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Mitochondria-targeting Polydopamine Nanocomposites as Chemo-photothermal Therapeutics for Cancer Zhuo Wang, Yuzhi Chen, Hui Zhang, Yawen Li, Yufan Ma, Jia Huang, Xiaolei Liu, Fang Liu, Tongxin Wang, and Xin Zhang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00325 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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Bioconjugate Chemistry

Mitochondria-targeting Polydopamine Nanocomposites as Chemo-photothermal Therapeutics for Cancer Zhuo Wang *,† , Yuzhi Chen,† Hui Zhang,† Yawen Li,† Yufan Ma,† Jia Huang,‡ Xiaolei Liu,‡ Fang Liu,‡ Tongxin Wang,¶ Xin Zhang *,†

†

State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Science, Beijing University of Chemical Technology, Beijing, 100029, China ‡

Department of Hepatobiliary Surgery, Department of Gastroenterology, China-Japan Friendship Hospital, Beijing, 100029, China ¶

College of Engineering and College of Dentistry, Howard University, Washington, DC, 20059, United States

Keywords: anticancer agents, apoptosis, chemo-photothermal therapeutics, mitochondriatargeting, polydopamine

Abstract: Mitochondria plays a key role in a variety of physiological processes, and the mitochondria-targeting drug delivery is helpful and effective in cancer therapy.Rhodamine123 (Rhod123) and Doxorubicin (Dox) are not new chemical molecules, and they both can inhibit the growth of cancerous cells. Here, we combine these two “old” chemicals with polydopamine nanoparticles (PDA NPs) to strengthen the antitumor effect with the aid of near infrared irradiation. PDA NPs carry these two chemicals tightly by hydrogen bonds and ππ stacking besides chemical bonds. The better antitumor profile of PDA-Rhod-Dox comes from the mitochondria-targeting delivery, which induce the decrease of ATP in living cells, cause the apoptosis of cancerous cells effectively and inhibit the growing of tumors in mice. The synergistic effect of PDA, Rhod123 and Dox improve the treatment effect of conventional chemotherapy drugs. Introduction As a "cell power plant", an important function of mitochondria is the generation of energy molecules ATP. More and more studies have found that the role of mitochondria in cells is far 1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

more than "cell energy station". In addition, mitochondria is involved in the production of reactive oxygen species (ROS), reducing small molecules and metabolites, regulating cell signal transduction, cell death and material metabolism, and is an important intracellular stress receptor.1 Many human diseases has link with the functional mitochondria.2 Mitochondrial function is related with human aging and cancer. Some literatures report that alteration in mitochondrial potential (△Ψm) is an important characteristic of cancer, and is caused by mitochondrial dysfunction, such as DNA mutation and oxidative stress.3,4 Moreover, mitochondria plays a different role in the different stages of cancer development. For example, mitochondria of cancer cells also provide some flexibility for tumor cells to cope with harsh growing conditions such as nutrient depletion, hypoxia, and cancer treatment, and play a role in the development and progression of cancer. Mitochondria in cancer cell metastasis, proliferation, metabolism and treatment process have different performance. The development of mitochondria-targeting therapeutics are still in the developing period.5 One of the most challenging problems in the development of drug therapy to target mitochondria is the distribution of these drugs to the mitochondria of cells. In the past years, various types of mitochondria-targeting vectors have been explored to deliver drug to this organelle. These vectors include organic molecular probes, nanomaterials and peptides. In these vectors, organic molecular probes with positive charge groups can reach the mitochondria, and usually light up the cells as effective dyes. The nanomaterials vectors are constructed by polymers with positive groups, such as triphenylphosphine, can target the mitochondria and deliver drugs in cells. Recently, the peptide vectors have been explored for the delivery of bioactive molecules to mitochondrial matrix.6,7 Although the development of the vectors for mitochondria is flourishing, few multifunctional and good biocompatible vectors are reported.8-15 The main challenge is how to realize mitochondria targeting, delivery drug, and effective therapy on one vector synergistically.16,17

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Nanomaterials provided the potential of improving some effective therapy, which lies in their ability to deliver molecules directly to the cells and simultaneously enhance stability and pharmacokinetics. Many kinds of nanomaterials are applied to deliver drugs, such as metallic nanomaterials,

inorganic

nanomaterials,

and

polymer

nanomaterials.18-22

Recently,

polydopamine (PDA) as a carrier and phototherapy agent is applied in treatment of tumors.2332

The various applications of PDA are triggered from the research of the adhesive protein of

mussels.33-35 The mussels show excellent affinity to all types of surfaces, including inorganic, organic, metallic surfaces, even polytetrafluooethylene materials. The strong sticky ability is due to the proteins of mussels with rich lysine animo acids. Dopamine is a kind of catechol compounds, and can convert to PDA under mild condition. The catechol structure of PDA supports the organic reaction with amines or thiols by Michael addition or Schiff base reactions.36,37 The catechol and phenyl structure of PDA can stick many substances by the hydrogen bond and π-π interaction. PDA shows good biocompatibility, negligible cytotoxicity, and can reduce the inflammatory response. These properties of PDA make it suitable as a biological carrier in vitro and in vivo. Moreover, PDA can be used in photothermal therapy (PTT). PTT as a minimally invasive therapeutic approach has attracted increasing attention in recent years.38-40 Materials with high near-infrared (NIR) optical absorbance can generate heat under laser irradiation to increase tumor local temperature and ablate cancer cells. So, PDA can work as a carrier and a PTT regent, which is consider as a drug for the cancerous cells. Here, we want to realize the above functions of PDA in the subcellular organellemitochondria. On the basis of the reaction of PDA with amines and sticky character, we link mitochondrial targeting molecules and drugs on the surface of PDA nanoparticles to realize delivery of drugs into the mitochondria. Rhod123 and Dox have been shown to inhibit mitochondrial function and to bind nuclear DNA and block DNA synthesis in vitro, respectively. Rhod123 is a commercial mitochondrial dye, and can imaging mitochondria specially. Rhod123 also shows some anti-cancer ability.41 For targeting mitochondria, 3 ACS Paragon Plus Environment


Rhod123 has an amine group, and can react with catechol structure, which give possibility to link Rhod123 with PDA. Doxorubicin (Dox) is a traditional drug for cancer, and also has an amine on the molecular structure. The reactions of Rhod123 and Dox with PDA are confirmed by Liquid Chromatograph-Mass Spectrometer (LC-MS), and the experimental details are discussed in the following context. PDA, Rhod123, and Dox have some phenyl, amine, hydroxyl groups in the molecular frame, so the hydrogen bond and π-π stacking exist among the three compounds. We proceed the theoretical calculation to prove the intermolecules force among PDA, Rhod 123 and Dox. A simplified model that contains four dopamine molecules (tetramer is a reliable model which can represent a part of the surface of PDA NPs), one Rhod123, and one Dox in water was employed. Water (with dielectric constant ε = 78.3553) is used as the solvent for the four complexes with SMD model.42 The quantum mechanical calculations of molecules are carried out with the Gaussian 09 software package. The geometry optimizations are carried out for Rhod123 and Dox (complex I), Dox and PDA (complex II), Rhod123 and Dox (complex III), Rhod123, Dox and PDA (complex IV) by PM6 semiempirical method.43 As shown in Table S1, all the binding energies are positive values indicating that the interactions of Rhod123/Dox and PDA are exothermic. It means Dox and Rhod123 tend to combine with the surface of the nanospheres rather than free in the solution. Dox has weak interaction with PDA, including hydrogen bonds and π-π stacking (as shown in Figure S1). With the participation of Rhod123, the binding energy with PDA is increased from 2.1 to 6.7 kcal/mol. The interaction between Rhod123 and PDA is relatively strong (the corresponding binding energy is 7.3 kcal/mol), because of one more hydrogen bond (between the amino group of Rhod123 and the hydroxyl group of PDA) is formed without Dox. (Figure S2) This also explains the presence of Rhod123 enhanced the interaction force between PDA and Dox. The Schematic diagram of the interaction among PDA, Rhod123 and Dox is shown in Figure 1. The theoretical data suggest that the formation


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of PDA-Rhod-Dox complex is based on the chemical reaction and the inter-molecular force, such as hydrogen bond and π-π stacking.

Figure 1. The chemical structure and intramolecular interaction of PDA, Rhod123 and Dox. Red dotted lines represent hydrogen bonding interactions (the numbers are the distance of OH or N-H, unit: Å), and orange solid lines represent π-π stacking. The magnified graphs show 5 ACS Paragon Plus Environment


representative structures, displaying π-π interactions between Dox and PDA, Rhod123 and PDA, respectively. Results and Discussion

Synthesis and characterization of PDA-Rhod-Dox nanocomposites. PDA-Rhod-Dox nanocomposites are synthesized in the following procedure. PDA nanoparticles are prepared according to the procedures which has been presented in previous reports. The synthesized PDA nanoparticles present a relatively narrow size distribution (average size is 112 nm, Figure 2b, Figure S4a). PDA-Rhod nanocomposites are synthesized by mixing the as-synthesized PDA NPs suspension and Rhod123 solution in aqueous solution and then stirred for 12 h at room temperature. PDA-Rhod nanoparticles still keep regularly circular shape (Figure S3). The hydrated particle size tested by Dynamic Light Scattering (DLS) changes from 138.61 nm to 159 nm. The Zeta potential has a significant reduction from -36.9 mV to -29.7 mV and the change is consistent with the property of Rhod 123 as a lipophilic cation. On the basis of DLS data, the particle size of PDA-Rhod looks increasing in some degree. While, the SEM image of PDA-Rhod indicates that the size is similar with PDA NPs. DLS gives a hydrodynamic size corresponding to the core and the swollen corona of the micelles. In the solution, functional groups on the surface of the particles will make the value of DLS larger than the exact size of the particles. The SEM image of PDA-Rhod indicates the actual size at an average diameter of 110 nm. Similarly, Dox is linked on the surface of PDARhod nanoparticles to form PDA-Rhod-Dox nanocomposites. The related characterization data is shown in Figure 2c, 2e and Figure S4. Compared with PDA-Rhod, PDA-Rhod-Dox shows a similar size and zeta potential. Besides, PDA-Rhod-Dox nanoparticles are stable in aqueous solution and FBS solution in two weeks (Figure S5). After 14 days, the samples show little precipitation observed by naked eyes. PDA-Rhod-Dox nanoparticles is confirmed by Fourier transform infrared (FT-IR) spectroscopy (Figure S4e). The FT-IR spectrum of 6 ACS Paragon Plus Environment

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PDA NPs shows strong absorption bands at 3400 cm-1, 1600 cm-1, 1507 cm-1, 1280 cm-1, which refer to O-H strech, C-C stretch (aromatic ring) and C-N stretch (aromatic amine) of PDA NPs respectively.44 After the modification of Rhod123 and Dox, new absorption bands at 1411 cm-1 and 989 cm-1 appear. The absorption bond at 989 cm-1 belongs to C-O strech (primary alcohol), which can be also found on the FT-IR spectra of PDA-Dox and PDARhod-Dox, but cannot be found on the spectra of PDA. The absorption band at 1411 cm-1 belongs to C-O strech (aromatic esters), which can be found on the spectra of PDA-Rhod and PDA-Rhod-Dox, but does not appear on the spectra of PDA. So the FT-IR can illustrate the existence of Rhod123 and Dox on the surfaces of PDA NPs. Thermogravimetric analysis (TGA) is introduced to determine whether the molecules are loaded onto PDA NPs. TGA analysis curves of PDA and PDA-Rhod-Dox are obtained by heating PDA NPs (Figure 2f) and PDA-Rhod-Dox (Figure 2g) from room temperature to 800℃ at a rate of 10℃/min under air environment. In Figure 2f, there are two mass losing intervals, the first interval (from room temperature to 120℃) is due to the water losing. In Figure 2g, there are three mass losing intervals on the curve and the second interval (from 120℃ to 270℃) attributes to the decomposition of the organic molecules on the surfaces of PDA NPs. The percentage of mass losing at the second stage reaches up to 18.75%. The loading content data obtained from the experiment of LC-MS test is 4.4% and 8.9% for Dox and Rhod123, respectively, by quantifying the content of drugs remaining in the supernatant after the synthesis of PDA-Rhod-Dox, and the loading content sum of Dox and Rhod 123 is 13.3%, which is relative with the data obtained from the TGA test (18.75%). The reactions between dopamine and Rhod123, dopamine and Dox are proved by LC-MS.45 By observing the chromatogram, we can see that the three compounds can be separated well with different retention time: 0.88 min, 4.07 min, 3.86 min for dopamine, Rhod123 and Dox respectively. Rhod123 and Dox are mixed with dopamine in methanol separately under stirring for 12h at room temperature. The addition product of dopamine and Rhod123 is found 7 ACS Paragon Plus Environment


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at m/z 241 with the retention time at 4.60 min, (Figure S6) and the addition product of dopamine and Dox is found at m/z 341 with the retention time at 4.80 min. (Figure S7) The addition products are imine compounds, and not very stable in the solution. The hydrogen bonding and π-π stacking can help to make PDA-Rhod-Dox more stable in aqueous environment. The above character data identify that PDA-Rhod-Dox Nanocomposite is constructed in the water solution successfully.

Figure 2. (a) Structure of PDA-Rhod-Dox. Rhod 123 and Dox are covered on the surface of PDA nanoparticles by chemical bonds and intramolecular interaction.

Structure

characterization of PDA-Rhod-Dox. Scanning electron microscope (SEM) images, zeta potential for PDA NPs (b, d), and PDA-Rhod-Dox (c, e); thermogravimetric analysis (TGA)


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curves for the heating of 4.35mg PDA (f) and 11.05mg PDA-Rhod-Dox (g) to 800℃ at a rate of 10℃/min.

Drug Loading and Laser- Controlled release

Dox and Rhod123 are loaded onto the surface of PDA NPs through the interaction of chemical reactions, hydrogen bonds and π-π stacking with a loading content of 4.2% and 8.9%, respectively. The release rate and efficiency of Rhod123/ Dox from the nanocomposites are important parameters to evaluate the nature of PDA-Rhod-Dox. PDA can absorb NIR irradiation and then result in an enhancement on the ambient temperature. As shown in Figure 3c, PDA-Rhod-Dox nanocomposites are dispersed in water with the concentration ranging from 100 to 400 µg/mL , and then the solution is irradiated with an 808 nm laser at 1 W/cm2 for 600 s. The PDA-Rhod-Dox solution with a relatively high concentration shows high temperature growth within the same irradiation time. The temperature of nanoparticles solution with a concentration of 400 µg/mL could reach up to 58℃ after 600s of NIR irradiation. The high temperature can induce the release of Rhod123 and Dox from the nanocomposite in some degree due to the attenuation of the inter-molecular force. The release rates of Rhod123 and Dox are detected by LC-MS at pH 7.4 and pH 5.4 with or without laser irradiation. In Figure 3a and 3b, the release rates of Rhod 123 and Dox at pH 7.4 are about 40% and 20% after 48h, respectively. While the release rate can reach up to nearly 60% at pH 5.4 for the two molecules. The pH-sensitive release of PDA-Rhod-Dox may be due to the imine structure of Rhod123 and Dox with PDA. The imine group is sensitive to acidic environment, which may induce a quick release of Rhod123 and Dox from the surface of PDA-Rhod-Dox nanocomposites. The release rate of Rhod123 and Dox can be significantly enhanced by NIR laser irradiation, because the laser-converted heat energy eliminates the 9 ACS Paragon Plus Environment


interaction between the molecules and PDA effectively. The release rates of Rhod123 and Dox molecules reach up to 95% and 80% over 48h respectively at pH 5.4 with NIR irradiation. The pH-sensitive and thermal-sensitive properties of PDA-Rhod-Dox are going to be helpful in killing cancerous cells.

Figure 3. Rhod123 (a) and Dox (b) release rates from PDA-Rhod-Dox nanocomposites in different pHs (5.4 and 7.4, ammonium acetate buffer) with or without NIR laser irradiation (NIR, near infrared). (c) Temperature elevation by different concentrations of PDA-RhodDox (100 µg/mL, 200 µg/mL, 400 µg/ mL in PBS, pH 7.4) under laser irradiation (808 nm, 1 W/cm2), PBS as a control experiment. PDA-Rhod-Dox behavior in living cells As an anti-tumor antibiotics, Dox is applied to treat many kinds of tumors by inhibiting the synthesis of nucleic acid.46 Rhod123 is a kind of delocalized lipophilic cations (DLCs) and has the ability of targeting mitochondria in response to negative inside transmembrane potentials.47 Rhod123 is reported to be accumulated and retained in carcinoma than in normal cells due to a higher mitochondrial or plasma transmembrane potential or both. Moreover, Rhod123 can inhibit oxidative phosphorylation in isolated mitochondria and have a cytostatic effect in some cancerous cells. In order to confirm the mitochondria-targeting of PDA-Rhod-Dox, the colocalization imaging with Mito-Tracker Red (a standard fluorescent probe for targeting mitochondria) is performed by the confocal microscopy (Figure 4a-c). In Figure 4d-f, the fluorescence of PDA-Dox (without Rhod 123 on PDA NPs) shows poor colocalization with Mito-Tracker Green. In 10 ACS Paragon Plus Environment

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Figure 4a, the clear fluorescence distribution (λEm: 510nm-530nm) which comes from Rhod123 of PDA-Rhod-Dox, shows well colocalization with that of Mito-Tracker Red (Figure 4b), indicating the successful targeting of PDA-Rhod-Dox into mitochondria. As a control group, the fluorescence image of PDA-Dox is taken under same conditions. The fluorescent intensity of PDA-Dox distributes in the whole cells including the nucleus and cytoplasm (Figure 4d). Figure 4e is the image of the cells stained by Mito-Tracker Green. Figure 4f is the merged image of Figure 4d and 4e and shows bad colocalization result. The similar targeting situation of Dox is gained after the incubation with HeLa cells (Figure S8). On the basis of these fluorescent images, we see that PDA-Rhod-Dox nanoparticles can be successfully internalized into the mitochondria of cells under the guide of Rhod123.

Figure 4. PDA-Rhod-Dox targets mitochondria in HeLa cells. (a) Fluorescence image of HeLa cells stained with PDA-Rhod-Dox (λEx: 488nm, λEm: 510nm-530nm). (b) Fluorescence image of HeLa cells stained with Mito-Tracker Red (λEx: 552nm, λEm: 580nm- 610nm). (c) Merged image of PDA-Rhod-Dox and Mito-Tracker Red. (d) Fluorescence image of HeLa cells stained with PDA-Dox (λEx: 488nm, λEm: 570nm-590nm). (e) Fluorescence image of



HeLa cells stained with Mito-Tracker Green (λEx: 488nm, λEm: 510nm-530nm). (f) Merged image of PDA-Dox and Mito-Tracker green. The scale bar is 25µm. Before the application of PDA-Rhod-Dox for tumors treatment in vivo, we apply the nanocomposites in cancer cells for cytotoxicity test. In Figure 5a, the cells treated with PDA NPs show good viability even at high concentrations, which certified that PDA NPs has good biocompatibility. Among these nanocomposites modified by small molecules on the surface of PDA NPs, PDA-Rhod-Dox exhibits the most substantial cytotoxicity compared to that of PDA-Rhod and PDA-Dox under the same condition. The cytotoxicity of PDA-Rhod-Dox comes from the synergetic treatment of PDA, Rhod123 and Dox. With the laser irradiation (808nm, 1W/cm2, 5min), PDA-Rhod-Dox represents high cytotoxicity for HeLa cells. Half maximal inhibitory concentration (IC50) is calculated to quantify the synergistic photothermalchemotherapy efficiency by Graphpad Prism 6 Software. IC50 of PDA is 160.20 µg/mL for the single photothermal therapy (PT) (Figure S9). On the basis of Dox concentration, IC50 values of PDA-Dox and PDA-Rhod-Dox are 12.21 µg/mL and 7.17µg/mL for the single chemotherapy (CT), respectively. The IC50 value of PDA-Rhod-Dox is lower than that of PDA-Dox for CT, which presents the effective coordinated treatment between Rhod123 and Dox. The synergetic photothermal-chemotherapy (PT-CT) of PDA-Rhod-Dox gives an IC50 of 65.57 µg/mL for PT and 2.88 µg/mL for CT, respectively. The combination index (CI) is utilized to evaluate the synergistic efficiency after the combination of different therapeutic drugs and different therapies. When CI ＜1, it demonstrates an efficient synergistic effect. The lower CI value represents the better the synergistic therapy. The CI value of PDA-RhodDox is 0.81, which indicates a well synergistic therapy effect of the photothermalchemotherapy combination. Cell apoptosis, as programmed cell death process, caused by PDA-Rhod-Dox is assessed using an Annexin V-kFluor647/ 7-AAD apoptosis detection kit.48 HeLa cells are incubated with PDA， PDA-Dox， PDA-Rhod-Dox at a concentration of 50 µg/mL for 24h respectively. 12 ACS Paragon Plus Environment

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The apoptosis rate is determined by Annexin V-kFluor647/ 7-AAD dual staining of the flow cytometry. The choice of Annexin V-kFluor647 apoptosis kit is based on the excitation and emission wavelength (Annexin V-kFluor647: λEx 650nm, λEm 665nm; 7-AAD: λEx 546nm, λEm 647nm), which can minimize the impact of fluorescence from Rhod123 and Dox. On the chart (Figure 5c-e), area R4 (Annexin V-kFluor647-negative and 7-AAD-negative cells) is identified as living cells. Area R5 (Annexin V-kFluor647-positive and 7-AAD-negative cells) is considered to be early apoptosis cells while area R3 (Annexin V-kFluor647-positive and 7AAD-positive cells) is related with late apoptotic cells. Area R2 (Annexin V-kFluor647negative and 7-AAD-positive cells) is considered to be dead cells. As shown in Figure 5c, most of cells (83.90%) are in R4, which indicates that PDA NPs has little influence for the living status of HeLa cells. When the cells are incubated with PDA-Dox, the apoptosis rate of cells is significantly enhanced and the early and late apoptosis rate reach up to 12.86% and 65.83% respectively due to existence of Dox (Figure 5d). When the cells are treated with PDA-Rhod-Dox for 24 h (Figure 5e), there are increments both in area R3 (from 65.83% to 69.05%) and area R2 (from 18.56% to 20.12%), and the fluorescent intensity of 7-AAD is also increased significantly. This indicates that PDA-Rhod-Dox brings HeLa cells a more efficient apoptosis process, and cause more damage to the cell membrane. PDA-Rhod-Dox can target mitochondria and induce the cell apoptosis effectively. We suggest that PDA-Rhod-Dox may disturb the mitochondrial function. One major function of mitochondria is to produce adenosine triphosphate (ATP) by oxidative phosphorylation, providing energy for various physiological activities in cells. So the content of ATP in cells can indicate the activity of mitochondria in some degree. An ATP Assay Kit is used to detect the intracellular ATP level and the quantified ATP level is obtained through the standard concentration curve. In Figure 5b, the cellular ATP amounts are different when HeLa cells are incubated with different nanocompsites. It should be noted that PDA-Rhod-Dox with or without NIR shows a relatively lower cellular ATP level than that of PDA, which indicated 13 ACS Paragon Plus Environment


that Dox and Rhod123 disorder ATP synthesis in mitochondria. With the enhancement of the nanocompsites concentration, the ATP level in cells gets lower. In the case of using 200 µg/mL of PDA-Rhod-Dox with 808 nm laser irradiation, the cellular ATP level decreases to nearly zero. PDA-Rhod-Dox can disturb the synthesis of ATP and induce the apoptosis of cancerous cell. Hemolysis assay is employed to investigate the interaction between PDA-Rhod-Dox nanoparticles and blood components. As shown in Figure 5f, even though the concentration of PDA-Rhod-Dox reaches up to 200µg/mL, there is still no obvious hemolysis phenomenon and the percentage of hemolysis keeps at a relatively low value. It can be concluded that PDARhod-Dox shows good biocompatibility to live cells and can be applied to biological applications as a relatively safe agent.

Figure 5. (a) Cell viability of HeLa cells treated with different concentrations of the nanocomposites (PDA, PDA-Rhod, PDA-Dox, PDA-Rhod-Dox) with or without laser irradiation (808nm, 1W/cm2, 5min).*: p < 0.05, **:p< 0.01, ***: p

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