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Pro-angiogenic properties of terbium hydroxide nanorods: Molecular mechanisms and therapeutic applications in wound healing Susheel Kumar Nethi, Ayan Kumar Kumar Barui, vishnusravan bollu, Bonda Rama Rao, and Chitta Ranjan Patra ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00457 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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ACS Biomaterials Science & Engineering

Pro-angiogenic properties of terbium hydroxide nanorods: Molecular mechanisms and therapeutic applications in wound healing Susheel Kumar Nethi, Ayan Kumar Barui, Vishnu Sravan Bollu, Bonda Rama Rao, Chitta Ranjan Patra*

Chemical Biology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad - 500007, Telangana State, India. Academy of Scientific and Innovative Research (AcSIR), Training and Development Complex, CSIR Campus, CSIR Road, Taramani, Chennai - 600113, India.

KEYWORDS: Terbium hydroxide nanorods, angiogenesis, mouse model, wound healing, nonimmunogenic.

ACS Paragon Plus Environment


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ABSTRACT: The process of angiogenesis, involving generation of new blood vessels from the existing ones, is vital for the supply of oxygen and nutrients to various tissues of body system. Angiogenesis is directly associated with several physiological and pathological processes. It is well established that impairment in angiogenesis process results in various fatal conditions. Recently, few research groups including ours demonstrated therapeutic angiogenesis through nanomedicine approach using metal oxide/hydroxide nanoparticles. However, there is still a thorough necessity for the development of novel, eco-friendly, pro-angiogenic nanomaterials. Hence, in the present study we demonstrate the in vitro and in vivo pro-angiogenic properties of terbium hydroxide nanorods (THNRs) synthesized using an advanced microwave irradiation method, along with the detailed molecular signaling cascade underlying THNRs induced angiogenesis. The in vivo wound healing and non-immunogenicity of the THNRs have been validated in the mouse models. We thus strongly believe that the present study establishing the pro-angiogenic properties of THNRs will aid in the development of alternative treatment strategies for wound healing along with cardiovascular and ischemic diseases, where angiogenesis is the chief target.


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INTRODUCTION The lanthanide metal-based nanomaterials have been demonstrated for their wide applications in the field of biology and medicine.1 Among several lanthanide nanomaterials, the terbium nanoparticles have been identified as promising candidates owing to its excellent fluorescence and biological properties. Researchers exhibited that lanthanide based nanomaterials can be employed for the detection of drug concentration in biological fluids2 as well as sensing of glucose molecules in presence of glucose oxidase (GOx).3 The luminescence property of terbium has widely extended the applications of terbium nanoparticles in molecular imaging and detection of enzymatic activity.4 One of the new areas, in which the lanthanide metal nanoparticles are widely investigated in recent years, is angiogenesis research.5 Angiogenesis, a complex biological process where new blood vessels originate from the already existing vessels, plays significant role in both physiological and pathological conditions.6 Physiologically, it is important for embryonic and skeletal growth, natural wound repair, hair growth etc. In pathological conditions, angiogenesis is related with various diseases including cancer, cardiovascular diseases (CVDs), diabetic retinopathy, age-related macular degeneration etc.7 The lack of proper angiogenesis (impaired angiogenesis) is well reported to play a keen role in several cardiovascular complications such as ischemic heart disease, ischemic limb disease and others.8 The recent reports of world health organization state that CVDs lead to the major cause of global deaths annually.9 The conventional therapeutic approaches for addressing CVDs include administration of natural pro-angiogenic growth factors such as VEGF, bFGF, PDGF etc.10 But the use of these growth factors has been demonstrated to end up with various limitations including thrombosis, fibrosis, tumorogenesis, non-specificity, high cost etc11 along



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with a possible worsening of the disease condition.12 All the above circumstances point out the critical need for the development of novel alternative approaches for the treatment of CVDs. In this context, our group has established the pro-angiogenic properties of different metal oxide/hydroxide nanoparticles such as europium hydroxide nanorods, zinc oxide nanoflowers, bio-synthesized gold nanoparticles, graphene oxide nanosheets in both in vitro and in vivo models.5a,b,13 Recently, a brief report demonstrated the pro-angiogenic properties of lanthanide hydroxide nanoparticles including terbium hydroxide nanoparticles.5c However the detailed molecular mechanism and therapeutic applications behind their pro-angiogenic properties especially for terbium hydroxide nanorods remain unclear and should be investigated in-detail. In the present study, we have reported pro-angiogenic properties of terbium hydroxide nanorods (THNRs) with special emphasis on detailed underlying signaling mechanisms, employing different in vitro (endothelial cells) and in vivo (chick embryo model) angiogenesis assays. The in vivo wound healing ability of the THNRs was revealed using the punch biopsy model in C57BL/6 mice. Furthermore, the THNRs were observed to be non-immunogenic in nature towards C57BL/6 and BALB/c mouse strains. Altogether, we hope that the pro-angiogenic and non-immunogenic THNRs could be developed as effective and alternate treatment strategies for angiogenesis related disease therapy. MATERIALS AND METHODS Materials. Terbium (III) nitrate hydrate was purchased from Alfa Aesar. Aqueous ammonium hydroxide (NH4OH), Dulbecco's Modified Eagle's Medium (DMEM), penicillin, streptomycin,

kanamycin,

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide), propidium iodide, RNase and vascular endothelial growth factor (VEGF) were procured from Sigma Aldrich, USA. PVDF membrane was bought from Merck Millipore, USA.


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[Methyl 3H]-Thymidine was purchased from Perkin-Elmer Life and Analytical Sciences, USA. Primary antibodies such as phospho-Akt (Ser 473), Akt, Phospho p38 MAPK (Thr180/Tyr182), p38 MAPK and secondary antibodies such as goat anti-rabbit IgG ALP or goat anti-mouse IgG ALP were procured from Cell Signaling Technology (CST), USA. TNF-α, IL-4, IL-6 and IFN-γ Quantikine ELISA kits were purchased from R&D systems, USA. Human umbilical vein endothelial cells were procured from Lonza, USA and cultured in basal media [EBM-2] supplemented with growth factors, antibiotics and 5% fetal bovine serum (FBS) [EGM-2]. ECV304 cells were a kind gift of Dr. Vijay Shah, Gastroenterology Department, Mayo Clinic, Rochester, MN, USA. EA.hy926 and ECV-304 cells were grown in DMEM supplemented with 10 % FBS and antibiotics. METHODS Synthesis of THNRs using an advanced microwave irradiation method. The synthesis of THNRs was carried out through the interaction of aqueous terbium (III) nitrate hydrate and NH4OH solutions using an advanced microwave oven system (SINEO-MAS II).13c In brief, 10 mL NH4OH (26-30 %) was added to 30 mL of 0.05 M aqueous solution of TbIII(NO3)2 at molar ratio [(OH/Tb) = 40] in a 100 mL round bottomed flask and stirred for 30 min. The resultant white colloidal solution was placed to the microwave system fitted with a reflux condenser and irradiated at 600W for 60 min at 100 ˚C. The thick white precipitate obtained at the end of the reaction process was collected and washed several times by centrifugation at 10,000 rpm using ultrapure water. The final obtained product was dried overnight in a hot air oven. Physico-chemical

characterization.

The

as-synthesized

fine

powdered

nanoparticles were characterized using several physico-chemical analytical tools such as XRD:



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X-ray Diffraction, TEM: transmission electron microscopy, FTIR: fourier transformed infrared spectroscopy, DLS: dynamic light scattering, TGA: thermogravimetric analysis etc. X-ray diffraction (XRD). The identification of the phase purity of the as-synthesized THNRs was determined by XRD method using a Bruker AXS D8 Advance Powder X-ray diffractometer (using Cu Kα λ = 1.5406 Å radiation). Fourier transformed infrared spectroscopy (FTIR). The functional group analysis of THNRs was carried out using FTIR spectroscopy recorded using a Thermo Nicolet Nexus 670 spectrometer in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets. Transmission electron microscopy (TEM): The shape and size of the nanoparticles were observed by TEM using a FEI Tecnai F12 (Philips Electron Optics, Holland) instrument operated at 100 kV. The selected area electron diffraction (SAED) pattern was also captured using this instrument. Dynamic light scattering (DLS). THNRs were dispersed in Millipore water followed by rigorous sonication and measurement of the hydrodynamic radius as well as zeta potential (surface charge) of the THNRs was performed using DLS, Kallipoe version, 1.4.4, Anton Paar, Serial No. 81962693. Further, in order to check the physical stability and aggregation, THNRs were incubated in DMEM containing serum (0.2% FBS and 10% FBS) at physiological pH. Initially, the nanoparticles were incubated in DMEM with 0.2% FBS and 10% FBS in a time dependent manner (0 h, 12 h, 1 day, 2 days, 5 days and 7 days). After incubation time, the dispersions were centrifuged and the nanoparticle pellet was resuspended in Millipore water and analyzed for hydrodynamic diameter (nm) and zeta potential (mV) using DLS.


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Thermogravimetric analysis (TGA). The thermal stability of the as synthesized THNRs was determined using TGA, recorded using a TA-SDT Q-600 thermal analyzer, USA, from 25-500 ºC under N2 atmosphere. MTT assay. The MTT assay was carried out according to our published report.14 Briefly, endothelial cells (HUVECs and EA.hy926) were seeded into 96-well plates at a density of around 8 X 103 cells per well. The cells were incubated with various concentrations of THNRs (1-20 µg/mL) for 24 h and 48 h. The media in each well was replaced with fresh media containing 0.5 % MTT and incubated for 4 h in dark. After this incubation period, the media in each well was replaced with DMSO: Methanol (1:1; v/v) solution to solubilize the formed formazan. The absorbance of the solution was measured at 570 nm using a Synergy H1 multimode plate reader system. All the treatments were performed in triplicates. [3H]-Thymidine cell incorporation assay. The effect of THNRs on DNA synthesis was determined using [3H]-Thymidine cell incorporation assay as per previous reports.15 The HUVECs were seeded in 24-well plate at a density of around 30,000 cells per well and grown. The cells were serum starved for overnight before performing the treatments. The cells were incubated with THNRs (5-10 µg/mL) for a period of 20 h and [3H]-Thymidine (1 µC) was added to cells and incubated for 4h. After the treatment time, the cells were washed with PBS twice and lysed using 0.1% SDS and scintillation fluid was added to it. After 1h incubation in dark, 200 µL of the mixture of cell lysate was transferred to 96-well plates and the radioactivity of the samples was measured as counts per minute. All the experiments were performed in triplicates and recorded. In vitro wound healing (scratch) assay. The scratch assay was performed as our earlier reported literature.14 EA.hy926 cells were seeded in 24-well plate and grown to



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confluence. A scratch was made in the middle of each well using a sterile tip to simulate an in vitro wound. Each well was washed using 1X PBS to remove the floating cells followed by addition of fresh growth medium. THNRs (5-10 µg/mL) treatments were performed in triplicate wells for individual concentration. VEGF was used as a positive control experiment. The brightfield images of cells in the scratch area were captured using a Nikon inverted microscope at periodic intervals of 0 h, 4 h and 8 h respectively. The closure of the wound area as a result of the migration of EA.hy926 cells was calculated using IMAGE J software (NIH, Bethesda, MD, USA). Cell cycle analysis. Cell cycle analysis was performed using the flow cytometry technique as per our previous protocols.13b Briefly, the endothelial cells (EA.hy926) were grown to confluence and incubated with THNRs at a dose range of 5-20 µg/mL for 24 h. After the incubation period, the cells were trypsinized, fixed with 70 % ethanol and stored at -20 °C for overnight. The cells were washed with PBS and stained with propidium iodide (PI) solution supplemented with RNase and triton-X for 40 min under dark condition. Finally, the cells were washed with PBS and run for cell cycle analysis in a BD FACS Canto flow cytometer. Cellular uptake study. Cellular uptake study was demonstrated as per our published report.16 ECV-304 cells and HUVECs were cultured and grown to confluence. The cells were treated with THNRs (20 µg/mL) in a time dependent manner (0-24 h) in separate flasks. The cells were harvested by trypsinization and counted using a cell counter (hemocytometer). The cell pellet was digested in 70 % nitric acid (HNO3) for 48-72 h at 50˚ C. The resultant solution was filtered using 0.22 µM Millex Millipore filter and analyzed for metal content (ppm) using ICP-OES technique.


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Detection of cellular reactive oxygen species (ROS). The detection of ROS was done as per our previous reports.13g,14 EA.hy926 cells were seeded into 6-well plates at a density of 5 X 106 cells per well and cultured. The cells were serum starved prior to treatments for overnight. Next day, the cells were incubated with THNRs (10 µg/mL) for 24 h. After the treatment time period, the cells were washed twice using 1X PBS and incubated with DCFDA (30 µM) for 30 min. The cells were again washed with PBS to remove the unbound dye and lysed using RIPA buffer. The cell lysate was centrifuged at 10,000 rpm for 8 min at 4 ºC and the supernatant was transferred into a black 96-well plate. The fluorescence of the plate was measured using a synergy H1X multimode plate reader at an excitation wave length of 480 nm. Determination of cellular total nitric oxide (NO). The total cellular NO levels were estimated as per our recent reports.14 In order to investigate the role of NO in THNRs mediated angiogenesis, a highly sensitive and quantitative colorimetric assay was performed using a NO detection kit (Arbor Assays; Catalog: K023-H1), for determining NO production in EA.hy926 cells in response to THNRs treatment. This assay is very sensitive and widely followed for the direct measurement of NO in biological systems.17 Briefly, EA.hy926 cells were seeded in 96well plates and grown to confluence. The cells were serum starved overnight prior to the treatments. The cells were incubated with THNRs (10 µg/mL) for different time points of 0, 4, 8 and 24 h. After the treatment time, the supernatant media was collected and the total NO concentration was measured using the nitric oxide detection kit. Chick embryo angiogenesis (CEA) assay. The fertile eggs of Vanaraja strain chicken variety were procured from ICAR-Directorate of Poultry Research, Hyderabad and the assay was performed as per our earlier literature.14,18 These fertile eggs were incubated in horizontal egg incubators (Southern) and maintained at 98.6 °F and 55-60 % RH for 4 days. On



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the fourth day of incubation, the eggs were opened by peeling the shell on the top creating a chick embryo-window model. To perform the detailed mechanistic study, the chick embryo was treated with THNRs in presence and absence of different pharmacological inhibitors such as diphenyl iodonium (DPI: inhibitor of NAPDH oxidase 2 or NOX 2), wortmannin (inhibitor of PI3K) and LY294002 (inhibitor of PI3K). The images of the chick embryo post-treatments were captured using a stereo microscope (Leica) at periodic time intervals. The growth of new blood vessels in terms of their length, size and junction was calculated using AngioQuant software (image analysis tool for angiogenesis). Western blotting. The western blot was done in order to investigate the effect of THNRs on the cellular protein expression in accordance with our previous reports.13g,14 The HUVECs were grown to confluence and incubated with THNRs (10 µg/mL) in a time (0-120 min) dependent manner. After the incubation time period, the cells were washed with PBS, scrapped in lysis buffer supplemented with protease inhibitor cocktail and centrifuged at 10,000 rpm at 4 °C for 14 min. The supernatant was collected and protein concentration was estimated using Bradford reagent. Equal amount of proteins (50 µg) were loaded and separated on a SDSPAGE as well as transferred to a PVDF membrane. The non-specific binding sites on the PVDF membrane were blocked using 5% bovine serum albumin solution (prepared in TBS-T) for 2 h at room temperature. After repeated washes with TBS-T, the membrane was incubated with primary antibodies [phospho-Akt (Ser 473), Akt, phospho p38MAPK (Thr180/Tyr182) and p38 MAPK] for overnight at 4 °C. Subsequently, the membrane was washed thoroughly with TBS-T and incubated with goat anti-rabbit IgG ALP or goat anti-mouse IgG ALP secondary antibodies for 1-2 h at room temperature. Ultimately, the blot was developed in dark employing the colorimetric method using pre-mixed NBT/BCIP solution.


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In vivo wound healing study. The wound healing study in mice was performed based

on

approval

of

the

Institutional

Animal

Ethics

Committee

(IAEC)

(IICT/CB/CRP/22/02/12/07), CSIR-IICT, Hyderabad, India. C57/BL6 mice were employed for establishing an in vivo wound healing model. Briefly, 3-4 week old C57/BL6 mice were purchased from National Institute of Nutrition (ICMR), Hyderabad. The mice (n=3) per experiment group were acclimatized for one week and later used for the experiments. The hair on the dorsal surface of each mouse was removed using commercially available hair remover cream (Veet). Further, a circular wound was created on the exposed surface of skin using a 6 mm punch biopsy and the skin was removed. THNRs- 1% treatment was pre-formulated using commercially available Vaseline cream. THNRs were weighed and mixed in Vaseline base cream (1% w/w) and made as a fine paste using a motor and pestle and applied (approximately 50 µL) around the mouse wound (made by punch biopsy). Based on our wound healing experiments in our laboratory using other nanomaterials in dose dependent manner (0.1% to 1%) and published literature, we have chosen THNRs (1% w/w) as the treatment concentration.19 The THNRs formulated cream was applied around the wound area in alternate days (day- 1, 3, 5 and 7) after creating the wound. Silverex (0.1%) was used as positive control treatment. The closure of the wound (mm) which represents the % wound healing was measured alternatively using digital Vernier calipers. Skin histological analysis. After the observation period, the mice were sacrificed and the skin region around the wound area were dissected and collected in 10 % formaldehyde solution. This was followed by embedding the skin tissues in paraffin wax blocks and thin sections (3 µm thicknesses) of the tissue were cut and mounted on to clean microscopic glass slides. The slides were washed thoroughly and stained with haematoxylin and eosin (H&E) as



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per our previous reports.20 Four sections of each skin sample were made and analyzed from each group. The sections for angiogenesis analysis were selected based on the primary wound closure data. The skin sections were paraffin embedded and slides were prepared and stained with H&E and submitted for analysis. The sections were randomly analyzed for angiogenesis measurement by a certified pathologist, by counting the number of vessels observed at 10 random locations of selected wound area section under HPF (40X) and the mean of these 10 observations was scored based on Abramov’s method.21 Based on this method: epithelialization was ranked as 0 (for none), 1 (for partial epithelialization), 2 (for complete but immature/thin epithelialization) and 3 (for complete and mature epithelialization). Angiogenesis was graded as 0 (None), 1 (≤ 5 blood vessels/HPF), 2 (6-10 blood vessels/HPF) and 3 (>10 blood vessels/HPF). Immunological response study. The study was performed based on approval of the Institutional Animal Ethics Committee (IAEC) (IICT/49/2016), CSIR-IICT, Hyderabad, India. The immunogenic response studies of THNRs in vivo were examined in 3-4 weeks aged male BALB/c mice. The mice were intra-peritoneally (i.p.) administered with THNRs (10 mg/kg b.w.) and blood was drawn from the retro-orbital plexus followed by serum isolation by centrifugation, after 24 h and 7 days of treatment period. The serum was used to check the expression of inflammatory and immune cytokines TNF-α and IL-4 using Quantikine ELISA kits, R&D systems. The procedure was followed as per the supplier’s protocol and finally the absorbance of the samples was measured at 450 nm and the values were subtracted from the values taken at 570 nm. A standard curve was plotted and the cytokines concentration (pg/mL) was extrapolated from the standard values. In a similar manner, the expression of IL-6 and IFN- γ cytokines was estimated in serum isolated from C57BL/6J mice treated with THNRs (10 mg/kg b.w.) for 24 h using the ELISA kit method.


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RESULTS AND DISCUSSION Synthesis and characterization of THNRs. THNRs have been synthesized using an advanced microwave system (MASS-II SINEO) as per our previous protocols,13c which include the interaction of aqueous TbIII(NO3)2 and NH4OH solutions under microwave reaction conditions. The as-synthesized THNRs were characterized by various physico-chemical techniques such as XRD, TEM, FTIR, DLS, TGA etc. The XRD pattern (Figure 1a) suggests the crystalline nature of THNRs with all the diffraction peaks indexed to the hexagonal phase which clearly matches with the published literature.22 The FTIR spectrum demonstrates the bond vibrational frequencies where the characteristic peaks at 3449.6 cm-1 and 636.25 cm-1 indicates the stretching of -OH bond and deformation of Tb-O bond, respectively (Figure S1). The TEM image clearly indicates the rod shaped structure of THNRs with an average length and width of around 340 nm and 65 nm, respectively (Figure 1b). The THNRs were found to possess a hydrodynamic diameter of around 545 nm (Figure 1b inset), with a zeta potential of 30.5±0.4 mV, measured by DLS analysis (Figure S2). The observation of a single peak in DLS size population indicates the monodispersed and uniform shape of THNRs. Furthermore, THNRs incubated in DMEM (0.2% and 10% FBS) illustrated no major increase in the hydrodynamic diameter (nm) of the nanorods incubated at various time points of 0 h, 12 h, 1 day, 2 days, 5 days and 7 days (Table S1). The zeta potential of THNRs was found to be negative when incubated in cell culture media containing serum (0.2% and 10% of FBS) proteins, which might be attributed to the protein corona formation around the nanoparticle surface as per the reported literature.23 The TGA analysis (Figure S3) shows the weight loss (4.36%) of THNRs from 31- 150 °C which might be due to the presence of residual water. The weight loss (12.12 ~ 12.33%) from 200-495 °C can be attributed to the decomposition of Tb(OH)3 to Tb2O3. Considering the TGA curve



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data, the as-synthesized nanomaterial can be considered as terbium hydroxide [Tb(OH)3] nanorods. Enhanced endothelial cell viability by THNRs. The cell viability assay is a basic assay performed to determine the nature of the treatment on the biological systems.13c The MTT assay was performed to evaluate the effect of THNRs on the growth and viability of endothelial cells (HUVECs and EA.hy 926). Figure 1c-d demonstrates that THNRs (1-10 µg/mL) enhanced the endothelial cell growth and proliferation upon incubation for 24 h compared to untreated (UT) control cells, suggesting the biocompatibility and pro-angiogenic behavior of THNRs. The positive control VEGF enhanced the cell proliferation as expected.

Figure 1(a-d). Physico-chemical and biological characterization of THNRs. (a) XRD and (b) TEM analysis of THNRs synthesized using advanced microwave technology (SINEO). The inset picture in (b) represents the hydrodynamic radius of THNRs measured using DLS. Determination of effect of THNRs on the viability of (a) HUVECs and (b) EA.hy926 cells in a dose-dependent manner (1-20 µg/mL),using MTT assay.


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All the cell culture experiments in endothelial cells were performed in starving media to remove the presence of serum growth factors and determine the exact effect of the treatments. However 0.2% of FBS was used to conserve the cell viability. It is in accordance with the previous reports published by several groups including ours where cell viability was determined using MTT in endothelial13c,24 and cancer cells25 under starving conditions for 24 h. We have also performed the cell viability experiments of endothelial cells (HUVECs and EA.hy926) in presence of THNRs using MTT for 48 h. The results of the assay demonstrate that HUVECs and EA.hy926 cells incubated with THNRs showed good viability even after 48 h of incubation time (Figure S4). Additionally, in order to investigate the effect of THNRs on DNA replication and cell proliferation [3H]-thymidine incorporation assay was performed, which demonstrated an increased uptake of [3H]-thymidine in HUVECs incubated with THNRs for 24 h compared to untreated control cells, suggesting the increased DNA replication and cell proliferation of HUVECs treated with THNRs (Figure 2). This result corroborates with results of cell viability assay.

Figure 2. [3H]-Thymidine incorporation assay of HUVECs incubated with THNRs (5-10 µg/mL) for 24 h. Cellular [3H]-Thymidine uptake expressed on Y-axis in counts per minute (CPM).

Promoted wound healing (in vitro) by THNRs. The scratch assay is the preliminary method to check the wound healing ability of any material in vitro.13c,14 After creating a wound



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(scratch), EA.hy926 cells were incubated with THNRs (5-10 µg/mL) to check the cell migration in the wound area. The results shown in Figure 3 suggest the enhanced migration of EA.hy 926 cells upon incubation with THNRs up to 8 h in comparison with control (UT) cells, indicating the wound healing properties of THNRs. Further, it was interesting to note that the THNRs promoted in vitro wound healing at lower concentrations (5 µg/mL), which was comparable with the positive control (VEGF).

Figure 3. (a) Determination of endothelial cells (EA.hy 926) migration induced by THNRs (5-10 µg/mL) treatment using wound healing (scratch) assay. (b) Histogram representation of the closure of the scratch area quantified using ImageJ software.

THNRs do not alter the endothelial cell cycle. The cell cycle analysis was performed in order to study the effect of THNRs on different phases of cell cycle (G0/G1, S and G2/M) in EA.hy926 cells using flow cytometer. Figure 4 exhibits the cell cycle analysis results, where a slight increase in the S-phase (DNA synthesis phase) cell population compared to control (UT)


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cells was observed which might support the pro-angiogenic properties of THNRs as per previously reported literature.13h Overall, the results suggest the biocompatible and proangiogenic nature of THNRs towards endothelial cells.

Figure 4. (a) Cell cycle analysis of EA.hy926 incubated with THNRs using flow cytometry. (b) Corresponding histogram representation of the cell population at various check points (G0/G1, S and G2/M) of cell cycle.

Cellular internalization of THNRs. The time-dependent cellular internalization study of THNRs was performed to determine their uptake kinetics in ECV-304 cells. Other than HUVECs and EA.hy926 cells, ECV-304 is another presumptive endothelial cell line which is widely used for angiogenesis research.26 Hence, we have used these cells as alternate source of endothelial cells for cellular uptake study and make our study more generalized for angiogenesis research. The cells were treated with THNRs (20 µg/mL) in a time-dependent manner (0-24 h) and harvested followed by counting and ICP-OES analysis. FigureS5 demonstrates that the uptake of THNRs by ECV-304 cells was increased up to 12 h, followed by a steady decrease up to 24 h,



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which corroborates with the reported literature.27 This time-dependent uptake kinetics of THNRs in endothelial cells clearly represents that the maximum internalization happens at 12 h post THNRs treatment. To further confirm the above observation, we have measured the cellularuptake of THNRs in HUVECs in a time dependent way (0-24 h) using ICP-OES analysis. The results of the assay showed a similar pattern of cellular uptake of THNRs as observed in ECV304 cells, supporting the above results (Figure S5). As explained in the experimental section, all the cell culture treatments were performed in serum starved media (0.2% FBS). THNRs induce intracellular ROS (H2O2) production. The ROS, especially H2O2 is well known to trigger the molecular signaling cascades inside the endothelial cells, promoting angiogenesis.14, 28 The role of H2O2 in the THNRs mediated angiogenesis was investigated by the fluorescence spectroscopy using DCFDA dye in EA.hy926 cells. It was observed that the THNRs treatments exhibited enhanced intracellular ROS (H2O2) levels compared to control (UT) experiment (Figure 5a). TBHP being a positive control experiment also induced the intracellular formation of H2O2. The overall results emphasize the role of ROS (H2O2) in THNRs induced pro-angiogenesis, which is in accordance with the previous reports.5c THNRs induce intracellular NO formation. As NO is a well-established pro-angiogenic signaling molecule,29 the role of NO in the THNRs induced angiogenesis was determined by evaluating the total cellular NO in EA.hy926 cells treated with THNRs in a time-dependent manner (4 h to 24 h). It was observed that THNRs induced enhanced cellular NO production even at 4 h post-treatment compared to untreated cells (Figure 5b). Henceforth, these observations highlight the vital role of NO, an important reactive nitrogen species (RNS) in THNRs induced angiogenesis.


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Figure 5. (a) Measurement of ROS (H2O2) in EA.hy926 cells treated with THNRs (10 µg/mL) using DCFDA fluorescence spectroscopy. (b) Detection of cellular nitric oxide (NO) produced by EA.hy926 cells treated with THNPs (10 µg/mL) using in a time-dependent manner using NO detection kit method.

In vivo angiogenesis induced by THNRs: Mechanistic pathway elucidation. The CEA assay was performed in order to investigate the effect of THNRs on blood vessel growth and development.14,30 The images of the chick embryo showed the accelerated growth of blood vessels by THNRs at 4 h post treatment compared to control (UT) experiment (Figure 6a). The positive control VEGF was also found to promote blood vessel growth. The quantification of the blood vessel development in terms of length, size and junction also supports the above observations (Figure 6b). Furthermore, to elucidate the mechanistic pathways involved in THNRs induced angiogenesis, the chick embryos were incubated with THNRs in presence and absence of DPI (NOX 2 inhibitor)31 and Wortmannin (PI3K inhibitor).32 Figure 6c-d illustrates that the treatment of these pharmacological inhibitors decreased the blood vessels growth, which was recovered by the addition of THNRs. To confirm the role of PI3K, LY294002 a potent PI3K inhibitor was employed for CEA assay and similar results were observed (Figure S6). Overall, the results indicate the significant role of NOX and PI3K signaling pathways underlying THNRs induced angiogenesis.



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Figure 6. In vivo chick embryo angiogenesis (CEA) assay: (a) Determination of the effect of THNRs on the formation of blood vessels in chick embryo model. (b) Corresponding quantification of the length, size and junction parameters of blood vessel growth using Angioquant software. (c) Mechanistic pathway study, where chick embryo is incubated with THNRs along with DPI (NOX-2 inhibitor) and Wortmannin (PI3K inhibitor) compounds. (b) Corresponding quantification of the length and junction parameters of angiogenesis using AngioQuant software. ACS Paragon Plus Environment

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Plausible angiogenic signaling mechanism of THNRs. The angiogenic signaling mechanism of THNRs at protein level in endothelial cells (HUVECs) was analyzed using western blot technique as done previously.13g The results of the assay demonstrate an increased phosphorylation of Akt (Ser473) [Figure 7a] and p38MAPK (Thr180/Tyr182) [Figure 7b] proteins in HUVECs incubated with THNRs compared to control (UT) experiment. The maximum Akt phosphorylation was observed at 30 min post THNRs treatments. The quantification of the protein bands using ImageJ software, also confirm the above observations (Figure S7). The results altogether suggest that THNRs induced angiogenesis through MAPK/Akt signaling pathway.

Figure 7. (a) Western blot analysis of (a) Phoshpho- Akt protein at Ser473 residue and (b) Phosphop38 MAPK protein at Thr180/Tyr182 residue, in HUVECs incubated with THNRs in a time dependent manner (0-120 min). VEGF (50 ng) is used as a positive control experiment.



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THNRs promote in vivo wound healing. The wound healing study was performed in C57/BL6 mice using punch biopsy model. The wound was created on the dorsal surface of skin and treatments were performed at alternate days (from day 1- day 7) and images were captured using a camera. The results of the study as shown in Figure 8a reveal the enhanced closure of wound in mice treated with THNRs (1%) compared to vehicle control group. Commercially available Silverex ointment treated mice were used as a positive control experiment. Measurement of the closure of the wound area in mm scale is represented as a histogram which supports the above results (Figure S8a). Altogether, the above results suggest the wound healing properties of THNRs towards mouse model.

Figure 8. (a) In vivo wound healing study of THNRs (1%) in C57/BL6 punch biopsy mouse model. (b) Histological analysis of isolated skin sections around the wound area stained with Haematoxylin and Eosin (H&E). V.C.: Vehicle control and P.C.: Positive control (Silverex).


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The anatomical changes of the skin section towards the wound in THNRs (1%) treated mice was determined by histological analysis. The H&E stained transverse skin section images of vehicle control, THNRs and positive control treated mouse are shown in Figure 8b and Figure S9. The Abramov`s score (histogram) of wound healing demonstrates enhanced angiogenesis and fibrosis in the skin tissues isolated from mice treated with THNRs compared to vehicle control group, suggesting the efficient wound healing properties of THNRs (Figure S8b). THNRs are non-immunogenic towards in vivo mouse model. The immunogenic response study of THNRs was performed in order to evaluate their immunotoxicity in mouse model.33 The determination of immune cytokines expression levels in serum of BALB/c mice, administered with THNRs (10 mg/kg b.w.) was performed using ELISA method. Figure 9 demonstrates that there is no significant elevation of serum concentrations (pg/mL) of TNF-α and IL-4 in mice injected with THNRs for 24 h and 7 days, in comparison with untreated group. Similarly no-significant difference in IFN-γ and IL-6 cytokine levels were observed in C57BL6/J mouse treated with THNRs compared to untreated mice (Figure S10). The above results suggest the non-immunogenic nature of THNRs towards in vivo systems and therefore could be safely and effectively delivered as a therapeutic agent without any significant immunotoxicity.

Figure 9. Estimation of the immune response cytokines (TNF-α and IL 4) expression in BALB/c mice intraperitoneally (i.p.) injected with THNPs (10 mg/kg. b.w.) for a period of 24 h and 7 days, using ELISA method. ACS Paragon Plus Environment


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The figures S1-S10 of the results and discussion part are provided in the supporting information. OVERALL DISCUSSION The field of nanotechnology has brought wide horizons together to deliver success in every aspect of science and engineering. The nanomaterials have surpassed the limitations of the conventional approaches and emerging as novel therapeutic and diagnostic tools in the area of biomedical research. Recently several research groups including ours demonstrated the proangiogenic metal oxide/hydroxide nanoparticles as observed by different in vitro and in vivo angiogenesis assays. The present work describes the pro-angiogenic properties of terbium hydroxide nanorods (THNRs) along with the detailed angiogenic signaling pathways. It is wellestablished that endothelial cell proliferation and migration are the hallmarks of angiogenesis. Therefore, the cell viability and scratch wound healing assays were performed to examine the effect of THNRs in endothelial cell proliferation and migration, respectively. These assays exhibited that THNRs induced enhanced endothelial cell proliferation and migration, indicating their pro-angiogenic properties. Cell cycle analysis also demonstrated the increased cell population in S-phase of cell cycle, which is an indication of enhanced DNA synthesis leading to angiogenesis.5a,13h The internalization studies of THNRs showed their maximum cellular uptake at 12 h of treatment time and further decreased up to 24 h. This study corroborates with the reported literature that cellular internalization of nanomaterials peaks at an optimum time point and decreases gradually.27 The published reports demonstrate the vital role of ROS (H2O2 and O2.-) and RNS (NO) in regulating physiological angiogenesis.34 In order to investigate the role of ROS and RNS in THNRs mediated angiogenesis, fluorimetric (ROS measurement using DCFDA) and advanced colorimetric (NO measurement) assays were performed, respectively. The nanorods incubated endothelial cells exhibited increased intracellular formation of H2O2 and


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NO which are the key signaling molecules for angiogenesis process. These observations were in agreement with our previous reports where metal oxide/hydroxide nanoparticles induced angiogenesis by elevating intracellular ROS and RNS.13c,13f The in vivo CEA assay revealed that THNRs could promote blood vessel growth, measured in terms of vessel size, length and junctions. The molecular mechanistic study employing CEA assay illustrated that NADPH oxidase (NOX) mediated PI3K/AKT signaling pathways might play an important role behind THNRs induced angiogenesis which is in accordance with our earlier published report.13c,14 Additionally, western blot analysis in HUVECs exhibited that THNRs induced the phosphorylation of Akt (Ser473) and MAPK (Thr180/Tyr182) proteins which might be the plausible angiogeneic signaling mechanism. Considering the pro-angiogenic activities of THNRs confirmed from various in vitro and in vivo angiogenesis assays, we hypothesized that these nanorods could be employed for therapeutic angiogenesis. Therefore we have chosen a punch biopsy wound healing model in C57BL6 mouse to establish the therapeutic angiogenic properties of THNRs. The results demonstrated the accelerated wound healing in C57BL6 mice administered with THNRs in a time-dependent manner compared to vehicle control experiment. Furthermore, several potent and pharmacologically active nanoparticles and nanoparticulate drug delivery systems trigger expression of several pro-inflammatory cytokines which ultimately confers to nanoparticle immunotoxicity.33 The in vivo therapeutic applications of THNRs is further strengthened by their non-immunogenic nature as evident by non-significant difference in TNF-α, IL-4, IL-6 and IFN-γ levels between THNRs administered and untreated mice groups. The overall plausible mechanistic pathway of THNRs mediated therapeutic angiogenesis has been represented in Scheme 1. Taken together all the above facts, THNRs induced NOX mediated H2O2 which activates PI3K/Akt/MAPK signaling pathways as well as intracellular



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formation of NO, ultimately triggering therapeutic angiogenesis in a downstream signaling manner. In the present study, we have demonstrated the pro-angiogenic properties of THNRs using several in vitro (cell-based) and in vivo (chick embryo angiogenesis) assays and investigated the underlying molecular mechanisms. As a proof of concept of our hypothesis that pro-angiogenic THNRs like other cytokines (VEGF, bFGF etc) can be useful for the wound healing. Therefore, we have evaluated the effect of THNRs on in vivo wound healing in mouse model by studying the wound closure measurement and histopathology (H&E staining) studies. In order to establish the role of THNRs as wound healing agent, rigorous molecular studies are to be carried out. In this context, we have initiated in depth molecular mechanistic study on the signaling pathways involved in THNRs induced wound healing by several assays including (i) gene and protein expression of the dissected skin sections using RT-PCR and western blot analysis, respectively, (ii) immunohistological analysis etc. in normal and drug-induced diabetic mouse which is currently beyond the scope of the present work. Although thorough toxicity, pharmacokinetics, bio-distribution and biosafety evaluation are needed, we hope that understanding the in-depth signaling cascade of pro-angiogenic THNRs might be helpful for their successful translation toward clinical applications.

Scheme 1. Overall schematic representation of the molecular signaling cascade governing the proangiogenic and wound healing properties of THNRs. ACS Paragon Plus Environment

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CONCLUSION We report the advanced microwave irradiation assisted synthesis of THNRs. These THNRs exhibited efficient pro-angiogenic response towards endothelial cells (HUVECs and EA.hy926) as evidenced by various cell based in vitro assays. In the chick embryo in vivo angiogenesis model, THNRs showed enhanced blood vessel growth, confirming their pro-angiogenic nature. Further mechanistic studies revealed that THNRs stimulate NOX mediated generation of ROS which further activates PI3K/Akt/MAPK signaling cascade, followed by the formation of intracellular NO, key signaling molecule for angiogenesis. The application of these nanorods for therapeutic angiogenesis in punch biopsy mouse model demonstrated the enhanced wound healing. The in vivo non-immunogenic nature of these THNRs was towards mice model is an essential factor which encourages the clinical translation of these nanorods. The present study involving keen understanding of the pro-angiogenic properties of THNRs along with molecular mechanisms and therapeutic applications would help in successful development of novel alternative therapeutic treatment strategies for cardiovascular diseases, wound healing etc in near future. AUTHOR INFORMATION Corresponding Author Chitta Ranjan Patra, Ph.D Chemical Biology Division, Associate Professor of Biological Sciences, Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad - 500007, Telangana State, India Tel: +91-40-27191480 Fax: +91-40-27160387 E-mail: [email protected]; [email protected].



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ACKNOWLEDGEMENTS CRP is grateful for financial support from DST-Nanomission project (SR/NM/NS-1252/2013; GAP 570). SKN, AKB and BRR are thankful to DST, UGC and CSIR, New Delhi, respectively for supporting with senior research fellowship. We are thankful to Steve Oglesbee, TCF, UNC Lineberger Comprehensive Cancer Center, NC, USA and Suvro Chatterjee, AU-KBC, Chennai, India for giving permission to use and sharing EA.hy926 cells, respectively. The authors are thankful to Mass and Analytical Division, CSIR-IICT for performing ICP-OES analysis, (AARF project, CSIR 12th FYP) for detection of metal content in biological samples. REFERENCES (1) Dong, H.; Du, S. R.; Zheng, X. Y.; Lyu, G. M.; Sun, L. D.; Li, L. D. Zhang, P. Z.; Zhang, C.; Yan, C. H., Lanthanide Nanoparticles: From Design toward Bioimaging and Therapy. Chem. Rev. 2015, 115 (19), 10725-815. (2) Tan, H.; Zhang, L.; Ma, C.; Song, Y.; Xu, F.; Chen, S.; Wang, L. Terbium-based coordination polymer nanoparticles for detection of ciprofloxacin in tablets and biological fluids. ACS Appl. Mater. Interfaces 2013, 5 (22), 11791-6. (3) Zeng, H. H.; Qiu, W. B.; Zhang, L.; Liang, R. P.; Qiu, J. D. Lanthanide Coordination Polymer Nanoparticles as an Excellent Artificial Peroxidase for Hydrogen Peroxide Detection. Anal. Chem. 2016, 88 (12), 6342-8. (4) (a) Zhou, Z.; Wang, Q.; Zhang, C. C.; Gao, J. Molecular imaging of biothiols and in vitro diagnostics based on an organic chromophore bearing a terbium hybrid probe. Dalton Trans. 2016, 45 (17), 7435-42; (b) Terai, T.; Ito, H.; Hanaoka, K.; Komatsu, T.; Ueno, T.; Nagano, T.; Urano, Y., Detection of NAD(P)H-dependent enzyme activity by time-domain ratiometry of terbium luminescence. Bioorg. Med. Chem. Lett. 2016, 26 (9), 2314-7. (5) (a) Patra, C. R.; Bhattacharya, R.; Patra, S.; Vlahakis, N. E.; Gabashvili, A.; Koltypin, Y.; Gedanken, A.; Mukherjee, P.; Mukhopadhyay, D. Pro-angiogenic properties of europium(III) hydroxide nanorods. Adv. Mater. 2008, 20 (4), 753-+; (b) Patra, C. R.; Kim, J.-H.; Pramanik, K.; d'Uscio, L. V.; Patra, S.; Katusic, Z. S.; Ramchandran, R.; Strano, M. S.; Mukhopadhyay, D., Reactive Oxygen Species Driven Angiogenesis by Inorganic Nanorods. Nano Lett. 2011, 11 (4992-4938); (c) Zhao, H.; Osborne, O. J.; Lin, S.; Ji, Z.; Damoiseux, R.; Wang, Y.; Nel, A. E.; Lin, S., Lanthanide Hydroxide Nanoparticles Induce Angiogenesis via ROS-Sensitive Signaling. Small 2016, 12 (32), 4404-11. (6) Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 2005, 438 (7070), 932-6. (7) (a) Folkman, J. Angiogenesis in Cancer, Vascular, Rheumatoid and Other Disease. Nat. Med. 1995, 1 (1), 27-31; (b) Kerbel, R. S., Tumor angiogenesis. N. Engl. J. Med. 2008, 358 (19), 2039-49. (8) Rivard, A.; Fabre, J. E.; Silver, M.; Chen, D.; Murohara, T.; Kearney, M.; Magner, M.; Asahara, T.; Isner, J. M. Age-dependent impairment of angiogenesis. Circulation 1999, 99 (1), 111-20. (9) (a) Wong, N. D. Epidemiological studies of CHD and the evolution of preventive cardiology. Nat. Rev. Cardiol. 2014, 11 (5), 276-89; (b) Lim, S. S.; Vos, T.; Flaxman, A. D.; Danaei, G.; Shibuya, K.; Adair-Rohani, H.; Amann, M.; Anderson, H. R.; Andrews, K. G.; Aryee, M.; Atkinson, C.; Bacchus, L.


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Pro-angiogenic Properties of Terbium Hydroxide ... - ACS Publications

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