeling techniques, the blood circulation of the redox polymer that forms the nanoparticles was confirmed 24 h after oral administration. This contrasted with its low molecular weight counterpart (NH2-

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TEMPO), which peaked 15 min post injection and was found to be cleared rapidly

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within minutes after the peak. We then tested its efficacy in the inflammatory skin model. Kud:Hr- hairless mice were irradiated with UVB (302 nm) to induce skin damage and inflammation. Throughout the entire period of UVB irradiation, RNPN was administered to mice by free drinking. NH2-TEMPO was used as the control. The results

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showed that oral supplementation of RNPN significantly improved the therapeutic effects of the core nitroxide radical compared with its low molecular weight counterpart. Furthermore, RNPN significantly reduced UVB-induced skin aging,

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epidermal thickening, edema, erythema, skin lesions, and various pathological skin inflammatory disorders in vivo. From the obtained data, we concluded that the use of

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long-circulating redox nanoparticles (RNPN) provided an effective treatment against the damaging effects of excessive ROS in the body.

Keywords: redox polymer, redox nanoparticles, drug bioavailability, ultraviolet radiation, skin inflammation, oral nanomedicines, oral drug delivery

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1. Introduction Inflammation is attributed to many pathological processes in the body. Clinical

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and experimental results have suggested that inflammation plays a crucial role in the progression of various diseases. Acute and chronic inflammation can be induced by ultraviolet (UV) rays from the sun. Much of the mutagenic and carcinogenic actions of

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sunlight have been attributed to the UVB portion of the solar spectrum. Prolonged and

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frequent exposure to UVB radiation may cause inflammation of the skin, which is characterized by edema and erythema [Matsumura and Ananthaswamy, 2002; Hsu et al., 2015; Wu et al., 2015]. Additionally, the damaging effects of UVB exposure accelerate skin aging. Reactive oxygen species (ROS) generated by UVB exposure play a

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substantial role in these inflammatory responses. UVB is considered as a mutagen and cytotoxic to the skin cells that causes its damages and aging [Hsu et al., 2015; Hu et al., 2016]. To prevent ROS-induced inflammation, versatile antioxidants such as vitamins C

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and E, synthetic compounds, and phytochemicals have been investigated. However, these low molecular weight (LMW) antioxidants internalize into healthy cells, which

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results in the dysfunction of important redox reactions, such as the electron transport chain [Vong et al., 2016]. To prevent such undesired adverse effects, a novel strategy must be developed. In addition to the prevention of the adverse side effects of LMW compounds, antioxidants with a long-term effect are preferable to prevent the sunlight-induced skin damage, aging, and inflammation that occur because of excessive exposure to ultraviolet radiation. To meet this objective, we have designed

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and developed self-assembling polymer antioxidant nanotherapeutics. One of the most important aspects of this synthesis is the covalent conjugation of the nitroxide radical compounds, which have the capacity to effectively eliminate ROS, into the

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hydrophobic segment of the amphiphilic block copolymers. The nitroxide radicalcontaining polymer spontaneously forms a polymeric micelle in aqueous media, named redox nanoparticles (RNPN), which has anti-inflammatory function that

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attenuates the damaging effects of UVB radiation. RNPN were synthesized following

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the previously described methods [Yoshitomi et al., 2009, 2011, 2014]. Because the diameter of RNPN was approximately 20–40 nm, as measured by dynamic light scattering (DLS) technique, the solution was completely transparent, and the mice could drink an amount equal to that of tap water when given free access to it. Kud:Hr-

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hairless mice were irradiated with UVB (302 nm) to induce skin damage and inflammation. Throughout the entire period of UVB irradiation, RNPN were administered to mice via free access to drinking, and LMW-nitroxide NH2-TEMPO was

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administered as control. The results showed that oral supplementation of RNPN significantly improved the therapeutic effects of the core nitroxide radical compared

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with LMW nitroxide radicals. RNPN significantly reduced UV-induced skin aging, edema, erythema, lesions, and various pathological skin inflammatory disorders in vivo. These results suggested that oral supplementation of RNPN provided an effective systemic approach for the protection of skin against the harmful effects of ultraviolet radiation.

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2.1. Design, synthesis, and characterization of RNPN

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2. Materials and methods

The design, synthesis, and characterization of RNPN can be found in our

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previously published paper (Feliciano et al., 2017). Here, we used the same RNPN

previously.

2.2. Animals and treatment groups

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which was produced in the same production batch that we have used and analyzed

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Four-week-old female Kud:Hr- hairless mice were obtained from Kyudo Co. Ltd, Japan, quarantined for microbial inspection for 3 weeks, and earmarked and acclimatized for an additional week in the caging facility of the University of Tsukuba.

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All mice were maintained under a controlled environment (12-h light/dark cycle) in special enclosed cages supplied with clean air. The mice received a sterilized rodent

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diet and drinking water to prevent opportunistic microbial infections. All the animal experiments were performed following animal protocols approved by the Animal Ethics Committee of the University of Tsukuba (Animal Plan Number: 15-328). The mice were randomly divided into four groups (n = 5–6 mice/group): control; no irradiation; UVB-irradiation; UVB-irradiation + NH2-TEMPO; and UVB-irradiation + RNPN.

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2.3. Dose mapping of the UVB irradiator Mice irradiation was performed using a handheld UV lamp (UVM-57; 6 W, 302 nm, 0.20 A, 100 V, 50–60 Hz; UVP, LLC, Upland, CA, USA). The UV intensity was

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measured routinely using a UV meter (UVX-31 radiometer; UVP, LLC, Upland, CA, USA). To assure the uniformity of the UVB irradiation, the intensity of UV radiation in the chamber was mapped. Briefly, the irradiation platform was raised to 13.5 cm from the

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bottom of the chamber. The radiometer (2.2 cm high) was set at 20 mW/cm2 and

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placed on top of the platform. The distance of the UV lamp from the surface of the radiometer was approximately 6.5 cm. UVX readings were recorded in triplicate after stabilization (a minimum of 1 min) at different specified points in the chamber. The irradiation dose was calculated following the standard formula: Dose (mJ/cm2) =

shown in Figure 1.

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exposure time (seconds) x UV intensity (mW/cm2). The dose mapping results are

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2.4. RNPN oral administration and UVB irradiation of Kud:Hr- hairless mice For the first ten days, 0.5 mg/ml of RNPN in sterile water were administered via

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free-drinking one day before the start of UVB irradiation. Subsequently, the concentration of RNP was increased to 1 mg/ml on day 11 and was continuously provided throughout the UVB treatment period. The total amount of water and RNP intake per mouse were calculated accordingly (~10 mL per mouse per day). An equivalent concentration of the low molecular weight nitroxide radicals 4-aminoTEMPO (NH2-TEMPO) was administered as the control.

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Mice irradiation was conducted following a previously published protocol (Goto et al., 2011) with some modifications. Mice were irradiated at the center of the irradiation chamber where the dose intensity was found to be uniform: ~1.59 mW/cm2

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at ~6.5 cm distance from the source. Kud:Hr- hairless mice were placed in a mice holder with a wire mesh cover. The mice heads were covered to provide protection from UVB exposure. The UV lamp was situated 6.5 cm above the dorsal skin of the

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mice. Then, the mice were exposed with 120 mJ/cm2 (2 times of the MED, minimum

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edematous dose) three times for the first week. For the four succeeding weeks (2nd to 5th), mice were irradiated only once per week using the same UVB dose (2-MED). The final irradiation was performed at the end of the 5-week treatment period, 24 h before sacrificing the mice.

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Mice skin were evaluated and scored before they were sacrificed. The total accumulated UVB dosage was 0.96 J/cm2 per mouse. While the total RNPN intake was 330 mg per mouse over the 5-week period or approximately 10 mg per day. A

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graphical illustration of the drug administration and UVB irradiation treatments are

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shown in Figure 1.

2.5. Skin evaluation

The skin condition of all mice was evaluated at the end of the treatment period.

The mice were anesthetized with isoflurane and the dorsal skin was examined and photographed. UVB-induced skin aging and wrinkling were assessed as previously described [Kong et al., 2015]. The number of skin lesions was also counted. Dorsal skin

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and ear erythema were scored by using previously described guidelines [OECD/OCDE 442B, 2016]. Skin edema, in terms of skin-fold and ear thickness, was determined using

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a digital Vernier caliper.

2.6. Histological examination

After macroscopic grading and evaluation, the dorsal skin of the mice was

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harvested. Skin samples were placed in 10% neutral-buffered formalin for 1–2 days,

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embedded in paraffin, and cut into 5-μm thick sections. The sections were then stained with hematoxylin and eosin (H&E), examined, and photographed under a bright field microscope (Keyence BZ-X700).

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2.7. Blood bioavailability of RNPN after oral injection using ESR method The blood bioavailability of RNPN (300 mg/kg of the redox polymer) was determined after oral injection into 8-week-old female ICR (IGS) mice (Charles River,

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Japan). Three hundred milligrams (300 mg) of RNPN contains 90 mg of NH2-TEMPO. While the known maximum tolerated dose (MTD) of NH2-TEMPO is 250 mg/kg (Hahn

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et al., 1998). Thus, 300 mg/kg of RNPN which is equivalent to 90 mg/kg of NH2-TEMPO is far less than half of the MTD. For safety reasons, we selected this dose. The test drugs were prepared in sterile physiological saline and administered by oral gavage to the mice. An equal volume (250 μL) of the suspending solution was administered for both drugs. At different time points (0, 0.25, 3, and 24 h) after drug administration, the mice were anesthetized using isoflurane inhalation, dissected, and blood was collected

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via cardiac puncture of the right ventricle using a heparinized needle. The blood samples were subsequently centrifuged at 3,000 × g for 10 min at room temperature (25°C). to separate the plasma. As a certain percentage of nitroxide radicals are

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reduced to hydroxylamine, which shows no ESR signal, by endogenous antioxidants in vivo, potassium ferricyanide (K3[Fe(CN)6]) was used to re-oxidize the hydroxylamines into nitroxide radicals to obtain the approximate total drug concentration (nitroxide

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radicals + hydroxylamines) in the plasma [Yoshitomi et al., 2013]. In brief, 10 μL 200

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mM K3[Fe(CN)6] was added to 90 μL plasma, mixed by pipette, and the signal intensities were measured immediately (< 30 min after re-oxidation) using an X-band ESR spectrometer (Bruker Bio-Spin, EMXPlus 9.5/2.7) with the following settings: frequency, 9.849143 GHz; center field, 3,500 G; sweep width, 100 G; receiver gain, 1 ×

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104; power, 0.2 mW; modulation frequency, 100 kHz; modulation amplitude, 4 G; time constant, 20.48 ms; and conversion time, 40 ms. Standard dilutions of RNPN and NH2TEMPO were prepared using plasma obtained from untreated mice and the ESR signal

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intensities were measured. To determine the amount of the test drugs in the blood at time 0, 17.3 μL RNPN (24 mg/mL) and NH2-TEMPO (7.2 mg/mL) were mixed with 100 μL

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whole blood, as the total blood volume of a mouse is approximately 72.25 mL/kg (1.445 mL/20 g mouse) [Diehl et al., 2001; Joslin, 2009; Raabe et al., 2011]. The drugspiked blood was centrifuged for 10 min at 3,000 x g to separate the plasma, serial dilutions were prepared using plasma as the diluent, and the ESR signal intensities were measured in the same manner as the other samples. The amount of RNPN and NH2-TEMPO in the plasma was expressed as a percentage of the injected dose (% ID).

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2.8. Blood bioavailability and biodistribution of radiolabeled RNPN after oral injection Next, the biodistribution of RNPN (and or redox polymer disintegrated from

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RNPN) and its blood bioavailability was verified using a radioactive labeling technique. RNPN were radioactively labeled with I125 using a previously described chloramine T method [Chonpathompikunlert et al., 2015], and 150 μL (equivalent to 77.4 kBq) was

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injected into mice via oral gavage (n = 3). Free radioactive iodine (I125, 139 kBq) was

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used as the control. The injected mice were sacrificed 24 h after drug administration. Whole blood and organs were collected and immediately measured for radioactivity. To determine the total radioactivity in each sample, 500 μL whole blood and whole organs were put in scintillation vials immediately after harvest and the total

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radioactivity in counts per minute (CPM) was measured using an Aloka GammaCounter. The amount of radiolabeled RNPN and I125 in the whole blood and organs was

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expressed as percentage of the injected dose (% ID).

2.9. Statistical analysis

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All data are presented as the mean ± SD. The results were analyzed for

statistically significant differences. For pairwise comparisons, a two-tailed t-test was used. For three or more treatments, a one-way analysis of variance (ANOVA) with Tukey-Kramer’s post-test was used to evaluate the differences between all the compared pairs.

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3. Results

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3.1. Skin evaluation

At the end of the drug and UVB treatment period, the skin condition of all mice was evaluated. UVB irradiation induced significant skin photoaging in mice (Fig. 2).

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However, treatment with RNPN significantly reduced the photoaging of mice (Fig. 2).

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Moreover, closer examination revealed that the skin of the UVB-irradiated control and the mice administered NH2-TEMPO exhibited observable skin lesions, large wrinkles, and dryness (Fig. 2). Clinical grading of the extent of erythema (skin redness due to inflammation) and edema (swelling) demonstrated that oral administration of RNPN

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significantly attenuated these skin disorders in vivo (Fig. 2-3).

3.2. Histological examination

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Figures 4-5 show the microscopic evaluation of the dorsal skin collected from the mice, which revealed a significant increase in UVB-induced skin inflammatory

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disorders, such as epidermal thickening, parakeratosis, hyperpigmentation, and lymphocyte infiltration. Closer examination of the histological samples revealed an abnormal increase in the epidermal cell number (hyperplasia), cell size (hypertrophy), and thickness of the stratum granulosum (hypergranulosis). These skin inflammatory disorders and abnormalities were significantly reduced when the mice were orally administered with RNPN.

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3.3. Blood bioavailability and biodistribution of RNPN after oral administration The blood circulation of the redox polymer RNPN was first determined using an

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ESR technique to quantify the approximate amount of the active nitroxide segment. The results showed the absorption of the redox polymer into the bloodstream after oral administration and confirmed the long-term blood circulation of RNPN for up to 24

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h after administration, which was a similar result to our previous observations

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[Chonpathompikunlert et al., 2015]. Conversely, the LMW NH2-TEMPO reached a peak concentration in the blood at only 15 min after oral gavage and failed to retain its high concentration in the blood flow 24 h later (Fig. 6). As we previously reported, RNPN disintegrated in an acidic environment [Yoshitomi et al., 2009; Marushima et al., 2011]

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and the liberated polymers were absorbed into the blood stream via mesentery. The adsorbed redox polymers interacted with serum proteins to improve the length of blood circulation. The slow internalization of the redox polymers in the blood stream

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owing to their high molecular weight may also contribute to their extended blood circulation time. As the LMW NH2-TEMPO was covalently conjugated to the polymer, it

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was hardly internalized in healthy cells, which maintains the normal redox reactions in the cells and thereby prevents any adverse side effects in the body [Vong et al., 2016]. The observed superior biocompatibility and long blood circulation time of RNPN

prompted us to examine the biodistribution using a more sensitive radioactive technique. The biodistribution was determined 24 h after oral gavage of the radiolabeled-RNPN in different organs and tissues using free radioactive iodine (I125) as

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the control. The results obtained revealed that the radiolabeled-RNPN circulated for significantly longer than the control LMW free radioactive iodine (Fig. 7), which confirmed the long-term blood bioavailability observed in the results obtained using

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the ESR method. Compared with I125, the levels of radiolabeled-RNPN were found to be higher in the lungs, although the difference was not statistically significant. In addition to the high concentration of RNPN in the blood, it was interesting to note that

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significant levels of the nanoparticles were localized in the skin, but very low

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accumulation was observed of the LMW radioactive iodine in this area. These results explained the efficacy of RNPN in the attenuation of the deleterious effects of excessive

4. Discussions

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UVB exposure on the skin.

Reactive oxygen species (ROS) induced by ultraviolet radiation in the skin are

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strongly involved in the development of skin damages, aging, and cancer progression. This leads to the development of many commercial sunscreen for skin protection.

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Most of commercially available sunscreens used for topical applications contain organic or inorganic ultraviolet (UV) filters. Despite its protecting function against the UV radiations, exposure of cells to UV filter pose some health effects. In fact, studies have been done to evaluate the possible adverse effects of UV filters to developing organs of fetuses and children and other potential endocrine disrupting properties [Krause et al., 2012]. This prompted the development of encapsulated UV filters to

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prevent the direct cellular exposure to UV filters when applied topically [Deng et al., 2015]. Also, many researchers also examined the possible use of natural antioxidants against UV radiation, both for topical and oral applications [Darvin et al., 2011; Korac

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and Khambholja, 2011; Perez-Sanchez et al., 2014; Sirerol et al., 2015; Hu et al., 2017]. However, the use of low molecular weight (LMW) antioxidants was determined to be not enough for skin protection. When used topically, LMW antioxidants are rapidly

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washed-out of the skin or remove due to desquamation, contact with clothing, and

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other environmental stress [Darvin et al., 2011]. Alternative route of antioxidant treatment is by oral administration, but due to its non-specific absorption and size, LMW antioxidants are rapidly cleared from the body, resulting to its low therapeutic effects. Other studies suggested the use of multiple or combinations of different LMW

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antioxidants with the hope of increasing its therapeutic efficacy [Korac and Khambholja, 2011]. But increasing the dose of an antioxidant is not logical for oral administration, because this may result to overdosing and may also bring adverse

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effects. Thus, alternative treatment strategies are sought. It has been shown that nitroxides compounds including NH2-TEMPO are sensitive scavenger of UV-generated

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free radicals and ROS in the skin [Herrling et al., 2003]. However, its poor bioavailability limits its therapeutic effects. Here, we demonstrated the use of oral nanotherapeutics as an alternative treatment for the safe and effective prevention of UV radiation-induced skin damages and inflammation. The drug was given by oral administration since it has a pH-sensitivity, this allows its gradual disintegration in the stomach due to its acidic condition. After its disintegration, this is then followed by its

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subsequent absorption into the bloodstream. Compared with topical application of sunscreens, oral administration of the redox nanoparticles makes it available in the critical areas of the skin (e.g. epidermis) due to its improved blood circulation, allowing

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its direct interaction with the UV-induced ROS for its effective scavenging and antiinflammatory effects (Fig. 8). At first, we wanted to reduce the possible adverse effects of low molecular weight antioxidants. However, we observed using an ESR technique

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the RNPN’s long blood circulation and prolonged retention in the skin area a result we

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confirmed by radioactive labelling. These observations lead us to conclude that the RNPN’s improved pharmacokinetics solved the poor bioavailability of LMW antioxidant

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NH2-TEMPO.

4. Summary and conclusions

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The unique characteristics of RNPN allow the gradual absorption of the redox

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polymer into the bloodstream after oral administration. Their blood circulation time was significantly longer compared with the LMW NH2-TEMPO, which experienced rapid absorption, oxidation, and clearance after administration. The blood levels of the redox polymer and control nitroxide were monitored for 24 h after administration by oral gavage in mice. The results revealed the long-term blood bioavailability of the redox polymer that contained the RNPN, even at 24 h post injection, in contrast to the LMW NH2-TEMPO. Moreover, a significant amount of the radiolabeled redox polymer

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was found in the skin samples of the treated mice even after 24 h of drug administration. The results obtained in this study clearly showed that the oral administration of RNPN significantly reduced the ROS-associated skin aging, epidermal

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thickening edema, erythema, skin lesions, and various pathological skin inflammatory disorders in vivo. These results proved that the long blood bioavailability of orally administered RNPN provided an effective systemic approach for the suppression of UVB

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radiation-induced skin inflammatory disorders. Therefore, we concluded that the use

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of redox nanoparticles (RNPN) can provide an effective anti-aging strategy for the protection of the skin against ROS and the reduction of the skin-damaging effects of

Acknowledgements

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prolonged UV exposure.

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This work was supported by a Grant-in-Aid for Scientific Research S (25220203) and the World Premier International Research Center Initiative (WPI Initiative) on

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Materials Nanoarchitectonics from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Mr. Chitho Feliciano would like to express his sincere gratitude to the Philippine Nuclear Research Institute, Department of Science and Technology (PNRI-DOST) of the Republic of the Philippines and to the Japanese Government for the Ph.D. scholarship he received under the MEXT (Monbukagakusho) Scholarship Program.

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Author contributions

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C. Feliciano conceived, designed, and performed all the experiments, analysed, summarized and interpreted the data, and wrote the paper. Y. Nagasaki. conceptualized the design and synthesis of the nanoparticles, edited the paper, and

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supervised the whole project.

Conflicts of interest

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None

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26. Yoshitomi T., Nagasaki Y. 2014. Reactive oxygen species-scavenging nanomedicines for the treatment of oxidative stress injuries. Advance Healthcare Materials, 3, 1149-1161.

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low pH circumstances. Bioconjugate Chemistry, 20 (9): 1792-1798 (2009).

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of low pH circumstance. Bioconjugate Chem, 20, 1792-1798.

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Figure 1. Oral administration of RNP via free-drinking and ultraviolet B (UVB) irradiation of Kud:Hr- hairless mice. a, Dose map of the UVB irradiation chamber. Mice irradiation was performed using a handheld UV lamp (UVM-57, 6 watt/302 nm/ 0.20 Amps/ 100 V/ 50-60 Hz from UVP, LLC, Upland, CA, USA). The UV intensity was measured routinely using UV meter (UVX-31 radiometer from UVP, LLC, Upland, CA, USA). The irradiation platform was raised to 13.5 cm from the bottom of the chamber. Then the radiometer (2.2 cm thickness in height) was 2 set at 20 mW per cm and placed on top of the platform. The distance of the UV lamp from the surface of the radiometer was approximately 6.5 cm. UVX readings (triplicate) were recorded

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after stabilization (at least 1 min) at different specified points in the chamber. The irradiation 2 dose was calculated following the standard formula: Dose (mJ/cm ) = exposure time (seconds) 2 x UV-intensity (mW/cm ). Mice irradiation was performed at the center of the chamber where 2 the dose intensity was found to be uniform: ~1.59 mW/cm at ~6.5 cm distance from the source. N N b, Oral administration of RNP and UVB irradiation of mice. RNP was given to mice one day before UVB (302 nm) irradiation. Mice were irradiated three times for the first week with 120 2 mJ/cm (2 times of the MED, minimum edematous dose) of UVB. For the four succeeding nd th weeks (2 to 5 ), mice were irradiated only once per week using the same UVB dose. The final irradiation was performed 24 h before the end of the 5-week treatment period. Mice skin were evaluated and scored before they were sacrificed. The total accumulated UVB dosage was 0.96 N J/cm2 per mouse. While the total accumulated RNP intake was 330 mg per mouse over the 5week period.

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Figure 2. RNP reduces UVB-induced skin photoaging in Kud:Hr- hairless mice. a, Photoaging grade. b, Skin appearances of representative mice from the treated groups. c, UVBinduced skin lesions in the dorsal area of the mice. d, UVB-induced dorsal skin erythema in mice. Values represent the mean ± SD (n = 3, control; 5-6, all other groups). One-way ANOVA # with post-test (Tukey-Kramer Multiple Comparisons Test) was used to compare the means. # ## indicates statistical difference as compared to the control group ( P<0.05, P<0.001). * indicates statistical difference as compared to the UV-B treated group (*P<0.05, **P<0.01, N ***P<0.001). NH2-TEMPO, 4-amino-TEMPO; RNP , nitroxide radical-containing redox nanoparticles; UVB, ultraviolet B.

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Figure 3. Edema characterized by (a) skin-fold and (b) ear thickness in Kud:Hr- hairless mice treated with UVB. Values represent the mean ± SD (n = 3, control; 5-6, all other groups). One-way ANOVA with post-test (Tukey-Kramer Multiple Comparisons Test) was used to compare the means. # indicates statistical difference as compared to the control group # ( P<0.05). *indicates statistical difference as compared to the UV-B treated group (*P<0.05). N NH2-TEMPO, 4-amino-TEMPO; RNP , nitroxide radical-containing redox nanoparticles; UVB, ultraviolet B.

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Figure 4. RNP reduces UVB-induced skin thickening in mice. a, Histological examination of the H&E stained dorsal skin samples taken from UVB-irradiated Kud:Hr- hairless mice (insert: magnified epidermis). b, Epidermal thickness. Values represent the mean ± SD (n = 3, control; 5-6, all other groups). One-way ANOVA with post-test (Tukey-Kramer Multiple Comparisons # Test) was used to compare the means. indicates statistical difference as compared to the control group (#P<0.05). *indicates statistical difference as compared to the UV-B treated group N (*P<0.05). NH2-TEMPO, 4-amino-TEMPO; RNP , nitroxide radical-containing redox nanoparticles; UVB, ultraviolet B. µm, micrometer.

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Figure 5. Histological examination of the H&E stained dorsal skin samples taken from UVB-irradiated Kud:Hr- hairless mice. Indications of skin inflammatory disorders are shown N (dash lined insert) NH2-TEMPO, 4-amino-TEMPO; RNP , nitroxide radical-containing redox nanoparticles; UVB, ultraviolet B. µm, micrometer.

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Figure 6. Blood bioavailability of RNP after oral injection into mice determined using N ESR method. 300 mg/kg of RNP was injected into 8-week old, female, ICR (IGS) mice N (Charles River, Japan), n = 3. NH2-TEMPO (90 mg/kg = Eq of NH2-TEMPO in the RNP ) was used as a control. Mice were sacrificed at different time points (0, 0.25, 3, and 24 h) after drug injection. The approximate total drug concentrations (nitroxide radicals + hydroxylamines) in the N plasma was measured using ESR method. The amount of RNP and NH2-TEMPO in the plasma were expressed as a percentage of the injected dose (% ID). ESR, electron spin N resonance; NH2-TEMPO, 4-amino-TEMPO; RNP , nitroxide radical-containing redox nanoparticles.

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Figure 7. Blood bioavailability and biodistribution of radiolabeled RNP after oral N injection. 150 µL of the radiolabeled RNP (Eq. to 77.4 kilo Becquerels, kBq) was injected into 125 mice via oral gavage (n = 3). Free radioactive iodine (I , 139 kBq) was used as control. The injected mice were sacrificed 24 h after drug administration. 500 uL of the whole blood and the whole organs were put in scintillation vials immediately after harvesting. The total radioactivity of each sample in counts per minute (CPM) was measured using Aloka Gamma-Counter. The N 125 amount of radiolabeled RNP and I in the whole blood and organs were expressed as the percentage of the injected dose (% ID). *Denotes significance difference (*P < 0.05, **P < 0.01).

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Figure 8. Design of oral nanoparticles, its delivery and absorption in the body including N the skin. a, Chemical structure of the pH-sensitive redox polymer, RNP . b, Graphical N illustration of the oral delivery of RNP and its absorption into the body and the skin area. The N unique characteristics of RNP allow the gradual absorption of the redox polymer into the bloodstream after oral administration, keeping its bioavailability high for its effective ROS scavenging effect.

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