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Enhancing Microcirculation on Multitriggering Manner Facilitates Angiogenesis and Collagen Deposition on Wound Healing by Photoreleased NO from Hemin-Derivatized Colloids Chia-Hao Su, Wei-Peng Li, Ling-Chuan Tsao, Liu-Chun Wang, Ya-Ping Hsu, Wen-Jyun Wang, Min-Chiao Liao, Chin-Lai Lee, and Chen-Sheng Yeh ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09417 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Enhancing

Microcirculation

on

Multitriggering

Manner Facilitates Angiogenesis and Collagen Deposition on Wound Healing by Photoreleased NO from Hemin-Derivatized Colloids Chia-Hao Su,§,† Wei-Peng Li,§,‡,∥ Ling-Chuan Tsao,§,‡ Liu-Chun Wang, ‡ Ya-Ping Hsu,‡,∥ WenJyun Wang,‡,∥ Min-Chiao Liao,† Chin-Lai Lee,† and Chen-Sheng Yeh,*,‡,∥,# †Institute

for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial

Hospital, Kaohsiung, 833, Taiwan. ‡Department

∥Center

of Chemistry, National Cheng Kung University, Tainan, 701, Taiwan.

of Applied Nanomedicine, National Cheng Kung University, Tainan, 701, Taiwan.

#Department

of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung,

807, Taiwan. §C.-H.

Su, W.-P. Li and L.-C. Tsao contributed equally.

*Correspondence author E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Deficiency of nitric oxide (NO) supply has been found to impair wound healing. Exogenous topical delivery of NO is a promising approach to enhance vasodilation and stimulate angiogenesis and collagen deposition. In this study, the CN groups on the surface of Prussian blue (PB) nanocubes were carefully reduced to -CH2-NH2 in order to conjugate with COOH group of hemin consisting of Fe-porphyrin structure with strong affinity toward NO. Accordingly, the NO gas was able to coordinate to hemin-modified PB nanocubes. The heminmodified PB carrying NO (PB-NO) can be responsible to NIR light (808 nm) exposure to induce thermo-induced liberation of NO based on the light-to-heat transformation property of PB nanocubes. The NO supply on the incisional wound sites can be readily topically dropped the colloidal solution of PB-NO for receiving NIR light irradiation. The enhanced blood flow was in a controllable manner whenever the wound sites containing PB-NO received NIR light irradiation. The promotion of blood perfusion following the on-demand multi-delivery of NO has effectively facilitated the process of wound closure to enhance angiogensis and collagen deposition. Keywords: prussian blue, hemin, nitric oxide, angiogenesis, wound healing


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It has been a burden on healthcare when the wounds are not healed in an orderly sequence leading to chronic wounds. Notably, the abnormal wound healing resulting in chronic wounds could be originated from the physiological defects. Underproduction of nitric oxide (NO) is considered as one of the physiological defects. For example, the reduced wound NO synthesis has caused the people with diabetes suffering from the impaired wound healing.1 The occurrence of the delayed wound closure was observed from the inducible NO synthase (iNOS) knockout mice.2 The animal studies indicated that angiogenesis was noticeably reduced in endothelial NO synthase (eNOS) knockout mice when compared to wild-type groups.3 The supply of NO thus has been suggested as the active approach to facilitate wound healing. NO is a pivotal regulation molecule to cause the positive effect for wound healing via multiple and different mechanisms, and to reveal the main benefits including inflammation, vasodilation, angiogenesis, cell proliferation and matrix deposition.4-6 For the inflammation phase of wound healing, NO has been shown to regulate the inflammation-associated cytokines, which include IL-8, transforming growth factor (TGF-β1), monocytes, and neutrophils, to further produce TNF-α and IL-1 on the wound causing a direct affect to inflammation condition.5 The NO gases diffuse into the vascular smooth muscle cells and then trigger the relaxation effect through the up-regulation of cyclic guanosine monophosphate (cGMP), leading to enhanced microcirculation around the wound sites. Vasodilation resulting from NO is an important process of wound healing, which enhances blood circulation to deliver nutrients to the lesions. For the proliferatiom phase of wound healing, NO can drive the expression of vascular endothelial growth factor (VEGF), bFGF, monocytes, substance P and TGF-β1, thereby stimulating angiogenesis.4,5,7 Inhibition of angiogenesis to the wound sites is the major reason to cause the development of chronic wounds. These NO-induced growth factors can also promote cells migration, adhesion and proliferation, that can enhance the growth of endothelial cells, fibroblast and keratinocytes to assist the repair of tissue and blood vessel.5 Regrading to the final remodeling stage, NO also plays a role in the stimulation of fibroblasts proliferation to increase collagen formation and deposition on the wound.5


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Delivery of exogenous NO topically on the wound sites has been found a readily and promising way to accelerate wound closure.6 To date, the topical application of NO has mainly used polymers as the substrates to incorporate with NO donors, e.g. organic nitrates, nitrites, diazeniumdiolates, nitrosothiols, or NO gas for wound healing. However, one concern has been brought up that the polymeric materials may cause the dermal toxicity and failure to wound healing. For example, linear polyethyleneimine (PEI) was found to experience the impaired wound healing because of its toxicity.8 Additionally, the formulated NO-conjugated polymers proceed NO delivery in the form of the sustained release. Unfortunately, the sustained release may also result in the increase inflammation because the presence of NO burst release at the beginning period causing the unfavorable treatments.8,9 Furthermore, the previous reports have evidenced the dramatic healing progression in the weekly NO application, which was similar to the daily application, indicating that a small amount of NO was sufficient for treatments.9-11 These studies clearly suggest that the continuous delivery of NO may not be necessary to facilitate wound healing. Therefore, considering the relatively short lifetime (6-50 s) in biological systems of NO and the burst release causing the toxicity at the site of application, the controlled release locally is an indispensable request in NO delivery. Although the nano-vehicles have been taken extensively to deliver NO in vitro and in vivo against bacteria and malignant tumors,12-14 there has lacked of the relevant design and study on wound healing based on NO-releasing nanostructures in a controllable mode. Herein, we have synthesized hemin derivatized Prussian blue (PB) nanocubes to topical delivery of NO on the incisional wounds upon near-infrared (NIR) light exposure. PB was chosen because of its biocompatibility that has been approved by FDA (USA) as antidote15 Another important consideration is based on the charge-transfer from Fe2+ to Fe3+ of the Fe3+-N≡C-Fe2+ composition in PB framework, developing NIR absorption that allows us using NIR light to initiate thermal process by the relaxation of the excited PB nanostructures. Previous reports in porphyrins have shown their characteristic optical and structural features to reveal the photobased applications including multimodal imaging, bio-sensing, drug delivery, radiotherapy and photodynamic therapy.16-19 In this study, hemin as one of the porphyrin’s derivatives has been elaborated as the attached sites to carry NO because of its large binding constant and high selectivity toward NO. To do that, the cyano (CN) groups on the surface of PB nanocubes were carefully reduced to -CH2-NH2, which can be further to form amide bonds with COOH group of


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hemin, using lithium aluminum hydride (LiAlH4) as reducing agent under ice bath condition. Through the thermo-induced NO liberation, the colloidal PB nanocubes were able to be topically triggered for multiple release of NO on wound sites, thereby enhancing blood circulation, promoting angiogenesis, and accelerating collagen deposition. RESULTS AND DISCUSSION Reduction of PB Surface from -CN to -CH2-NH2 Group to Conjugate with Hemin for Carrying NO. PB nanocubes were prepared, followed by the reduction of the surface group from -CN to -CH2-NH2 in order to proceed amide formation with hemin grafted on PB surface. The exposed hemin on PB has strong binding toward NO to form NO-carried PB (PB-NO) nanocubes. The PB-NO colloids were topically dropped on the incisional wounds to receive a NIR light exposure for NO release. Through a short period (10 min) and low intensity (0.5 W/cm2) of NIR irradiation, multiple NO-delivery has enhanced blood circulation, accelerated vessels formation, and increased collagen deposition (Scheme 1). Figure 1a taken from TEM reveals the cubic shapes with the edge of ~100 nm for the particles. Through a 36° tilting operation supported a cubic structure (Figure S1a,b). Following the mapping in elemental analysis identified the presence of Fe (Figure S1c). X-ray diffraction (XRD) characterization showed the standard PB diffraction peaks (Figure 1e), indicating the successful synthesis of PB nanocubes. The clear crystal planes of (200) and (220) and the single crystal feature can be seen from high resolution TEM and selected area electron diffraction analysis, respectively (Figure S1d,e). PB nanocubes have CN groups exposed on the surface. The LiAlH4 was used to reduce CN forming -CH2-NH2. Because LiAlH4 is a strong reduction reagent, this reduction reaction needed to be carried out carefully at low temperature under iced bath for a short period of 10 min. Under this circumstance, the NH2-functionalized PB (PB-NH2) can retain the size and morphology without aggregation (Figure 1b). The zeta potential measurements indicated the increase of the surface charge from -26 mV of PB to -17 mV of PB-NH2 because of the existence of NH2 on the surface (Figure 1f). When the effort was intended to complete surface reduction by taking a longer reaction time, the destruction of PB structure was seen concomitant of aggregation (Figure S2). The Fourier-transform infrared (FTIR) spectroscopy was conducted to characterize


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functional groups of PB-NH2. In addition to the peaks at 2060, 603, and 503 cm-1 associated with C≡N, Fe-C, and Fe-N, respectively,20 two signals at 1080 and 733 cm-1 respectively corresponding to C-N and N-H vibrations appeared,21 giving the evidence of -CH2-NH2 tethered on PB surface (Figure 1g, Figure S3). Therefore, the -NH2 can then be readily to conjugate with COOH through carbodiimide-mediated conjugation reaction. Notably, a vibration signal of C=O was seen around 1646 cm-1 due to the polyvinylpyrrolidone (PVP) on the surface of PB.22 PVP was employed in the preparation of PB nanocubes. The hemin molecule is an Fe-containing porphyrin composed of chlorine that can coordinate with NO at the axial position of the six-coordination hemin (Scheme 1). Fe-porphyrin has stronger binding toward NO compared to the binding energies with carbon monoxide (CO) and oxygen.23,24 A stronger bond between Fe-porphyrin and NO also suggests a greater stability of Fe-porphyrin carrying NO to avoid the unspecific release. Accordingly, the hemin with COOH exposed was taken to graft on PB-NH2 yielding hemin-modified PB (PB-Hemin). Figure 1c shows the TEM of PB-Hemin nanocubes. The FTIR spectrum of PB-Hemin revealed the signals of 1210 and 1300 cm-1 related to the vibrations of C-O,21 supporting the successful modification of hemin (Figure 1h). UV-vis spectra provide additional evidence of hemin conjugated on PB showing the appearance of 400 nm band which is the characteristic peak of hemin (Figure 1i). The amount of hemin was calculated to be 1.2 × 104/nanocube (Detailed calculation in Supporting Information). In addition, a strong broad band covering from 550 to 1000 nm is attributed to the charge-transfer band between Fe2+ and Fe+3 in PB. To carry NO on PB-Hemin, NO gas was purged into the bottle of the PB-Hemin colloidal solution for 1 min, and then the bottle was sealed to remain still for 6 h generating NO-carried PB-Hemin (PB-NO) nanocubes without the change of size and morphology (Figure 1d). FTIR characterized the N-O vibration at 1385 cm-1 and an enhanced broad band on 1580-1600 cm-1 overlapping with the C=O vibrations (Figure S4).21 NO Liberation Upon NIR Light Irradiation. Because of the strong NIR absorption band of PB, a low diode laser intensity of 0.5 W/cm2, following the class IIIb limitation of FDA and NIH (USA) when 808 nm used for clinical treatments, was used to examine photothermal effect as a function of nanocube concentration (Figure 2a). The temperature of the colloidal solutions elevated as particle dosage increase, while the water alone receiving 808 nm of light nearly


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unchanged in temperature. This temperature elevation performance has allowed us to choose a 100 ppm of PB-NO for study of NO-liberation upon NIR diode laser (808 nm) exposure at 0.5 W/cm2. The irradiation of 100 ppm colloids using 0.5 W/cm2 has created an elevation temperature of 21 oC that could be an appropriate condition to avoid burn and scald for skin because of overheating. Thermographic analysis revealed around 20 oC for the body temperature of mice in our animal studies. The binding of myoglobin-NO and heme-NO were found to be 22.8 and 21.6 kcal/mol, respectively.25 Additionally, our previous report in metal carbonyl conjugated on PB was able to demonstrate the thermal-induced CO liberation upon NIR light exposure.22 It has been known that the binding energy of Fe-CO from metal carbonyl is 31 kcal/mol.26 Thus, we speculate that NO-release process could follow a light-to-heat conversion mechanism mediated by PB-Hemin. A NO assay kit following the Griess test27 was used to quantify NO release when PB-NO received NIR light irradiation at 0.5 W/cm2 for 10 min. Figure 2b shows that NO coordinated PB-Hemin can be cleaved following NIR light illumination and the release amount of NO revealed a linear behavior as PB concentration increase. The release quantity of NO from 100 ppm of PB-NO was estimated as 0.22 μmole following 10 min exposure at 0.5 W/cm2. A parallel experiment was conducted using PB without the hemin modification to carry NO. Different from the NO-release increase as colloidal concentration in Figure 2b, a little absorbance of azo compound, a product from the Griess diazotization reaction, was seen and remained unchanged up to 500 ppm of colloidal concentration, indicating no NO-release from NO-loaded PB colloids. That is PB colloids cannot be purely used to adsorb NO (Figure S5). An important parallel examination was conducted to understand the stability of NO carried by PB-Hemin without laser irradiation. The PB-NO were respectively dispersed in H2O, serum, phosphate-buffered saline (PBS) at pH 5 and PBS at pH 7 for a week of observation. Figure 2c shows the good stability of NO on PB-Hemin that less than 20% of NO release was seen after 7 days at different storage conditions. These experiments present an appreciable stability compared to the reported NO delivery systems, which showed rapid NO-release behavior at room temperature.28,29 and also indicate that PB-NO can be triggered by NIR light to conduct a controllable NO-release fashion. Further to consider the structural stability, TEM measurements and the macroscopic observation of the colloidal solutions displayed no morphology and size change and no precipitation, which has given a blue appearance in the course of storage under different mediums (Figure S6). The


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spectral and physical (surface charge) stability of PB-NO in serum at 37°C was also monitored through UV-vis spectra (Figure S7) and dynamic light scattering (DLS) measurements (Figure S8), respectively. The absorbance at 808 nm for PB-NO showed no obvious change and no abnormal variation in surface charge was seen for 7 days. In Vitro Evaluation. Because the normal cell line has less tolerance to the stress when the materials possess certain degree of toxicity, MRC-5 cells, which are the normal fibroblast cell type, were tested for biocompatible studies. The cell viability was evaluated by (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following a 24-h of incubation with PB-NO colloids at different dosages (based on iron concentration). PB-NO exhibited great biosafety with cells survival rate even up to 90% when the dosage ranged between 50 and 200 ppm (Figure 2d). The controllable NO release upon NIR light exposure was then demonstrated for intracellular liberation. The nuclei were stained with Hoechst (blue fluorescence) and the NO was detected using a fluorescent sensor of DAF-2 DA (green fluorescence) (Figure 2e). The green fluorescence is generated based on the reaction of the cellpermeable NO sensor with NO to hydrolyze forming DAF-2 by intracellular esterase.30,31 For cells alone and cells treated with PB and PB-NO colloids (100 ppm) without NIR light, no discernible green fluorescence was seen (Figure 2e). In contrast, a strong green fluorescence clearly became evident for the PB-NO treated cells subjected to NIR light irradiation (10 min exposure at 0.5 W/cm2), suggesting a reliable PB-Hemin carrier to achieve NIR light-induced intercellular NO release. The results of cell viability showed no sign of cell damage after treatments with colloids (PB and PB-NO) with laser exposure (Figure S9). Topical Delivery of PB-NO on Incisional Wounds. For in vivo studies, a 2 cm (in length) wound was created on the back of the B6 mice. Firstly, we have employed the Doppler flowmetry to assess red cells velocity in the circulation of wound sites in the very early period (1-5 h). Five different groups of H2O alone (as control), H2O + Laser (10 min exposure at 0.5 W/cm2), PB colloids (100 ppm), PB colloids (100 ppm) + Laser, and PB-NO colloids (100 ppm) + Laser were topically delivered on open wounds. As seen in Figure 3a,b, the wounds treated with PB-NO + NIR light appeared greater blood flow. In fact, the large area for blood flow signal in the capillary around the wounds was seen. In particular, the post-treatment at 1 h has significant effect. Notably, the wound sites receiving laser alone did not cause any sign of


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enhancing blood flow. The higher blood perfusion in group of PB + Laser at pre-treatment was seen owing to the inborn difference of body factor in mice. Importantly, no enhanced signal in Doppler imaging was detected after laser exposure. Figure 3b corresponds to the quantification of blood perfusion of Figure 3a. To further investigate this NO-enhanced blood microcirculation, PB-NO + Laser was respectively repeated in 3 consecutive days on wounds, referred as PB-NO + Laser*3 (Figure 3c,d). The blood flow was then monitored by Doppler flowmetry at 1 h after NO delivery. Clearly, the microcirculation can be promoted following each treatment. Once again, the blood perfusion images displayed no observable change for laser exposure only. This demonstration importantly indicates the multi-delivery of PB-NO to achieve controllable promotion of microcirculation that might facilitate for the later wound closure process. The wound closure on mice was then conducted by topically dropping colloids on lesions. Eighteen mice were randomly divided into six groups of H2O alone (as control), Laser*3 (referred to H2O + laser for operation in 3 consecutive days), PB colloids, PB colloids + Laser, PB-NO colloids + Laser, and PB-NO colloids + Laser*3 (referred to PB-NO colloids + Laser for operation in 3 consecutive days) (Figure 3e,f). It turned out that the group of PB-NO + Laser*3 showed better would healing efficacy and revealed the statistical significance (Please see on 5th, 7th, and 9th days) compared to the control groups in the course of wound closure. The results from Laser*3, PB, PB + Laser and PB-NO + Laser exhibited the same behavior as that of H2O alone. Clearly, the administration of the single dose of PB-NO + Laser was not effective to cause discernible efficacy. The body weight of all mice were monitored in the period of 20 days showing no unusual change after each treatment (Figure S10). Although there was no cells damage from PB colloids-induced photothermal effect (Figure S9), the in vivo evaluation in photothermal effect was conducted. The treatments of PB colloids + Laser (without NO release with only PB colloids-induced photothermal effect) were performed in the infrared thermographic analysis (Figure S11). No temperature was elevated above 40 oC when PB colloids received NIR laser irradiation, indicating no photothermal effect. As the cells or tissues were exposed to electromagnetic radiation, the DNA could be damaged and results in rapid phosphorylation of H2A.X at Ser139 by PI3K-like kinases.32,33 H2A.X is required for DNA fragmentation during apoptosis and is phosphorylated by various kinases in response to apoptotic signals and the phosphorylated H2A.X at Ser139 by DNA-PK could indicate the cell death.34-36


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We have used phospho-histone H2A.X (Ser139) antibody to evaluate the DNA damage response following PB colloids + Laser. The H&E (Figure S12 a) and phospho-histone H2A.X staining (Figure S12 b) images of wound regions at pre-treatment, post 1h, and post 5h indicated that no obvious change was seen in lymphocyte or macrophage in H&E staining in the dermis regions. The phospho-histone H2A.X staining at pre-treatment, post 1h, and post 5h also did not exhibit signal change in epidermis and dermis areas even in 40x scale. Clearly, no photothermal effect in damage of DNA was observed when PB colloids received laser exposure. Immunohistochemical Analysis on Wounds. The tissues were collected at different periods and analyzed by the immunohistochemical stain of the wound regions for the different treatments of H2O alone (as control), Laser*3, PB colloids, and PB-NO colloids + Laser*3. The skin sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains for the observation of the collagen deposition at the connective tissues in neodermis. At early stages of the post-immediately and -5 h, no observable difference was seen from 4 different treatments (Figure S13). The extended analysis of tissues was exercised on postwound day 2, 3, 5, 9 and 15. The wounds began to enclose on day 2 after wounding and the thicken epidermis was format under the scabs on the margin of wounds on day 3 (Figure 4). The trichrome staining showed that the collagen (blue color) began to form in connection region on day 3 day after wounding. Particularly, the wound treated with PB-NO colloids + Laser*3 has gained more collagen deposition in connective tissues on day 5 and 9. The connective tissues were further magnified on day 5 and 9 (Figure 5a,c). The collagen appeared in blue color showing no statistical significance in the treatments of H2O (control), Laser*3, and PB colloids. On the other hand, the significant collagen formation and deposition was observed in the group of PB-NO colloids + Laser*3, giving 24% more on day 5 and 81% increase on day 9 compared to the control. The angiogenesis is also an indicator to reflect the efficacy of the treatments. The endothelial cells revealing as brown color were stained with CD31 to evaluate the angiogenesis in the course of wound healing (Figure 5b). It was found that the microvessels were increased on day 9 with 2.6 times greater relative to other groups (Figure 5d). The signal of endothelial cells did not show clear discrepancy on day 5 for all of treatments. Overall, these results indicate that the delivery of NO following the multiple triggering fashion effectively resulted in the


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acceleration of collagen deposition and enhanced endothelial cells proliferation leading to microvessel formation. Biocompatibility Evaluation for in Vivo. For the bio-secure studies, the blood analysis, the biodistribution study, and the haematoxylin and eosin (H&E) staining were performed immediately after treatment and on postwound day 7. H2O (control) and PB-NO colloids were dropped on the wounds. The liver and kidney function indices displayed within normal limits (Figure S14). In the results of biodistribution, no observable elevated iron in heart, liver, spleen, lung, kidney, and skin around the wound, suggesting a low systemic absorption of the PB through the wounds (Figure S15). No morphology change, visible damage, or inflammatory lesion was found in all organ tissues (Figure S16). These findings suggest the PB-NO nanocarriers with no acute toxicity for external administration. CONCLUSIONS We successfully reduced PB surface from -CN into -CH2-NH2 group to conjugate with hemin. Thus, NO can be readily coordinated to hemin-modified PB nanocubes. NO was quite stabilized on PB until exposure of NIR light. The NIR light irradiation of PB resulting in the thermo-induced NO liberation from PB-NO. Therefore, this PB-NO was able to behave NIR light-triggering NO-release on demand to avoid burst release as seen in many NO-releasing polymeric materials. The burst release can cause overdose effect resulting in an unnecessary inflammation on lesions. The PB-NO colloids can be just easily to topically drop on the wound sites to receive NIR light. The augmented blood microcirculation can be achieved in a controllable fashion following multiple administration of PB-NO colloids receiving NIR light on wounds, thereby effectively enhancing angiogensis and collagen deposition in the course of wound healing. Contrarily, single delivery is not sufficient to provide efficacy.

METHODS Preparation of PB Nanocubes. 6.0 g of polyvinylpyrrolidone (PVP) was dissolved in 60 mL 0.01M of HCl solution. The solution was stirred until the PVP powder completed dissolved. And then, 0.2 g of kaliumhexacyano ferrat(III) was added into the solution. The solution was heated


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at 80 ℃ for 1 h. The solution was collected, and then centrifuged at 14000 rpm for 5 min. The supernatants were removed. The precipitates were repeatedly washed for at least three times before re-dispersed into ethanol. Preparation of Amine-Functionalized PB Nanocubes (PB-NH2 Nanocubes). First, 1000 ppm of PB nanocubes was dissolved in 9 mL of solution containing 50% THF and 50% ethanol under an ice bath condition. 1 M of LiAH4 was diluted to 0.01 M of THF. Then, 10 mL of LiAH4 (0.01 M) solution was added into 9 mL of PB nanocubes-containing solution. The mixture was stirred at ice bath for 10 min. Finally, the solution was collected and centrifuged at 14,000 rpm for 5 min. The supernatants were removed. The precipitates were repeatedly washed for at least three times before re-dispersed into ethanol. Preparation of Hemin-Modified PB Nanocubes (PB-Hemin Nanocubes). 1000 ppm of PBNH2 nanocubes was dispersed in 8 mL of DMSO, and then the 0.5 mL of EDC (1.5 mM) and 0.5 mL of NHS (1.5 mM) were added into PB-NH2 nanocubes-containing solution to stir for 10 min. Then, the 1 mL of hemin (0.7 mM), dissolved in 50% DMSO, was added into mixture to react for 16 h. After the reaction, the solution was collected and centrifuged at 14,000 rpm for 5 min. The supernatants were removed. The precipitates were repeatedly washed for at least three times before re-dispersed into H2O. Preparation of NO-Carried PB Nanocubes (PB-NO Nanocubes). The NO gas was directly purged into the 10 mL solution containing 1000 ppm of PB-Hemin nanocubes for 1 min, and then the bottle was sealed to stand still for 6 h. After the NO-loaded process, the solution was collected and centrifuged at 14,000 rpm for 5 min. The supernatants were removed. The precipitates were repeatedly washed for at least three times before re-dispersed into H2O. NO Release from PB-NO Nanocubes Upon Laser Irradiation. The NO colorimetric assay kit is used to quantify the NO concentration in the solution. The solutions containing PB-NO nanocubes in 100, 200, and 500 ppm of iron concentrations were prepared. These solutions were irradiated with 808 nm laser at power density of 0.5 W/cm2 for 10 min. After laser exposure, the solutions were collected and centrifuged at 14,000 rpm for 10 min to obtain supernatants. And then, the supernatants were immediately mixed with Griess reagent, and were stood still for 1 h. The 540 nm absorbance of mixtures were measured by ELISA reader (Thermo Scientific


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Multiskan EX) to evaluate NO amounts in the supernatants. The sodium nitrite solutions in different concentrations as the standards were measured to obtain the calibration curve. All of the data were measured in triplicate. Heating Performance of PB Nanocubes Upon Laser Irradiation. 100 μL of PB nanocubes with different concentrations (0, 100, 200, and 500 ppm in iron concentration) were exposed to an 808 nm diode laser at 0.5 W/cm2 power density for 15 min. The change of temperature in solutions was recorded by digital thermometer (TES 1319A-K type). Stability Studies of PB-NO Nanocubes. 100 ppm (Fe concentration) of PB-NO nanocubes were dispersed in H2O, serum, PBS (pH = 5.0) and PBS (pH = 7.0) at 37 °C for 7 days. The amount of NO release, UV-Vis spectra, morphology of PB-NO nanocubes, and photographs of colloidal solutions were monitored to evaluate the stability of PB-NO nanocubes in these mediums. Cell Culture. MRC-5 cells (human fetal lung fibroblast cell lines) were cultured in MEM/EBSS containing penicillin/streptomycin (PS, 1%), and fetal bovine serum (FBS, 10%) in the incubator at 37 °C and 5% CO2. Cytotoxicity Studies of PB-NO Nanocubes. MTT assay weas used to evaluate the cytotoxicity of PB-NO nanocubes. MRC-5 cells were seeded in a 96-well plate (10000 cells/well) and were incubated for 24 h at 37 °C with 5% CO2. Subsequently, the medium was removed, and then the fresh medium was added to the culture with PB-NO nanocubes in different iron concentrations. After another 24 h incubation, the uninternalized materials were removed, followed by a wash with PBS for at least 3 times. MTT reagent (10%) was added into cell cultures following the standard method of MTT assay. Finally, an ELISA reader (Thermo Scientific Multiskan EX) was used to measure the absorbance of formazan to evaluate the cell viability. All of the data were measured in quadruplicate. In Vitro Fluorescence Imaging. The fluorescence dye of DAF-2 DA was used as the NO sensor. 5.0 × 104 MRC-5 cells/well were grown in 8-well chamber slides for 24 h, followed by the treatment of DAF-2 DA dye for another 1 h. Subsequently, the medium was removed, and then the cultures were washed by PBS for at least three times. The fresh medium was added to the wells with nanocubes in 100 ppm iron concentration. The medium without the nanocubes was added in the well as the control group. For the group of PB-NO nanocubes + Laser, the cells


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were exposed to an 808 nm diode laser at 0.5 W/cm2 for 10 min. After laser treatment, the medium was removed, and then the cells were treated with Hoechst 33342 for nuclei staining subjected to laser scanning confocal imaging. Animals and Wound Model. Female C57BL/6 (B6) mice aged 4-5 weeks were obtained from the laboratory animal center at National Cheng Kung University (NCKU) and Chang Gung Memorial Hospital (CGMH) in Taiwan. All animal treatments and surgical procedures were performed in accordance with the guidelines of NCKU Laboratory Animal Center (Tainan, Taiwan) and CGMH Laboratory Animal Center (Kaohsiung, Taiwan). Each mouse had free access to water and food ad libitum. A whole skin layer longitudinal midline incision for 2 cm wound was created by a scalpel after anesthesia. Mice were housed individually after experiments. In Vivo Wound Treatment. Eighteen mice with 2 cm wounds were randomized into six groups (n = 3) including control, PB, Laser *3, PB + Laser*3, PB-NO + Laser, and PB-NO + Laser *3. 40 μL of H2O (control) and PB nanocubes (100 ppm in Fe concentration) were dropped on the wounds without laser exposure. For the Laser* 3 (referred to H2O + laser), the wounds were treated with laser exposure (0.5 W/cm2) for 10 min and this procedure was repeated for 3 consecutive days. For PB-NO + Laser, the PB-NO nanocubes (100 ppm in Fe concentration) were dropped on the wounds, followed by the laser irradiation at 0.5 W/cm2 for 10 min. For PB + Laser*3 and PB-NO + Laser *3, the wounds were treated with PB and PB-NO + Laser for 3 consecutive days. Wound sizes were measured and recorded with a digital camera on the day of operation, followed by every 2 days until the wounds healed completely. Laser Doppler flowmetry was used to monitor the blood flow on the wounds. The blood flow signals on the wound sites were collected to quantify the perfusion units. Each data group was evaluated from three mice studies. In Vivo Hyperthermia Efficacy of PB Nanocubes Under 808 nm NIR Laser Irradiation. The mice with 2 cm wounds were treated with PB nanocubes with laser irradiation. The PB nanocubes (100 ppm in Fe concentration) were dropped on the wounds, and then the wounds were treated with laser exposure (0.5 W/cm2) for 10 min. The digital near-infrared thermal camera was used to evaluate and record the temperature change in the course of 808 nm NIR irradiation.


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Immunohistochemistry of DNA Damage with Phospho-Histone H2A.X Expression. The formaldehyde-fixed paraffin-embedded sections were dried at 65 oC for 1 h, then deparaffinized slides and hydrated to the deionized water. Slides immersed in the preheated citrate buffer (Sigma-Aldrich) at 100 °C for 20 min, followed by cooling in cold water for 20 min. After that, washed twice with TBST washing buffer (TBS with 0.025% Triton X-100) for 5 min with agitation and continued the following steps according to the instruction of UltraVision Quanto Detection System HRP DAB (Thermo Scientific). Briefly, incubated slides in Hydrogen Peroxide Block for 10 min to reduce nonspecific background staining. After washed by TBST washing buffer, applied Ultra V Block for 5 min and incubated slides in diluted primary antibody at 4 °C overnight. Anti phospho-histone H2A.X (Ser139) antibody (Cell Signaling, #9718) was used to determine DNA damage response following NIR laser irradiation. Washed slides by TBST washing buffer and incubated in Primary Antibody Amplifier Quanto for 10 min. After that, washed slides, applied HRP Polymer Quanto for 10 min and avoided from light exposure. Then, washed by TBST washing buffer, mixed DAB Quanto Chromogen to DAB Quanto Substrate and applied to tissue for 5 min. Finally, rinsed slides in deionized water. The slides were counterstained with hematoxylin, dehydrated through alcohol, cleared in xylene and mounted. Images were acquired using 3D HISTECH Pannoramic MIDI. Masson’s Trichrome Staining for Skin Sections. Trichrome staining for determination of collagen fibers was performed using the Masson’s Trichrome staining kit (Sigma-Aldrich). All of the formaldehyde-fixed paraffin-embedded sections were dried at 65 oC for 1 h, then deparaffinized slides and hydrated to deionized water. Then, the slides were immersed in preheated bouin’s solution at 56 °C for 15 min, followed by washing in running tap water to remove yellow color from sections. After that, the slides were stained in working weigert’s iron hematoxylin solution for 5 min., and washed in running tap water for 5 min and rinsed in deionized water. In addition, the slides were stained in biebrich scarlet-acid fucshin for 5 min, and rinsed in deionized water followed by place slides in working phosphotungstic/ phosphomolybdic acid solution for 5 min, aniline blue solution for 5 min, 1% acetic acid for 2 mi. and rinsed in deionized water. Finally, all slides were dehydrated through alcohol, cleared in xylene and mounted. The images were acquired by using 3D HISTECH Pannoramic MIDI and viewer. The imaging signal intensities were measured using ImageJ 2.0.0-rc-68/1.52h software.


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Immunohistochemistry of Blood Vessels with CD31 Expression. All of the formaldehydefixed paraffin-embedded sections were dried at 65 oC for 1 h then deparaffinized slides and hydrated to deionized water. Slides immersed in the preheated citrate buffer (Sigma-Aldrich) at 100 °C for 20 min, followed by cooling in cold water for 20 min. After that, washed twice with TBST wash buffer (TBS with 0.025% Triton X-100) for 5 min with agitation and then continued by following steps according to the instruction of UltraVision Quanto Detection System HRP DAB (Thermo Scientific). Briefly, incubated slides in Hydrogen Peroxide Block for 10 min to reduce nonspecific background staining. After washing by TBST wash buffer, applied Ultra V Block for 5 min and incubated slides in diluted primary antibody at 4 °C overnight. Anti-CD31 antibody (Arigo, ARG52748) was used to determine blood vessels distribution by CD31 expression. Washed slides by TBST wash buffer and incubated in primary antibody amplifier quanto for 10 min. After that, washed slides and applied HRP polymer quanto for 10 min., avoided from light exposure. Then, washed by TBST wash buffer, mixed DAB quanto chromogen to DAB quanto substrate and applied to tissues for 5 min. Finally, rinsed slides in deionized water, dehydrated through alcohol, cleared in xylene and mount. Images were acquired using 3D HISTECH Pannoramic MIDI and viewer. The imaging signal intensities were measured using ImageJ 2.0.0-rc-68/1.52h software. In Vivo Biodistribution, H&E Staining and Blood Analysis. 40 μL of PB-NO nanocubes (100 ppm in Fe concentration) or H2O were dropped on the wounds. On the 0th and 7th day, the mice were sacrificed, and the tissues (heart, liver, spleen, lung, kidney, and skin) were collected. The tissues were washed with PBS and 4% pare-formaldehyde. For the biodistribution analysis, the tissues were soaked with aqua regia for a week. The iron content in each tissue was measured by AA spectrometer. The H&E results were stained from the tissue bank at National Cheng Kung University Hospital (Taiwan). For blood analysis, the mice blood was obtained from the heart, and then the heparin sodium was added immediately. The blood biochemistry analysis (AST, ALT, ALP, T-BIL, CRE, BUN, and UA) was determined by a biochemical analyzer (FUJI DRICHEM 4000i). Triplicate measurements were conducted for the studies. Statistical Analysis. Quantitative data was expressed as means ± SD. Statistical analysis of values at different time points were performed by one-way ANOVA followed by post hoc multiple comparisons with the Tukey-Kramer test. Cross-sectional studies were analyzed by


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using the Student t test at each time point for the experimental wound model in mice with different treatments. Statistical analysis was performed using Graphpad prism version 7 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). A probability value

Enhancing Microcirculation on Multitriggering ... - ACS Publications

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