Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Lysozyme-Assisted Photothermal Eradication of MethicillinResistant Staphylococcus aureus Infection and Accelerated Tissue Repair with Natural Melanosome Nanostructures Jun Li, Xiangmei Liu, Ziao Zhou, Lei Tan, Xianbao Wang, Yufeng Zheng, Yong Han, Da-Fu Chen, Kelvin Wai Kwok Yeung, Zhenduo Cui, Xianjin Yang, Yanqin Liang, Zhaoyang Li, Shengli Zhu, and Shuilin Wu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b03982 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 78 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

ACS Nano

Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant

Staphylococcus

aureus

Infection and Accelerated Tissue Repair with Natural Melanosome Nanostructures

Jun Li,† Xiangmei Liu,*,‡ Ziao Zhou,‡ Lei Tan,‡ Xianbao Wang,‡ Yufeng Zheng,§ Yong Han,∥ Da-Fu Chen,⊥ Kelvin Wai Kwok Yeung,# Zhenduo Cui,† Xianjin Yang,† Yanqin Liang,† Zhaoyang Li,† Shengli Zhu,† Shuilin Wu*,†,‡

†

The Key Laboratory of Advanced Ceramics and Machining Technology by the Ministry

of Education of China, School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China

ACS Paragon Plus Environment

1

ACS Nano 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

‡

Page 2 of 78

Hubei Key Laboratory of Polymer Materials, Ministry-of-Education Key Laboratory for

the Green Preparation and Application of Functional Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China

§

College of Engineering, State Key Laboratory for Turbulence and Complex System,

Department of Materials Science and Engineering, Peking University, Beijing 100871, China

∥ State

Key Laboratory for Mechanical Behavior of Materials, School of Materials

Science and Engineering, Xi’an Jiaotong University, Xi'an, Shaanxi, 710049, China

⊥ Beijing

JiShuiTan Hosp, Beijing Res Inst Orthopaed & Traumatol, Lab Bone Tissue

Engn, Beijing 100035, Peoples R China.

#

Department of Orthopaedics & Traumatology, Li Ka Shing Faculty of Medicine, The

University of Hong Kong, Pokfulam, Hong Kong 999077, China

* To whom correspondence should be addressed:


2

Page 3 of 78 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

ACS Nano

E-mail: [email protected]; [email protected] (S.L. Wu);

[email protected] (X.M. Liu)

ABSTRACT

Patients often face the challenge of antibiotic-resistant bacterial infections and lengthy tissue reconstruction after surgery. Herein, human hair-melanosome derivatives (HHMs), comprising keratins and melanins, are developed using a simple "low-temperature alkali heat" method for potentially personalized therapy. The mulberry-shaped HHMs have an average width of ~270 nm and an average length of ~700 nm, and the negatively charged HHMs can absorb positively charged Lysozyme (Lyso) to form the HHMs-Lyso composites through electrostatic interaction. These naturally derived biodegradable nanostructures

act

as

exogenous

killers

to

eliminate

methicillin-resistant

Staphylococcus aureus (MRSA) infection with a high antibacterial efficacy (97.19 ±


3

ACS Nano 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

Page 4 of 78

2.39%) by synergistic action of photothermy and "Lyso-assisted anti-infection" in vivo. Additionally, HHMs also serve as endogenous regulators of collagen alpha chain proteins through the "protein digestion and absorption" signaling pathway to promote tissue reconstruction, which was confirmed by quantitative proteomic analysis in vivo. Notably, the 13 upregulated collagen alpha chain proteins in the extracellular matrix (ECM) after HHMs treatment demonstrated that keratin from HHMs in collagendependent regulatory processes serves as a notable contributor to augmented wound closure. The current paradigm of natural material-tissue interaction regulates the cellECM interaction by targeting cell signaling pathways to accelerate tissue repair. This work may provide insight into the protein-level pathways and the potential mechanisms involved in tissue repair.

KEYWORDS: human hair, biodegradable, antibiotic-resistant bacterial infections,

collagen alpha chain proteins, tissue repair


4

Page 5 of 78 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

ACS Nano

Bacterial infectious diseases exhibit an increasing threat to global healthcare, which is the predominant factor of morbidity and final mortality worldwide.1,2 For instance, approximately 15 million deaths are directly caused by bacterial infection and the associated complications every year.1,2 Additionally, the long-period abuse of antibiotics has driven the rapid evolution, rise and spread of antibiotic-resistant bacteria at alarming rates, where the rapid emergence of infections related to drug-resistant bacteria have led to an enormous public medical and financial burden.1,3,4 Particularly, the rising prevalence of methicillin-resistant S. aureus (MRSA) strains in chronic or recurrent infections is the huge challenge because MRSA has high levels of tolerance to clinically available antibiotics and conventional antibiotics are not effective against MRSA. Moreover, the number of deaths related to MRSA is more than that induced by hepatitis, human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS), and influenza combined.5 Mortality rates are predicted to rise more than tenfold by 2050 due to untreatable infections in the absence of new therapies.6 Although the new classes of efficacious antibiotics can significantly decrease the mortality caused by bacterial infections, the development of new antibiotics often


5

ACS Nano 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

Page 6 of 78

need several years and even more to be available in clinical practice, while it usually takes several weeks for bacteria to generate antibiotic resistance.1,7 Consequently, the development of effective antibacterial therapeutics is a top priority to safely and rapidly eliminate drug-resistant bacterial infections, including MRSA infections.

Generally,

bacterial

infections

often

occur

during

the

recovery,

repair

or

reconstruction of damaged tissues, trauma, acute illness, or chronic disease conditions, which also present a tremendous threat to global health.8,9 Demographically, more than 114 million patients worldwide who develop wounds from surgical procedures annually need wound repair devices.8 Notably, the total share of the global wound care market reached $15.6 billion in 2014 and is expected to increase to $18.3 billion by 2019.8 Hence, more rapid and effective strategies for bacterial elimination and simultaneous tissue repair are quite urgent to be developed to improve patient outcomes, preferably non-surgical interventions.10

Recently, utilizing exogenous triggers, including photothermal therapy (PTT) based on near-infrared (NIR) light irradiation, has been widely applied for biomedicine.11-13


6

Page 7 of 78 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

ACS Nano

Many photothermal materials have been developed, including gold nanoparticles, carbon-based nanomaterials, melanin.12-14 Local heat triggered by a photothermal effect will effectively damage the bacterial membrane and cause bacterial death through hyperthermia.13-16 PTT has broad-spectrum antibacterial efficiency by physical heat, and relatively brief treatment (only a few minutes) can rapidly and efficiently kill bacteria.14-16 Simultaneously, NIR light during PTT demonstrates excellent capability of tissue penetration and minimal damage to healthy tissues.13,14,16 Consequently, PTT for bacterial infections using a biocompatible photothermal material (such as melanin) by NIR light irradiation has tremendous advantages to eliminate drug-resistant bacterial infections, including rapid MRSA eradication.

However, no appropriate PTT materials are available to combat MRSA while promoting tissue reconstruction. Nature possesses a wealth of extraordinary materials, which have evolved for billions of years and have become a continuing source of inspiration for scientists and engineers.1,6,17 Natural biocompatible polymers acquired from natural materials, such as keratin, collagen, melanin, gelatin, chitosan and


7

ACS Nano 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

Page 8 of 78

alginate, are regarded as the most promising materials for safe and effective therapy against various diseases in vivo.17 For instance, keratin, fibrous structural protein, can be extracted from hairs, nails, epithelial cells, etc.17 Collagens consist of ~25% of human dry body weight and constitute approximately one-third of all proteins in the body.18 These natural polymers have raised great interest for tissue repair in response to injury.19,20 However, the cellular mechanisms underlying tissue repair are still poorly understood.21 Advanced understanding of these mechanisms will be beneficial to the development of excellent biomaterials.

In this study, we evaluated the naturally derived HHMs nanostructure, comprising structural keratins and functional melanins, which combine biodegradability and biocompatibility with high photothermal-conversion efficiency. The melanin in HHMs can eradicate MRSA infection by "Lysozyme (Lyso)-assisted photothermy" in vitro and in

vivo. The keratin in HHMs can regulate collagen alpha chain proteins through the "protein digestion and absorption" signaling pathway to accelerate tissue repair, which is confirmed by the quantitative proteomic analysis in vivo. Notably, the 13 upregulated


8

Page 9 of 78 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

ACS Nano

collagen alpha chain proteins in the extracellular matrix (ECM) after HHMs treatment indicate that keratin from HHMs is an important regulator of collagen homoeostasis to augment wound closure. The current paradigm of natural material-tissue interaction can regulate cell-ECM interaction by targeting cell signaling pathways to accelerate tissue repair. This work may bring more insight and understanding into the protein-level pathways and potential mechanisms involved in tissue repair.

RESULTS AND DISCUSSION

Characterization of HHMs and HHMs-Lyso. Human hair has a natural hierarchical structure on the micro-/nano-scales that is similar to other natural materials, such as wool and bone.12,22 A typical hair fiber with a diameter of ~100 μm (Figure S1A) is composed of the core medulla (Figure S1B1), middle cortex (Figure S1B2), and outermost cuticle sheath (Figure S1B3), as observed by sectional human hair fibers (Figure 1A and Figure S1B), where the melanosomes are uniformly inlaid in the middle


9

ACS Nano 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

Page 10 of 78

cortex and core medulla (red arrows in Figure 1B). Based on the above analysis, the hierarchical structure of human hair is schematically illustrated in Figure S1C.

As schematically illustrated in Figure S1D, HHMs are easily extracted by a simple "low-temperature alkali heat" method, where 0.5 mol L-1 sodium hydroxide (0.5 M NaOH) and clean human hair are placed in a hydrothermal reactor at 50 °C for 12 h to obtain HHMs. This technique is easily available, ecofriendly and low cost. The mulberryshaped HHMs in Figure S1E and Figure 1C have an average width of ~270 nm and an average length of ~700 nm, in which the schematic illustration of HHMs and molecular structure of melanin within HHM are shown in Figure 1D. Additionally, black hair is chemically composed of structural keratin and functional melanin, where the melanin granules are uniformly distributed in the keratin matrix.12,22 Therefore, the HHMs are the perfect combination of structural keratins and functional melanins. Generally, melanin, a naturally occurring pigment, exists not only in hair but also in eye, inner ear, and skin of human body.12,22 Besides, the types and contents of amino acids within keratin of HHMs were systematically tested by using an amino acid analyzer machine in Figure 1E,


10

Page 11 of 78 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

ACS Nano

where the contents of 17 amino acids have a range from 2% to 18%. The specific contents of the 17 amino acids were shown in the supplementary discussion in Supporting Information. Generally, four main amino acids existing in keratin within HHMs are glutamic acid (Glu), cystine (Cys), aspartic acid (Asp), and serine (Ser). Importantly, keratin contains several beneficial peptide-binding motifs, including GluAsp-Ser and Leu-Asp-Val, which endows keratin with the capacity of cell adhesion by protein-ligand interactions.23

These natural amino acids endow keratin with excellent biocompatibility. Keratin contains several beneficial peptide-binding motifs to form specific binding sites to improve cell adhesion and cell growth by protein-ligand interactions.19 Many other wellknown extracellular matrix (ECM) proteins, including collagen, show the same capacity.20 Therefore, HHMs containing keratin might create a suitable environment for better cell adhesion and proliferation by cell-ECM interaction.

Additionally, the composites between HHMs and Lyso can be prepared by electrostatic interaction. The zeta potential measurements in Figure 1F show that the


11

ACS Nano 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

Page 12 of 78

negatively charged HHMs (-30.3 ± 1.2 mV) can absorb positively charged Lyso (20.1 ± 1.5 mV) to form the HHMs-Lyso composites (4.2 ± 0.3 mV). After Lyso loading, the surface potential of HHMs increased from -30.3 ± 1.2 mV to 4.2 ± 0.3 mV because the negatively charged HHMs are bonded and neutralized by the positively charged Lyso through electrostatic interaction.

Compared with the bare HHMs (Figure 1G and Figure 1G1), the TEM images of HHMs-Lyso (Figure 1H and Figure 1H1) indicate that Lyso is obviously adhered on the surface of HHMs after the modification. As shown in Figure 1I, the hydrodynamic average sizes of HHMs-Lyso (804.6 nm) are larger than those of HHMs (570.0 nm) measured by dynamic light scattering (DLS), which may be attributed to the fact that Lyso-attached HHMs lead to a slight conglomeration of HHMs. Additionally, the hydrodynamic average sizes of HHMs in PBS (583.8 nm) and in serum (556.9 nm) are performed by DLS in Figure S2. The digital photographs in Figure 1J display that the asprepared HHMs and HHMs-Lyso can be well dispersed in water, where the typical Tyndall effect of HHMs and HHMs-Lyso in water indicates their ability to scatter light. In


12

Page 13 of 78 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

ACS Nano

Figure 1K, the UV absorbance spectra of aqueous suspensions of dispersed Lyso at various concentrations (100, 200, 300, 400, 500, 1000, and 2000 ppm) were measured from 240 nm to 320 nm, and the maximum absorbance of dispersed Lyso lies at 281 nm. Besides, the corresponding values of absorbance at 281 nm exhibited excellent linear dependence (y = 1.30671x - 0.00627, R2 = 0.99477) with the corresponding concentrations in Figure 1L. This standard linear relationship can be used to calculate the amount of Lyso adsorbed to the HHMs. In Figure S3, the different mass ratios (Lyso:HHMs = 1, 2, 4, 8, 16) were set to determine the maximum amount of loading of Lyso, where the amount of HHMs was constant and the amount of Lyso varied (the details were shown in Supporting Information). The mass ratios of adsorbed Lyso:HHMs ranged from 0.51 to 0.63, so the maximum amount of loading was 1:0.63 (HHMs:Lyso, mass ratio) and the mass ratio of HHMs:Lyso (1:0.33) was selected for further study.

Biodegradation behavior of HHMs. Proper biodegradability in a timely manner is necessary for biomedical applications because poor biodegradability will cause the


13

ACS Nano 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

Page 14 of 78

long-term preservation of foreign substances in vivo, which may accentuate the risk of deleterious effects and trigger unpredictable adverse impacts.24,25 The relatively stable behavior of the HHMs in deionized (DI) water was evaluated without shaking at room temperature. The biodegradable behavior of the HHMs in phosphate-buffered saline (PBS; pH 7.4) was explored in a horizontal shaker at 37 °C, which is a suitable model to study biodegradation in vitro.26 As shown in Figure S4, HHMs in PBS in a horizontal shaker at 37 °C underwent relatively rapid degradation compared with those in water without shaking at room temperature over 8 weeks, which have the time-dependent biodegradable behavior. In Figure S4A and Figure S4B, over time, the absorbance of the HHMs gradually deteriorated for 8 weeks, accompanied by color fading and obvious morphological changes due to the gradual degradability of HHMs. In Figure S4C, the hydrodynamic sizes of HHMs measured by DLS gradually decreased over time, indicating the slow degradation of HHMs. Comparatively, in Figure S4D and Figure S4E, the relatively rapid biodegradability of the HHMs in PBS over 8 weeks was confirmed by the faster color fading, more obvious morphological changes, and lower absorbance than those in DI water. Besides, in Figure S4F, the degradability of the HHMs into


14

Page 15 of 78 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

ACS Nano

smaller nanoparticles in PBS was more rapid than that in DI water in Figure S4C, where the medium of PBS and the mechanical shake at 37 °C would accelerate the degradability of the HHMs. HHMs can be degraded into the smaller nanoparticles in

vitro in the absence of proteinase. Similarly, as shown in Figure S5, the stability of HHMs-Lyso has the same tendency with that of HHMs. The stability of HHM-Lyso will be further discussed below.

Based on the above analysis of the results, the possible process of biodegradability of the HHMs in a physiological environment is illustrated in Figure S4G. In Figure S6, the intensities of absorption spectra of the HHMs (200 ppm) in H2O2 solutions gradually decrease with the increase of the concentration of H2O2 solutions (1, 10, 100, 1000 mM) on Day 2, confirming that the melanin in HHMs has oxidation-induced degradation in the presence of oxidizing agents. Consequently, the HHMs have proper biodegradation behavior in a time-dependent manner with the characteristics of short-term relative stability and long-term biodegradation. Importantly, the more details of discussion about Figure S4, Figure S5, and Figure S6 are shown in Supporting Information.


15

ACS Nano 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

Page 16 of 78

Photothermal property of HHMs. It is worth noting that the internal melanins in HHMs have many promising functions. Commonly, melanin, a major component of the naturally occurring biopolymer, is also widely distributed in the human body and shows excellent biocompatibility.24 Moreover, the excellent photothermal-conversion efficiency of melanin has been reported.24 Therefore, the photothermal property of HHMs was comprehensively investigated (Figure 2). As shown in Figure 2A, the intensities of VisNIR absorbance curves (400-1000 nm) of the HHMs solution gradually increased in a concentration-dependent manner. Additionally, the temperature of HHMs solutions gradually increased in a power-dependent and concentration-dependent manner under 808 nm NIR light in 10 min. Specifically, the maximum temperature (Figure 2B) of 200 ppm HHMs solution in 10 min at various power densities (0.5, 1.0, and 1.5 W cm-2) increased to 54.7 °C, 67.7 °C, and 94.1 °C, respectively. The maximum temperature (Figure 2C) of HHMs solutions in 10 min at various concentrations (100, 200, and 400 ppm) at 1.0 W cm-2 increased to 53.2 °C, 67.7 °C, and 77.8 °C, respectively. Moreover,


16

Page 17 of 78 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

ACS Nano

the excellent photothermal reversibility and cycling stability of HHMs can be confirmed in Figure 2D. The recycling temperature variations of 200 ppm HHMs were recorded under 808 nm NIR laser radiation for 10 min (laser on), followed by natural cooling to room temperature for 20 min (laser off) for five laser on/off cycles. The photothermal performance of HHMs exhibited no significant deterioration during five on/off cycles, demonstrating the high stability and potential of HHMs as durable photothermal treatments. In Figure 2E, the photothermal-conversion efficiency (η) of HHMs (41.65%) was quantitatively calculated by the results of the time constant (τs = 241.5) and maximum steady-state temperature, further suggesting that the HHMs possess a high photothermal-conversion efficiency and can rapidly and efficiently convert NIR energy into thermal energy. In addition to the melanin in HHMs extracted from black hair, the HHMs extracted from brown hair also contained melanin, which had good photothermal effect. As shown in Figure S7, the maximum temperatures of 200 ppm HHMs solutions extracted from black hair and brown hair in 10 min at 1.0 W cm-2 increased to 67.7 °C and 62.5 °C, respectively.


17

ACS Nano 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

Page 18 of 78

Antibacterial activity in vitro. Figure 3A shows that HHMs and HHMs-Lyso at various concentrations (200 and 400 ppm) have low antibacterial activity (less than 30%) in the dark for 2 h (D2: Dark for 2 h). Based on the excellent photothermal-conversion efficiency of HHMs shown in Figure 2, the safe and appropriate PTT for bacterial infection was performed under 808 nm NIR light irradiation at a relatively low power density to maintain at 50 °C for 10 min (this treatment was marked as "L": Light). The locally increased temperature (50 °C) generated by 808 nm NIR light irradiation can lead to denaturation of bacterial proteins, lethality through hyperthermia, and irreversible bacterial destruction, eventually killing bacteria.15,27 The 400 ppm HHMs-Lyso (84.96 ± 5.63%) displayed greater bacterial killing efficiency than the 200 ppm HHMs-Lyso (75.72 ± 1.61%), 400 ppm HHMs (65.88 ± 3.21%), and 200 ppm HHMs (61.85 ± 6.77%) against MRSA under 808 nm NIR light irradiation to maintain at 50 °C for 10 min, as shown in Figure 3B. Lyso is an antimicrobial glycoside-hydrolase enzyme that can specifically attack the protective cell walls of bacteria and widely exists in avian egg


18

Page 19 of 78 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

ACS Nano

white and mammalian secretions (tears, saliva, milk and mucus).28 However, the use of Lyso alone to kill bacteria is often compromised by the limited stability and poor reusability of the enzyme.28,29 The way that HHMs improve the stability and reusability of the Lyso is to immobilize Lyso on the surface of HHMs and form high enzymatic loadings. As shown in Figure S8, free Lyso at various concentrations (25, 50, 100, and 200 ppm) for 10 min and 2 h incubation shows low antibacterial activity (less than 30%). Additionally, the activities of free Lyso and adsorbed Lyso (HHMs-Lyso) are compared in the case of the same concentration of Lyso (100 ppm) in Figure S9, where the relative activity of adsorbed Lyso has higher than that of free Lyso (control group). Besides, in order to evaluate the effect of temperature on the activity of Lyso, the Lyso solutions (100 ppm) are firstly incubated for 2 h at range of temperatures (25, 40, 50, 60, 70 °C), and then its relative activities are measured in Figure S10. Obviously, when the incubation temperature of Lyso is no more than 60 °C, the relative activities of Lyso have no significant differences compared with that at 25 °C (control group). Therefore, the local heat by photothermal effect of HHMs (50 °C for 10 min) will not decrease the activity of Lyso in HHMs-Lyso group.


19

ACS Nano 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

Page 20 of 78

There has a hypothesis that heat and Lyso from HHMs-Lyso have the optimized synergistic antibacterial action. In order to comprehensively verify this hypothesis, 808 nm NIR light was utilized to irradiate bacterial medium with 400 ppm HHMs-Lyso to maintain at 50 °C for 10 min at a specific setting point after culturing for 0, 1, and 2 h, as shown in Figure 3C. Compared with the control group, all the experimental groups with 400 ppm HHMs-Lyso showed definite antibacterial efficiency against MRSA, as shown in Figure 3D. Specifically, the D2 group exhibited the lowest antimicrobial activity, with antibacterial rates of 25.61 ± 10.18% against MRSA. The L+D2 group (97.90 ± 2.19%) displayed greater bacterial killing efficiency against MRSA than the D1+L+D1 group (90.50 ± 1.38%) and the D2+L group (87.29 ± 2.46%), demonstrating that the treatment of L+D2 has the optimized synergistic antibacterial action of heat and Lyso from HHMsLyso. Additionally, in the L+D2 group, the antibacterial activity of HHMs (67.21 ± 3.28% for 200 ppm and 69.63 ± 3.62% for 400 ppm) was lower than that of HHMs-Lyso (93.06 ± 3.06% for 200 ppm and 97.90 ± 2.19% for 400 ppm) in Figure 3E, further suggesting the synergistic antimicrobial action of heat and Lyso. This enhanced efficiency of bacterial inactivation by different treatment can be attributed to "Lyso-assisted


20

Page 21 of 78 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

ACS Nano

photothermy". That is, the photothermal effect of HHMs-Lyso will effectively damage the bacterial membrane and increase its permeability, leading to further enhancement of Lyso efficiency to destroy bacterial cell walls and disturbance of the intracellular metabolic pathways of bacteria. The related mechanism will be discussed later. The stability of HHMs-Lyso was further evaluated by the corresponding antibacterial activity of HHMs-Lyso by the treatment of L+D2 after 1, 3, and 7 days without shaking at room temperature in Figure S11, indicating the characteristics of short-term relative stability of HHMs-Lyso.

Antibacterial mechanism. The morphologies of MRSA interacting with HHMs and HHMs-Lyso in the D2 groups and L+D2 groups were qualitatively evaluated by SEM observation. In Figure 3F, the morphologies of MRSA in the control groups (D2 and L+D2) were regular spherical shapes with intact bacterial membranes. Additionally, the bacteria in the D2 groups had tight contacts with HHMs and HHMs-Lyso (marked by white arrows). By contrast, the morphologies of MRSA treated with HHMs and HHMs-


21

ACS Nano 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

Page 22 of 78

Lyso in the L+D2 groups became irregular, corrugated, or even partly lysed (marked by blue arrows). Moreover, the degree of bacterial destruction of HHMs-Lyso was more serious than that in the L+D2 group. Moreover, the broad-spectrum bactericidal action of HHMs and HHMs-Lyso was confirmed in Figure S12. Similarly, compared with E. coli in the control groups (D2 and L+D2), E. coli interacting with HHMs and HHMs-Lyso in the L+D2 groups were corrugated, distorted or even partly lysed. Moreover, the bacteria treated with HHMs-Lyso showed more serious destruction than those treated with HHMs (Figure S12A), where the antibacterial rate of 400 ppm HHMs-Lyso (99.11 ± 0.49%) was higher than that of 400 ppm HHMs (74.94 ± 1.69%) for L+D2 treatment in Figure S12B.

The possible mechanism of "Lyso-assisted photothermy" is schematically illustrated in Figure 3F. First, the HHMs and HHMs-Lyso have tight contact with bacteria, and the local heat generated by the photothermal effect of HHMs under NIR light irradiation will effectively damage the bacterial membrane and increase its permeability, further favoring the Lyso-bacteria interactions. Additionally, Lyso can damage the structure of


22

Page 23 of 78 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

ACS Nano

bacterial cell walls by catalyzing the hydrolysis of the β-1,4 glycosidic bonds between Nacetylmuramic acid (NAM) and N-acetylglucosamine (NAG), which are characteristic peptidoglycans in the bacteria cell wall.28 The destruction of the bacterial membrane and bacterial cell walls will conversely lead to the reduction of the heat resistance of bacteria. To sum up, the accelerated bacteriolysis of HHMs-Lyso by the synergistic effect between local photothermy and biocatalytic hydrolysis of glycosidic linkages by Lyso-bacteria interactions can greatly enhance the bactericidal efficiency. The collaborative action of local photothermy and biocatalytic hydrolysis of glycosidic linkages by Lyso provides a broad-spectrum antibacterial strategy and brings more insight and understanding of antibacterial application.

In vivo therapeutic efficacy for MRSA wound infections. Encouraged by the above antibacterial mechanism, the in vivo therapeutic efficacy of HHMs and HHMs-Lyso was assessed using an animal MRSA wound model, and the related scheme is shown in Figure 3G, where the synergistic antibacterial action of heat and Lyso of HHMs-Lyso


23

ACS Nano 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

Page 24 of 78

can rapidly eradicate MRSA in wounds. MRSA-infected mice were exposed to the 808 nm NIR laser at a power density of less than 0.35 W cm−2, which meets the requirement within the scope of maximum permissible exposure (MPE) for skin exposure formulated by the American National Standards Institute (ANSI Z136.1-2000), and the infected wound-site temperature was maintained at 50 °C for 10 min of laser irradiation. In comparison, the temperature of the control group under only 808 nm NIR laser irradiation showed no significant change (Figure S13). At 1 and 2 days post infection, the bacterial counts in MRSA-infected wounds were quantitatively evaluated using the spread plate method in Figure 3H. Obviously, the HHMs-Lyso groups exhibited greater bacterial killing efficiency on day 1 (96.14 ± 3.38%) and day 2 (97.19 ± 2.39%) than the HHMs groups on day 1 (70.80 ± 3.94%) and day 2 (81.01 ± 4.50%) against MRSA in

vivo, which is well consistent with the antibacterial activity in vitro. Additionally, the treatment with vancomycin, the gold standard for current clinical MRSA treatment, is set as a positive control for conventional antibiotic therapy. In Figure S14, the treatment of vancomycin shows the relatively low antibacterial rates (78.10 ± 3.73% on day 1 and 87.75 ± 2.02% on day 2) against MRSA infection compared with the HHMs-Lyso group,


24

Page 25 of 78 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

ACS Nano

demonstrating the potential advantage of HHMs-Lyso over the conventional antibiotic therapy.

Many lobulated neutrophils indicated by hematoxylin and eosin (H&E) staining (red arrows in Figure S15) can be observed in the wounds in the control groups, where many neutrophils in wounds represent serious bacterial infection because neutrophils will migrate rapidly from circulating blood to infected sites.30 In comparison, the percentage of neutrophils (versus all cells) in the HHMs (23.64 ± 5.03%) and HHMsLyso (20.13 ± 2.81%) groups was fewer than that in the control (49.94 ± 7.89%) groups on day 2 in Figure 3I by corresponding H&E staining (Figure S15), illustrating the relatively minor infection and effective antibacterial ability of HHMs and HHMs-Lyso in

vivo. Over time, the percentage of neutrophils gradually decreases, and the HHMs and HHMs-Lyso groups still comprise a lower percentage of neutrophils than the control groups. In Figure S15, compared with the control group, a lot of round and pink tissues in the HHMs and HHMs-Lyso groups are normal muscle tissues, indicating that the treatments by HHMs and HHMs-Lyso have negligible damage to normal tissues.


25

ACS Nano 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

Page 26 of 78

Besides, increased connective tissues (loose structure) in subcutaneous area on Day 12 reflect the process of wound healing after infection.

The representative photographs of the wound-healing process exhibit significant differences between the experimental groups and control groups in Figure S16. By contrast, the HHMs and HHMs-Lyso groups clearly show accelerated wound closure compared with the control groups, demonstrating that the HHMs and HHMs-Lyso can significantly promote wound healing for MRSA infections. In Figure 3J, the quantitative analysis of the above images of wounds over time also confirmed that the wound closure rate of the HHMs and HHMs-Lyso groups was significantly faster than that of the control groups. The ability of HHMs to improve wound healing in vivo will be systematically discussed later.

In vivo toxicology. The in vivo toxicology was examined by the corresponding histological analyses of major organs (heart, liver, spleen, lung, and kidney) through H&E staining on day 12, as shown in Figure S17. No significant differences and no sign


26

Page 27 of 78 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

ACS Nano

of organ damage between the control group and experimental groups were found, indicating that HHMs and HHMs-Lyso have no apparent histological toxicology in vivo.

In vitro cytocompatibility evaluation. As shown in Figure S18, the cell morphologies were evaluated by staining with FITC and DAPI to visualize the F-actin and nuclei, respectively. Compared with the control group, the cells spread well and exhibited a polygonal morphology in all the experimental groups (200 and 400 ppm) after incubation for 1 day without 808 nm NIR light. The abundant cellular extension and polygonal shape with a large number of filopodia and lamellipodia in the HHMs and HHMs-Lyso groups indicated the excellent cytocompatibility without appreciable cytotoxicity, suggesting that HHMs favor the growth and proliferation of cells. By contrast, the cells in the HHMs and HHMs-Lyso groups showed a relatively spherical morphology without obvious filopodia extensions under 808 nm NIR light irradiation to maintain at 50 °C for 10 min, followed by incubation for 24 h at 37 °C compared with the control group,


27

ACS Nano 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

Page 28 of 78

indicating that local heat triggered by the photothermal effect of HHMs has certain cytotoxicity to cells.

The viability of cells treated with HHMs and HHMs-Lyso is shown in Figure S19. Generally, the HHMs and HHMs-Lyso groups at various concentrations (200 and 400 ppm) exhibited excellent cytocompatibility. Specifically, the largest cell viability was 181.42 ± 9.86% in the 400 ppm HHMs group on day 7, and the lowest cell viability was 99.44 ± 8.28% in the 400 ppm HHMs-Lyso group on day 1. Over time, the experimental groups, especially the HHMs group, gradually showed increased cell viability compared with that in the control group, suggesting that HHMs can promote cell proliferation. Additionally, the cell viability of bare Lyso was assessed in Figure S20, indicating that Lyso has no significant effect on cell proliferation over time. According to the above results in Figure S19 and Figure S20, HHMs but not Lyso, improved cell proliferation. The ability of HHMs to improve cell proliferation was attributed to the external keratin of HHMs, where keratin contains several beneficial peptide-binding motifs to form specific binding sites to improve cell adhesion and cell growth by protein-ligand interactions.19


28

Page 29 of 78 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

ACS Nano

Therefore, biodegradable HHMs containing keratin will create a suitable environment for better cell adhesion and proliferation by cell-ECM interaction.

After 808 nm NIR light irradiation to maintain at 50 °C for 10 min, followed by incubation for 1, 3, and 7 days at 37 °C, as shown in Figure S21, the HHMs and HHMsLyso groups exhibited definite cell toxicity on day 1 compared with that in the control group. The maximum cytotoxicity of the 400 ppm HHMs-Lyso groups on day 1 revealed a cell viability of 68.30 ± 10.03%. However, the cell viabilities of the HHMs group (116.68 ± 10.27% for 200 ppm and 119.96 ± 3.80% for 400 ppm) and HHMs-Lyso group (111.88 ± 2.44% for 200 ppm and 114.62 ± 2.08% for 400 ppm) on day 3 are much higher than those on day 1, confirming that the cytotoxicity induced by the local heat of photothermy can be significantly relieved with the increase in incubation time.

Related mechanism of tissue repair by HHMs-tissue interaction. According to the above cytocompatibility evaluation in vitro, the ability of bare Lyso to perform tissue repair was first assessed in Figure S22, indicating that bare Lyso cannot perform tissue repair. A


29

ACS Nano 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

Page 30 of 78

hypothesis that HHMs can favor tissue repair was proposed. Therefore, the ability of bare HHMs to promote tissue repair was carefully evaluated. First, the representative photographs of the wound-healing process are shown in Figure S23. By contrast, the HHMs groups clearly exhibited accelerated wound closure compared with the control groups, demonstrating that the HHMs can significantly promote wound healing. In Figure 4A, quantitative analysis of the images corresponding to Figure S23 over time also showed that the wound closure rate of the HHMs groups is much faster than that of the control groups. The ability of HHMs to improve tissue repair in vivo was attributed to the external keratin of HHMs that represents one of the intermediate filaments involved in regulating cell behaviors, including cell adhesion, migration, and proliferation. Therefore, regulating cell-ECM interaction by the keratin of biodegradable HHMs will create a suitable environment to accelerate tissue repair in vivo, and the related mechanism will be comprehensively and systematically explored subsequently by proteomics.


30

Page 31 of 78 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

ACS Nano

Next, Masson's trichrome staining and Sirius red staining were used to detect collagen deposition (blue color for Masson's trichrome staining and red color for Sirius red staining, respectively) in the wounds.31 Collagen is essential for the proliferative and remodeling phases of the tissue repair process, which offers a foundation for matrix formation. Obviously, the HHMs groups have significantly higher levels of collagen deposition than the control groups on day 12, as shown in Figure S24. To further evaluate collagen accumulation within the wound, the relatively quantitative areas of collagen, as detected by Masson's trichrome staining and Sirius red staining (corresponding to different high magnifications in Figure S24) are shown in Figure 4B and Figure 4C. Consistently, the ratios of collagen-occupied regions of the HHMs groups (88.16 ± 1.22% for Masson's trichrome staining and 65.36 ± 2.38% for Sirius red staining) were higher than those of the control groups (49.58 ± 8.80% for Masson's trichrome staining and 44.04 ± 5.91% for Sirius red staining) on day 12. Collagen in tissues possesses many inherent properties, including cell recognition signals, ability to form 3D scaffolds of various physical conformations, controllable mechanical properties, and biodegradability.32 Therefore, collagen is indispensable for maintaining the


31

ACS Nano 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

Page 32 of 78

structural and biological integrity of tissues, and the architecture of the collagen matrix is central to tissue function.33

To further explore the mechanism of accelerated tissue repair of mouse skin by HHMs treatment, quantitative proteomic analysis by Tandem mass tag (TMT) technology was performed to study the protein changes in the wounds on the dorsum of C57BL/6 mice without and with HHMs treatment. The results of the volcano plot (Figure 4D) of the TMT-based proteomics showed 3,940 differential proteins (P < 0.05; additional details are shown in Supplementary Table S1 to Supplementary Table S3), where the 231 downregulated proteins (cutoff value of 0.83-fold for expressed variation) and 243 upregulated proteins (cutoff value of 1.2-fold for expressed variation) represented a wide range of biological changes due to HHMs treatment. These differential proteins were shown in the heatmap analysis (Figure 4E and Supplementary Table S4), indicating excellent intra-group and inter-group parallelism in general.

As shown in Figure 4F and Supplementary Table S5, according to Gene Ontology (GO) classification, all differential proteins were typically divided into three classes


32

Page 33 of 78 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

ACS Nano

(biological process, molecular function, and cellular component), which were further enriched in the top 20 terms for detailed investigation. Obviously, a higher number of identified proteins focused on cellular component (especially extracellular region, extracellular space, and extracellular region part) in GO enrichment analysis, suggesting that the significant and essential changes occurred in the extracellular region. Various components in ECM provide the physical and biochemical signals for directing cell fate, such as cell recognition, cell attachment, cell spreading, cell growth, cell proliferation, cell migration, and cell differentiation, particularly in wound healing and tissue regeneration.20,33 Some structural proteins and integrin-binding proteins in the ECM, including collagen, possess cell adhesion motifs such as the RGD (Arg-Gly-Asp) motif that are key components in cell-cell, cell-ECM, and cell-soluble molecule interactions.20

Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations of identified proteins were used to explore the underlying signaling pathways and processes following HHMs treatment (Supplementary Table S6), and the enriched


33

ACS Nano 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

Page 34 of 78

KEGG pathways in the top 5 terms are shown in Figure 4G. Among them, the most prominent pathway was the "protein digestion and absorption" signaling pathway. In the signaling pathway, protein is central to nutritional homeostasis, and ingested protein normally undergoes a complex series of degradative processes following the action of various enzymes, where the product reacting with the proteolytic enzymes is a mixture of small peptides and amino acids. Amino acids are transported into diverse cells by various amino acid transporters and small peptides can be absorbed into the specific cells. Additionally, these resulting amino acids can be absorbed by amino acid transporters into blood.

In the current study, the activated "protein digestion and absorption" signaling pathway plays an essential role in the control of protease-mediated degradation and protein synthesis. That is, the external keratin of HHMs reacting with specific proteolytic enzymes will be degraded into amino acids and small peptides, where amino acids will be transported into diverse cells by various amino acid transporters and small peptides will be absorbed into specific cells. Furthermore, this process may regulate protein


34

Page 35 of 78 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

ACS Nano

synthesis in the damaged tissue. Therefore, the protein-protein interactions (PPI) network analysis located in the "protein digestion and absorption" signaling pathway was evaluated in Figure 4H. Each correlation degree of 13 proteins from the PPI network was more than 11, suggesting that these proteins in the "protein digestion and absorption" signaling pathway have very strong interactions with each other. These 13 proteins are all collagen alpha chain proteins, including collagen alpha-4(IV) chain (Col4a4), collagen alpha-1(IV) chain (Col4a1), collagen alpha-1(I) chain (Col1a1), collagen alpha-2(I) chain (Col1a2), collagen alpha-1(XXIV) chain (Col24a1), Collagen alpha-1(II) chain (Col2a1), collagen alpha-1(III) chain (Col3a1), collagen alpha-3(IV) chain (Col4a3), collagen alpha-1(V) chain (Col5a1), collagen alpha-1(XI) chain (Col11a1), collagen alpha-2(V) chain (Col5a2), collagen alpha-1(XV) chain (Col15a1), and collagen alpha-1(XIV) chain (Col14a1).

As shown in Figure 5A, the enrichment of 13 collagen alpha chain proteins in the HHMs group was increased, varying from 189.69 ± 46.13% to 121.65 ± 1.58% compared with that of the control group. The 13 upregulated collagen alpha chain


35

ACS Nano 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

Page 36 of 78

proteins further confirm strong interactions with each other in the activated signaling pathway of "protein digestion and absorption", consistent with increased collagen deposition in Figure 4B and Figure 4C. To further confirm upregulation of the collagen alpha chain proteins, the Col14a1 protein, with a minimum upregulated fold (121.65 ± 1.58%), was selected for more accurate analysis based on the results of the proteomics and PPI network analysis. Parallel reaction monitoring (PRM) technology was performed to measure quantitative Col14a1 protein enrichment. First, the MS spectra of the specific peptide sequences in Col14a1 protein are shown in Figure S25, and the corresponding MS/MS spectra are also exhibited (NLVVDDETATSLR in Figure S26, IGILITDGK in Figure S27, and VTVTPVYTVGEGVSVSAPGK in Figure S28) As shown in Figure 5B, the enrichment of the three peptide sequences were quantitatively calculated to comprehensively evaluate of the enrichment of the Col14a1 protein. Additional details are shown in Figure S29 to Figure S32. Similar to the results of proteomics, the HHMs have a stronger increase (232.26 ± 25.43%) in the Col14a1 protein enrichment than the control, fully confirming upregulation of the collagen alpha chain proteins corresponding to the results of proteomics.


36

Page 37 of 78 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

ACS Nano

Functionally, the 13 collagen alpha chain proteins, as an ECM structural constituent, are central to many biological processes, including cell adhesion, cell migration, cell spreading, cell differentiation, skin development, and wound healing. In general, collagen is the most abundant extracellular matrix protein and a fundamental structural component of connective tissues, which will self-assemble into cross-striated fibrils to provide support for cell growth and mechanical resilience of connective tissues.32 Collagens are important for almost all cell-cell interactions and cell attachment, which play key roles in tissue repair.34 Collagen-dependent cellular interactions are revealed by cell adhesion on collagen through specific receptors on the cell surface (collagenbinding integrin receptors).34-37

In Figure 5C, the potential mechanism involved in tissue repair after HHMs treatment is systematically shown. That is, the external keratin of HHMs containing several beneficial peptide-binding motifs can form specific binding sites to regulate cell behaviors by protein-ligand interactions.19 Moreover, the external keratin of natural biodegradable HHMs by specific proteolytic enzymes will be degraded into amino acids


37

ACS Nano 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

Page 38 of 78

and small peptides, where amino acids will be transported into diverse cells by various amino acid transporters and small peptides will be absorbed into the specific cells. Furthermore, this process will activate the "protein digestion and absorption" signaling pathway to regulate collagen alpha chain proteins synthesis. Next, the increased collagen synthesis will promote collagen fibrils in ECM,38 where the self-assembled collagen fibrils provide both specific mechanical properties to handle external stresses on the damaged tissues and well-defined protein binding sites on collagen fibrils to interact with cells by collagen-binding integrin receptors in the cell-ECM interaction.36 Finally, the improvement of cell behaviors (cell recognition, adhesion, spreading, growth, proliferation, and differentiation) will accelerate tissue repair.

In summary, keratin from HHMs is an important regulator of collagen homoeostasis through the "protein digestion and absorption" signaling pathway and promotes collagen synthesis by the corresponding cell-mediated processes. Conversely, cell-collagen interaction can further accelerate the process of tissue repair. Our proteomic analysis provides insight into the protein-level pathways involved in natural material-tissue


38

Page 39 of 78 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

ACS Nano

interactions and the potential mechanisms involved in tissue repair. Therefore, the advantage of the current design is that HHMs can not only rapidly eradicate MRSA infection and but also efficiently accelerate tissue repair compared with other photothermal materials in many researches.39-41 This study may inspire the potentially personalized therapy for bacterial elimination and simultaneous tissue repair by using patients' hairs themselves.

CONCLUSION

HHMs, naturally derived biodegradable nanostructures, contain structural keratins and functional melanins, combining biocompatibility with high photothermal-conversion efficiency. These mulberry-shaped HHMs possess an average width of ~270 nm and an average length of ~700 nm, and the negatively charged HHMs can absorb positively charged Lyso to form the HHMs-Lyso composites through electrostatic interaction. Eradication of MRSA infection can be achieved by synergistic action of photothermy and "Lyso-assisted anti-infection". Accelerated tissue repair after HHMs treatment can


39

ACS Nano 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

Page 40 of 78

be realized by regulation of collagen alpha chain proteins. Therefore, HHMs offer both challenges and opportunities as a natural biomaterial of choice to eradicate drugresistant bacterial infections and drive enhanced tissue repair. The identification of keratin from HHMs as a collagen regulator through the signaling pathway of "protein digestion and absorption" provides further understanding of protein-level pathways and the potential mechanisms involved in tissue repair.

EXPERIMENTAL SECTION

Materials. Human hair was mainly collected from the first author in this paper, and partly obtained from the other members of our research group. Sodium hydroxide (NaOH) was purchased from Sinopharm Chemical Reagent Co. (China). Lysozyme was purchased from Shanghai Yeasen Biotechnology Co., Ltd.

Extraction of HHMs from human hair. The human hair was cut into small pieces, and then ultrasonically cleaned for15 min in deionized water for three times in order to remove contaminants. The cleaned hair was added into 60 mL NaOH (0.5 M) and then


40

Page 41 of 78 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

ACS Nano

hydrothermally treated at 50 °C in 100 mL autoclave for 12 h. After reaction, the autoclave was cooled down naturally to room temperature. The resulting solution was centrifuged at 9500 rpm for 15 min to obtain HHMs. The HHMs were ultrasonically washed with deionized water and centrifuged at 9500 rpm for 15 min for three times, and then dried in 40 °C under vacuum. Subsequently, the HHM powders were stored under argon (Ar) atmosphere until use.

Preparation of HHMs-Lyso. First, the maximum Lyso-loading amounts on HHMs through electrostatic interaction were characterized. Specifically, the as-prepared 4 mg HHMs powders were uniformly dispersed in 5 mL deionized water. Simultaneously, the 4, 8, 16, 32, 64 mg Lyso powders were dissolved in 5 mL and then were mixed with the above HHMs solutions. The mixed solution was stirred at room temperature for 6 h and centrifuged at 15000 rpm for 30 min to obtain a supernatant of free Lyso and a precipitate of HHMs-Lyso. The HHMs-Lyso was ultrasonically washed with deionized water and centrifuged at 15000 rpm for 30 min three times. The corresponding supernatants of free Lyso were collected and filtered through a 220 nm filter, then the


41

ACS Nano 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

Page 42 of 78

concentration of unbounded Lyso was calculated by spectrophotometric protein measurement at 281 nm using a UV-Vis spectrophotometer (UV-3600, Shimadu, JP). Thus, the amount of Lyso attached to the HHMs was calculated for the initial and final Lyso concentrations of the immobilization medium.

The as-prepared 30 mg HHM powders were uniformly dispersed in 10 mL deionized water. Simultaneously, the 10 mg Lyso powders were dissolved in 10 mL and then mixed with the above HHMs solution to obtain HHMs-Lyso solution (2 mg/mL). The HHMs-Lyso solution was stirred at room temperature for 6 h for immediate use and not stored.

Characterization. The surface morphology was investigated by field-emission SEM (JSM7100F, JEOL, JP) and SEM (JEOL-820 and JSM6510LV). A high resolution transmission electron microscope (HRTEM; JEM-2100F) was employed to observe the microstructure of the samples. The zeta potential and hydrodynamic size of the samples were measured using a Zeta Sizer Nano series (Nano-ZS, Malvern Instruments Ltd.).


42

Page 43 of 78 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

ACS Nano

UV-Vis-NIR absorption was examined using a microplate reader (SpectraMax I3MD USA).

Amino acid analysis of HHMs. The HHMs sample was dissolved in 6 mol/L HCl (Sinopharm Chemical Reagent Co., China) and was hydrolyzed at 110 °C for 22 h under nitrogen protection. After cooling, the solution was diluted with water. Thereafter, the treated sample was dried at 55 °C under nitrogen atmosphere, and then distilled water was added to dissolve the treated sample again. This process was repeated three times. Next, the treated sample was fully dissolved in 0.02 mol/L HCl and filtered with 0.45 μm filter, and then the filtered liquid was injected into the amino acid analyzer machine (L8900) for amino acid analysis.

Measurements of photothermal performance. The temperature trends of HHMs with different concentrations (100, 200, and 400 ppm) at various power densities (0.5, 1.0, and 1.5 W cm-2) were measured under irradiation using an 808 nm laser (Hi-Tech Optoelectronics Co., Lid, China). Specifically, 0.5 mL HHMs solution in an EP tube (Eppendorf tube) was irradiated under an 808 nm NIR laser for 10 min. The temperature


43

ACS Nano 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

Page 44 of 78

of the HHMs solutions was monitored and recorded using an FLIR thermal camera (FLIR-E64501, Estonia). The photothermal conversion efficiencies (η) of HHMs can be calculated by the following equation:



hS (Tmax  Tamb )  Q0 I (1  10 A ) ,

where h indicates the heat transfer coefficient, S represents the surface area of the container, Tmax is the equilibrium temperature, Tamb indicates the surrounding ambient temperature, Q0 represents heat absorption of the EP tube (Eppendorf tube), I is the laser power, and A represents the absorbance of HHMs at 808 nm.

When the heat input of the system is equal to the heat output,

hS 



i

mi C p ,i

S



mH 2O CH 2O

S

,

in which mH2O is the weight of water, CH2O represents the specific heat capacity of water, and s indicates the time constant of HHMs. At the cooling period,

t   S ln    S ln

T  Tamb Tmax  Tamb .


44

Page 45 of 78 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

ACS Nano

Therefore, s can be calculated by the linear regression curve through the above equation.25

Antibacterial experiments in vitro. The in vitro antibacterial activity of HHMs against Gram-positive MRSA and Gram-negative E. coli was quantitatively assessed by the spread plate method. The bacteria were cultured in standard Luria-Bertani (LB) culture medium. Next, 200 μL bacterial suspensions (5 × 106 CFU/mL) were incubated with control (PBS), HHMs, and HHMs-Lyso in a 96-well plate without and with an 808 nm laser to maintain at 50 °C for 10 min. After these treatments, 20 μL of appropriate diluted bacterial solution was plated on standard LB agar and incubated at 37 °C for another 24 h. The bacterial colonies were counted, and the corresponding antibacterial ratio was calculated by the formula: Antibacterial ratio (%) = (A − B)/A *100%, where A is the average number of bacteria colonies in the control group (CFU/sample) and B is the average number of bacteria colonies in the experimental group (CFU/sample).

The morphology of bacteria (MRSA and E. coli) interacting with HHMs and HHMsLyso by various treatments was qualitatively evaluated by SEM observation. The


45

ACS Nano 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

Page 46 of 78

treated bacteria were fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., China) solution for 2 h and were rinsed with sterile PBS twice. Next, the treated bacteria were sequentially dehydrated in ethanol (Sinopharm Chemical Reagent Co., China) solution with different concentrations (30, 50, 70, 90 and 100%, v/v) for 15 min. Next, the treated bacteria were air-dried overnight before SEM observation.

Measurements of Lyso activity. The enzymatic activity of Lyso was measured by bacteriolysis, which has biocatalytic hydrolysis of glycosidic linkages by Lyso-bacteria interactions. Therefore, the Lyso activity has the same tendency with the corresponding antibacterial activity.28 Briefly, the bacterial suspensions (5 × 106 CFU/mL) were incubated with free Lyso or adsorbed Lyso (HHMs-Lyso) solution for 2 h at 37 °C. Then, 20 μL of appropriate diluted bacterial solution was plated on standard LB agar and incubated at 37 °C for another 24 h. The bacterial colonies were counted, and the corresponding antibacterial ratio was calculated by the formula as mentioned above. Then, the relative activities of Lyso (%) = D/C *100%, where C is the antibacterial ratio in the control group and D is the antibacterial ratio in the experimental group. Besides,


46

Page 47 of 78 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

ACS Nano

in order to evaluate the effect of temperature on the activity of Lyso, the Lyso solutions (100 ppm) are firstly incubated for 2 h at range of temperatures (25, 40, 50, 60, 70 °C), and then its relative activities are measured.

In vitro cytocompatibility evaluation. NIH-3T3 cells (mouse embryonic fibroblast cell line) were cultured in MEM/EBSS medium (HyClone) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution (HyClone), and 1% amino acid in an atmosphere of 5% CO2 and 95% humidity at 37 °C. The culture medium was changed every 3 days.

For fluorescent morphology of cells, NIH-3T3 cells were first incubated in 96-well plates for 1 day at 37 °C, and were further incubated with HHMs and HHMs-Lyso with various concentrations (200 and 400 ppm) without and with an 808 nm laser to maintain at 50 °C for 10 min. The treated cells were cultured for another 1 day, 3 days, and 7 days. After co-incubation, the culture medium was removed, and the treated cells were washed with PBS three times and fixed with 4% formaldehyde (Sinopharm Chemical Reagent Co., China) for 10 min, followed by rinsing with PBS three times. Next, the


47

ACS Nano 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

Page 48 of 78

treated cells were stained with fluorescein isothiocyanate (FITC; YiSen, Shanghai) for 30 min in a dark environment, washed with PBS three times, and further stained with 4',6-diamidino 2-phenylindole (DAPI; YiSen, Shanghai) for 30 s in a dark environment and washed with PBS three times. Cytoskeletal actin (green fluorescence) was stained by FITC, and the cell nuclei (blue fluorescence) were stained with DAPI. Finally, the cell morphology was photographed using a fluorescence microscope (IFM, Olympus, IX73).

For the in vitro cytotoxicity assay, NIH-3T3 cells were first seeded for 1 day in 96-well plates. Next, HHMs and HHMs-Lyso at various concentrations (200 and 400 ppm) were co-incubated with cells in 96-well plates without and with an 808 nm laser to maintain at 50 °C for 10 min, followed by co-culture for another 1 day, 3 days, and 7 days. The standard MTT assays were performed to measure the relative cell activity. Specifically, after co-incubation for specific periods, the medium was removed and 0.5 mg/mL MTT (Aladdin Reagent Co., China) solution was added, followed by incubation for 4 h at 37 °C. Next, MTT solution was removed, and 200 μL DMSO was added to dissolve the crystals under vibration for 15 min. The 96-well plates were centrifuged at 500 rpm for 5


48

Page 49 of 78 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

ACS Nano

min. Next, the absorption of the corresponding supernatant at 570 nm was examined on a microplate reader (SpectraMax I3MD USA). Finally, the cell viability (%) was calculated by comparing the absorbance values of the experimental group with those of the control group.

Mouse cutaneous wound model and MRSA wound infection model. All the animal experiments and procedures in this study were approved by the Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All animals were kept and utilized in accordance with the Animal Management Rules of the Ministry of Health of the People's Republic of China and the Guidelines for the Care and Use of Laboratory Animals of China. Eightweek-old C57BL/6 mice were obtained from the Hubei Provincial Centers for Disease Prevention & Control. Sixty C57BL/6 mice were randomly divided into three groups as follows: control, HHMs, and HHMs-Lyso. First, prior to surgery, the mice were anaesthetized by the intraperitoneal injection of 3% pentobarbital (1 mL/kg) and were placed on a sterile drape to provide sterile conditions during surgery. Next, the dorsal


49

ACS Nano 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

Page 50 of 78

side of the mice was shaved, depilated and disinfected. Next, two symmetrical round wounds with 6 mm in diameter were made on the dorsum of each mouse using a biopsy punch. The mouse cutaneous wound model was accomplished.

For the mouse cutaneous MRSA wound infection model, each wound of the mice from each group was firstly infected with an MRSA dose of 107 CFU per wound. After 30 min, each wound in control goups was directly treated with 20 μL PBS solution, then irradiated with an 808 nm laser for 10 min. After 30 min, each wound in experimental groups was directly administered with 20 μL HHMs (4 mg/mL) or 20 μL HHMs-Lyso (4 mg/mL), then irradiated with an 808 nm laser to maintain at 50 °C for 10 min. Comparatively, the treatment with Vancomycin (40 mg/kg/day) is chosen as positive controls for conventional antibiotic therapy of MRSA-infected wound through intravenous injection. Next, the treated wounds were covered with nonwoven fabrics and fixed with a surgical adhesive. To evaluate the therapeutic effect of HHMs and HHMs-Lyso for MRSA wound infection, the bacterial counts in each wound were


50

Page 51 of 78 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

ACS Nano

assessed by the spread plate method at different times (day 1 and day 2), and the wounds were photographed at regular time intervals.

For histomorphological analysis, the mice were sacrificed, and the corresponding skin tissues of wounds were obtained and fixed with 4% paraformaldehyde solution, followed by paraffin embedding. Hematoxylin and eosin (H&E) staining was performed to examine bacterial infection in wounds on days 2, 4, 8, and 12. Additionally, Masson's trichrome staining on day 12 and Sirius red staining on day 12 were used to detect collagen deposition. For in vivo biosafety, the major organs (heart, liver, spleen, lung, and kidney) were stained with H&E staining on day 12.

Tandem mass tag (TMT) technology for quantitative proteomic analysis. In the current study, the tissue samples for proteomic analysis were provided by the corresponding skin tissues of wounds on day 12 without or with HHMs treatment. The tissue samples were prepared by homogenate and SDT Lysis.42 Briefly, SDT buffer (1 mM DTT, 100 mM Tris-HCl, 4% SDS, pH 7.6) was mixed with the tissue samples, followed by transfer to tubes with quartz sand (MP 6910-050) and other ceramic beads (MP 6540-034). The


51

ACS Nano 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

Page 52 of 78

corresponding lysate was homogenized using an MP Fastprep-24 automated homogenizer (MP Biomedicals). The homogenate was sonicated (1 cycle: 80 W for 10 s ，remaining cycles for 15 s; 10 cycles) by ultrasonic liquid processors and then boiled for 15 min. Next, after centrifugation for 40 min at 14000 g, the supernatant was filtered with 0.22 µm filters and the corresponding filtrate was quantified with the BCA Protein Assay Kit. The sample was stored at -80 °C.

SDS-PAGE separation was operated to isolate and visualize the corresponding proteins. The enzymolysis process of protein was performed by Filter-aided sample preparation (FASP) method.43 The peptide mixture of each sample was marked using the TMT 6/10 plex Isobaric Label reagent (Thermo). TMT-labeled samples were fractionated and digested into 15 fractions using the Pierce high pH reversed-phase peptide fractionation kit (Thermo scientific) and an increasing acetonitrile step-gradient elution according to the instructions. Each fraction was performed using nano LCMS/MS analysis (Q Exactive mass spectrometer, Thermo Scientific) that was connected with Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific). MS/MS spectra


52

Page 53 of 78 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

ACS Nano

were investigated by using MASCOT engine (Matrix Science, London, UK; version 2.2) inserted into Proteome Discoverer 1.4.

Bioinformatic Analysis. For Gene Ontology (GO) annotation and mapping,44,45 the retrieved sequences were locally explored against the SwissProt database (mouse). For KEGG pathway annotation, the Fast Adaptive Shrinkage/Thresholding Algorithm (FASTA) protein sequences of differential proteins were managed against the online KEGG database (http://www.kegg.jp/) to acquire their KEGG Orthologs (Kos) and were subsequently plotted to pathways in KEGG.46 The protein-protein interaction information (PPI)

was

acquired

from

the

IntAct

molecular

interaction

database

(http://www.ebi.ac.uk/intact/) by STRING software (http://string-db.org/).

Parallel reaction monitoring (PRM) analysis. To confirm the expression level of the corresponding protein collected by the TMT-based proteomic analysis,47 the protein expression level was further quantified by LC-PRM/MS analysis in Shanghai Applied Protein Technology Co., Ltd. The Skyline (MacCoss Lab, University of Washington) was used to evaluate and analyze the raw data.48


53

ACS Nano 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

Page 54 of 78

Statistical analysis. All the quantitative data were analyzed by the one-way ANOVA program and expressed as mean values ± standard deviations. A student t-test was used to evaluate the statistical significance of the variance. Values of *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Supporting Information contains Figure S1-S32, Supplementary discussion about Figure 1 and Figure S4-S6, and Supplementary Table S1-S6.

The authors declare no competing financial interest.


54

Page 55 of 78 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

ACS Nano

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected].

*E-mail: [email protected].

*E-mail: [email protected].

Author Contributions J.L., L.T. and S.W. conceived and designed the concept of the experiments. J.L. and Z.Z synthesized the materials and conducted the material characterizations. J.L., X.L. and S.W. analysed the experimental data and co-wrote the manuscript. X.L., X.W., Y.Z., Y.H., D.C., K.W.K.Y., Z.C., X.Y., Y.L., Z.L., S.Z. and S.W. provided important experimental insights. All the authors discussed, commented and agree on the manuscript.


55

ACS Nano 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

Page 56 of 78

ACKNOWLEDGMENT This work is jointly supported by the National Natural Science Foundation of China, Nos. 51671081, 51871162, 51801056 and 51422102, and the National Key R&D Program of China No. 2016YFC1100600 (sub-project 2016YFC1100604), and Natural Science Fund of Hubei Province, 2018CFA064.

REFERENCES (1) Smith, P. A.; Koehler, M. F. T.; Girgis, H. S.; Yan, D.; Chen, Y.; Chen, Y.; Crawford, J. J.; Durk, M. R.; Higuchi, R. I.; Kang, J.; Murray, J.; Paraselli, P.; Park, S.; Phung, W.; Quinn, J. G.; Roberts, T. C.; Rougé, L.; Schwarz, J. B.; Skippington, E.; Wai, J.

et al. Optimized Arylomycins Are a New Class of Gram-Negative Antibiotics. Nature 2018, 561, 189-194.


56

Page 57 of 78 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

ACS Nano

(2) Brochado, A. R.; Telzerow, A.; Bobonis, J.; Banzhaf, M.; Mateus, A.; Selkrig, J.; Huth, E.; Bassler, S.; Beas, J. Z.; Zietek, M.; Ng, N.; Foerster, S.; Ezraty, B.; Py, B.; Barras, F.; Savitski, M. M.; Bork, P.; Göttig, S.; Typas, A. Species-Specific Activity of Antibacterial Drug Combinations. Nature 2018, 559, 259-263. (3) Liu, Y.; Bai, P.; Woischnig, A. K.; Hamri, G. C. E.; Ye, H.; Folcher, M.; Xie, M.; Khanna, N.; Fussenegger, M. Immunomimetic Designer Cells Protect Mice from MRSA Infection. Cell 2018, 174, 259-270.e11. (4) Baker, S.; Thomson, N.; Weill, F. X.; Holt, K. E. Genomic Insights into the Emergence and Spread of Antimicrobial-Resistant Bacterial Pathogens. Science 2018, 360, 733-738. (5) Kim, W.; Zhu, W.; Hendricks, G. L.; Van Tyne, D.; Steele, A. D.; Keohane, C. E.; Fricke, N.; Conery, A. L.; Shen, S.; Pan, W.; Lee, K.; Rajamuthiah, R.; Fuchs, B. B.; Vlahovska, P. M.; Wuest, W. M.; Gilmore, M. S.; Gao, H.; Ausubel, F. M.; Mylonakis, E. A New Class of Synthetic Retinoid Antibiotics Effective against Bacterial Persisters. Nature 2018, 556, 103-107. (6) Hover, B. M.; Kim, S. H.; Katz, M.; Charlop-Powers, Z.; Owen, J. G.; Ternei, M. A.;


57

ACS Nano 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

Page 58 of 78

Maniko, J.; Estrela, A. B.; Molina, H.; Park, S.; Perlin, D. S.; Brady, S. F. CultureIndependent Discovery of the Malacidins as Calcium-Dependent Antibiotics with Activity against Multidrug-Resistant Gram-Positive Pathogens. Nat. Microbiol. 2018,

3, 415-422. (7) Baym, M.; Stone, L. K.; Kishony, R. Multidrug Evolutionary Strategies to Reverse Antibiotic Resistance. Science 2016, 351, aad3292. (8) Lee, E. J.; Huh, B. K.; Kim, S. N.; Lee, J. Y.; Park, C. G.; Mikos, A. G.; Bin Choy, Y. Application of Materials as Medical Devices with Localized Drug Delivery Capabilities for Enhanced Wound Repair. Prog. Mater. Sci. 2017, 89, 392-410. (9) Eming, S. A.; Martin, P.; Tomic-Canic, M. Wound Repair and Regeneration: Mechanisms, Signaling, and Translation. Sci. Transl. Med. 2014, 6, 265sr6. (10)

Kurita, M.; Araoka, T.; Hishida, T.; O’Keefe, D. D.; Takahashi, Y.; Sakamoto, A.;

Sakurai, M.; Suzuki, K.; Wu, J.; Yamamoto, M.; Hernandez-Benitez, R.; Ocampo, A.; Reddy, P.; Shokhirev, M. N.; Magistretti, P.; Delicado, E. N.; Eto, H.; Harii, K.; Belmonte, J. C. I. In Vivo Reprogramming of Wound-Resident Cells Generates Skin Epithelial Tissue. Nature 2018, 561, 243-247.


58

Page 59 of 78 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

ACS Nano

(11)

Liu, G.; Zhang, S.; Shi, Y.; Huang, X.; Tang, Y.; Chen, P.; Si, W.; Huang, W.;

Dong, X. "Wax-Sealed" Theranostic Nanoplatform for Enhanced Afterglow ImagingGuided Photothermally Triggered Photodynamic Therapy. Adv. Funct. Mater. 2018,

28, 1804317. (12)

Zheng, D. W.; Hong, S.; Xu, L.; Li, C. X.; Li, K.; Cheng, S. X.; Zhang, X. Z.

Hierarchical Micro-/Nanostructures from Human Hair for Biomedical Applications.

Adv. Mater. 2018, 30, 1800836. (13)

Tan, L.; Li, J.; Liu, X.; Cui, Z.; Yang, X.; Zhu, S.; Li, Z.; Yuan, X.; Zheng, Y.;

Yeung, K. W. K.; Pan, H.; Wang, X.; Wu, S. Rapid Biofilm Eradication on Bone Implants Using Red Phosphorus and Near-Infrared Light. Adv. Mater. 2018, 30, 1801808. (14)

Yang, Y.; Ma, L.; Cheng, C.; Deng, Y.; Huang, J.; Fan, X.; Nie, C.; Zhao, W.;

Zhao, C. Nonchemotherapic and Robust Dual-Responsive Nanoagents with OnDemand Bacterial Trapping, Ablation, and Release for Efficient Wound Disinfection.

Adv. Funct. Mater. 2018, 28, 1705708. (15)

Yang, Y.; He, P.; Wang, Y.; Bai, H.; Wang, S.; Xu, J. F.; Zhang, X.


59

ACS Nano 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

Page 60 of 78

Supramolecular Radical Anions Triggered by Bacteria In Situ for Selective Photothermal Therapy. Angew. Chem. Int. Ed. 2017, 56, 16239-16242. (16)

Hu, D.; Li, H.; Wang, B.; Ye, Z.; Lei, W.; Jia, F.; Jin, Q.; Ren, K. F.; Ji, J. Surface-

Adaptive Gold Nanoparticles with Effective Adherence and Enhanced Photothermal Ablation of Methicillin-Resistant Staphylococcus aureus Biofilm. ACS Nano 2017, 11, 9330-9339. (17)

Wang, B.; Yang, W.; McKittrick, J.; Meyers, M. A. Keratin: Structure, Mechanical

Properties, Occurrence in Biological Organisms, and Efforts at Bioinspiration. Prog.

Mater. Sci. 2016, 76, 229-318. (18)

Ewald, C. Y.; Landis, J. N.; Abate, J. P.; Murphy, C. T.; Blackwell, T. K. Dauer-

Independent Insulin/IGF-1-Signalling Implicates Collagen Remodelling in Longevity.

Nature 2015, 519, 97-101. (19)

Kim, S.; Wong, P.; Coulombe, P. A. A Keratin Cytoskeletal Protein Regulates

Protein Synthesis and Epithelial Cell Growth. Nature 2006, 441, 362-365. (20)

Hernandez-Gordillo, V.; Chmielewski, J. Mimicking the Extracellular Matrix with

Functionalized, Metal-Assembled Collagen Peptide Scaffolds. Biomaterials 2014, 35,


60

Page 61 of 78 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

ACS Nano

7363-7373. (21)

Linehan, J. L.; Harrison, O. J.; Han, S. J.; Byrd, A. L.; Vujkovic-Cvijin, I.; Villarino,

A. V.; Sen, S. K.; Shaik, J.; Smelkinson, M.; Tamoutounour, S.; Collins, N.; Bouladoux, N.; Dzutsev, A.; Rosshart, S. P.; Arbuckle, J. H.; Wang, C. R.; Kristie, T. M.; Rehermann, B.; Trinchieri, G.; Brenchley, J. M. et al. Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair. Cell 2018, 172, 784-796.e18. (22)

Cho, S.; Shim, T. S.; Kim, J. H.; Kim, D. H.; Kim, S. H. Selective Coloration of

Melanin Nanospheres through Resonant Mie Scattering. Adv. Mater. 2017, 29, 1700256. (23)

Lin, Y. H.; Huang, K. W.; Chen, S. Y.; Cheng, N. C.; Yu, J. Keratin/Chitosan UV-

Crosslinked Composites Promote the Osteogenic Differentiation of Human Adipose Derived Stem Cells. J. Mater. Chem. B 2017, 5, 4614-4622. (24)

Liu, Y.; Ai, K.; Lu, L. Polydopamine and Its Derivative Materials: Synthesis and

Promising Applications in Energy, Environmental, and Biomedical Fields. Chem.

Rev. 2014, 114, 5057-5115.


61

ACS Nano 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

(25)

Page 62 of 78

Lin, H.; Gao, S.; Dai, C.; Chen, Y.; Shi, J. A Two-Dimensional Biodegradable

Niobium Carbide (MXene) for Photothermal Tumor Eradication in NIR-I and NIR-II Biowindows. J. Am. Chem. Soc. 2017, 139, 16235-16247. (26)

Shao, J.; Xie, H.; Huang, H.; Li, Z.; Sun, Z.; Xu, Y.; Xiao, Q.; Yu, X. F.; Zhao, Y.;

Zhang, H.; Wang, H.; Chu, P. K. Biodegradable Black Phosphorus-Based Nanospheres for in Vivo Photothermal Cancer Therapy. Nat. Commun. 2016, 7, 12967. (27)

Li, J.; Liu, X.; Tan, L.; Liang, Y.; Cui, Z.; Yang, X.; Zhu, S.; Li, Z.; Zheng, Y.;

Yeung, K. W. K.; Wang, X.; Wu, S. Light-Activated Rapid Disinfection by Accelerated Charge Transfer in Red Phosphorus/ZnO Heterointerface. Small

Methods 2019, 3, 1900048. (28)

Kiristi, M.; Singh, V. V.; de Ávila, B. E. F.; Uygun, M.; Soto, F.; Uygun, D. A.;

Wang, J. Lysozyme-Based Antibacterial Nanomotors. ACS Nano 2015, 9, 92529259. (29)

Song, H.; Nor, Y. A.; Yu, M.; Yang, Y.; Zhang, J.; Zhang, H.; Xu, C.; Mitter, N.;

Yu, C. Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition. J.


62

Page 63 of 78 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

ACS Nano

Am. Chem. Soc. 2016, 138, 6455-6462. (30)

Li, J.; Tan, L.; Liu, X.; Cui, Z.; Yang, X.; Yeung, K. W. K.; Chu, P. K.; Wu, S.

Balancing Bacteria-Osteoblast Competition through Selective Physical Puncture and Biofunctionalization of ZnO/Polydopamine/Arginine-Glycine-Aspartic AcidCysteine Nanorods. ACS Nano 2017, 11, 11250-11263. (31)

Lim, C. H.; Sun, Q.; Ratti, K.; Lee, S. H.; Zheng, Y.; Takeo, M.; Lee, W.; Rabbani,

P.; Plikus, M. V.; Cain, J. E.; Wang, D. H.; Watkins, D. N.; Millar, S.; Taketo, M. M.; Myung, P.; Cotsarelis, G.; Ito, M. Hedgehog Stimulates Hair Follicle Neogenesis by Creating Inductive Dermis During Murine Skin Wound Healing. Nat. Commun. 2018,

9, 4903. (32)

Sorushanova, A.; Delgado, L. M.; Wu, Z.; Shologu, N.; Kshirsagar, A.;

Raghunath, R.; Mullen, A. M.; Bayon, Y.; Pandit, A.; Raghunath, M.; Zeugolis, D. I. The

Collagen

Suprafamily:

From

Biosynthesis

to

Advanced

Biomaterial

Development. Adv. Mater. 2019, 31, 1801651. (33)

Kubow, K. E.; Vukmirovic, R.; Zhe, L.; Klotzsch, E.; Smith, M. L.; Gourdon, D.;

Luna, S.; Vogel, V. Mechanical Forces Regulate the Interactions of Fibronectin and


63

ACS Nano 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

Page 64 of 78

Collagen I in Extracellular Matrix. Nat. Commun. 2015, 6, 8026. (34)

Malhotra, V.; Erlmann, P. The Pathway of Collagen Secretion. Annu. Rev. Cell

Dev. Biol. 2015, 31, 109-124. (35)

Elkhatib, N.; Bresteau, E.; Baschieri, F.; Rioja, A. L.; van Niel, G.; Vassilopoulos,

S.; Montagnac, G. Tubular Clathrin/AP-2 Lattices Pinch Collagen Fibers to Support 3D Cell Migration. Science 2017, 356, eaal4713. (36)

Goldberga, I.; Li, R.; Duer, M. J. Collagen Structure-Function Relationships from

Solid-State NMR Spectroscopy. Acc. Chem. Res. 2018, 51, 1621-1629. (37)

McCall, A. S.; Cummings, C. F.; Bhave, G.; Vanacore, R.; Page-McCaw, A.;

Hudson, B. G. Bromine Is an Essential Trace Element for Assembly of Collagen IV Scaffolds in Tissue Development and Architecture. Cell 2014, 157, 1380-1392. (38)

Stegen, S.; Laperre, K.; Eelen, G.; Rinaldi, G.; Fraisl, P.; Torrekens, S.; Van

Looveren, R.; Loopmans, S.; Bultynck, G.; Vinckier, S.; Meersman, F.; Maxwell, P. H.; Rai, J.; Weis, M.; Eyre, D. R.; Ghesquière, B.; Fendt, S. M.; Carmeliet, P.; Carmeliet, G. HIF-1α Metabolically Controls Collagen Synthesis and Modification in Chondrocytes. Nature 2019, 565, 511-515.


64

Page 65 of 78 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

ACS Nano

(39)

Ma, H.; Zhou, Q.; Chang, J.; Wu, C. Grape Seed-Inspired Smart Hydrogel

Scaffolds for Melanoma Therapy and Wound Healing. ACS Nano, 2019, 13, 43024311. (40)

Fan, B.; Yang, X.; Li, X.; Lv, S.; Zhang, H.; Sun, J.; Li, L.; Wang, L.; Qu, B.; Peng,

X.; Zhang, R. Photoacoustic-Imaging-Guided Therapy of Functionalized Melanin Nanoparticles: Combination of Photothermal Ablation and Gene Therapy against Laryngeal Squamous Cell Carcinoma. Nanoscale, 2019, 11, 6285-6296. (41)

Kim, M.; Kim, H. S.; Kim, M. A.; Ryu, H.; Jeong, H. J.; Lee, C. M.

Thermohydrogel Containing Melanin for Photothermal Cancer Therapy. Macromol.

Biosci., 2017, 17, 1600371. (42)

Zhu, Y.; Xu, H.; Chen, H.; Xie, J.; Shi, M.; Shen, B.; Deng, X.; Liu, C.; Zhan, X.

Peng, C. Proteomic Analysis of Solid Pseudopapillary Tumor of the Pancreas Reveals Dysfunction of the Endoplasmic Reticulum Protein Processing Pathway.

Mol. Cell. Proteomics 2014, 13, 2593-2603. (43)

Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal Sample

Preparation Method for Proteome Analysis. Nat. Methods 2009, 6, 359-362.


65

ACS Nano 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

(44)

Page 66 of 78

Götz, S.; García-Gómez, J. M.; Terol, J.; Williams, T. D.; Nagaraj, S. H.; Nueda,

M. J.; Robles, M.; Talón, M.; Dopazo, J.; Conesa, A. High-Throughput Functional Annotation and Data Mining with the Blast2GO Suite. Nucleic Acids Res. 2008, 36, 3420-3435. (45)

Quevillon, E.; Silventoinen, V.; Pillai, S.; Harte, N.; Mulder, N.; Apweiler, R.;

Lopez, R. InterProScan: Protein Domains Identifier. Nucleic Acids Res. 2005, 33, W116-W120. (46)

Moriya, Y.; Itoh, M.; Okuda, S.; Yoshizawa, A. C.; Kanehisa, M. KAAS: An

Automatic Genome Annotation and Pathway Reconstruction Server. Nucleic Acids

Res. 2007, 35, W182-W185. (47)

Peterson, A. C.; Russell, J. D.; Bailey, D. J.; Westphall, M. S.; Coon, J. J. Parallel

Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics. Mol. Cell. Proteomics 2012, 11, 1475-1488. (48)

MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.;

Frewen, B.; Kern, R.; Tabb, D. L.; Liebler, D. C.; MacCoss, M. J. Skyline: An Open Source Document Editor for Creating and Analyzing Targeted Proteomics


66

Page 67 of 78 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

ACS Nano

Experiments. Bioinformatics 2010, 26, 966-968.


67

ACS Nano 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

Page 68 of 78

Figure 1. Morphology and structure. SEM images of (A) low and (B) high magnification (red arrows: melanosomes) of sectional human hair fiber. (C) SEM images of relatively high magnification of HHMs. (D) Schematic illustration of HHMs and molecular structure of melanin within HHM. (E) Types and contents of amino acids of keratin within HHM examined using an amino acid analyzer machine (n = 3). (F) Zeta potential measurements of HHMs, Lyso, and HHMs-Lyso. TEM images of (G) low and (G1) high magnification of HHMs. TEM images of (H) low and (H1) high magnification of HHMsLyso. (I) Hydrodynamic sizes of HHMs and HHMs-Lyso. (J) Digital photographs and of


68

Page 69 of 78 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

ACS Nano

H2O, HHMs, and HHMs-Lyso, including typical Tyndall effect. (K) UV absorbance spectra (240-320 nm) of aqueous suspensions of dispersed Lyso at various concentrations (100, 200, 300, 400, 500, 1000, and 2000 ppm), and the maximum absorbance of dispersed Lyso at 281 nm. (L) Standard linear relationship calculated by corresponding values of absorbance at 281 nm.

Figure 2. (A) Vis-NIR absorbance spectra (400-1000 nm) of aqueous suspensions of dispersed HHMs at varied concentrations (10, 20, 50, 100, and 200 ppm). (B)


69

ACS Nano 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

Page 70 of 78

Photothermal heating curves of aqueous suspensions of dispersed HHMs under 808 nm laser irradiation at varied power densities (0.5, 1.0, and 1.5 W cm-2). (C) Photothermal heating curves of aqueous suspensions of dispersed HHMs under 808 nm laser irradiation at varied concentrations (100, 200, and 400 ppm). (D) Recycling heating profiles of 200 ppm HHMs under 808 nm laser irradiation (1.0 W cm-2) for five on/off cycles. (E) Calculation of the photothermal-conversion efficiency (η) of HHMs at 808 nm. Black line: photothermal effect of HHMs for certain periods, and then the laser is turned off. Red line: time constant (τs) from the cooling period by utilizing the linear time data.


70

Page 71 of 78 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

ACS Nano


71

ACS Nano 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

Page 72 of 78

Figure 3. Antibacterial activity in vitro and in vivo. (A) Antibacterial activity of HHMs and HHMs-Lyso at various concentrations (200 and 400 ppm) in the dark for 2 h (D2: Dark for 2 h, n = 3). (B) Bacterial killing efficiency of HHMs and HHMs-Lyso at various concentrations (200 and 400 ppm) under 808 nm NIR light irradiation to keep 50 °C for 10 min (this treatment is marked as "L": Light, n = 3). (C) Scheme of 808 nm NIR light irradiation of bacterial medium with HHMs-Lyso to maintain at 50 °C for 10 min at a specific setting point after culturing for 0, 1, and 2 h. (D) Antimicrobial activity of HHMsLyso (400 ppm) for MRSA under different treatments, including D2, L+D2, D1+L+D1 and D2+L (n = 3). (E) Antibacterial activity of HHMs and HHMs-Lyso at various concentrations (200 and 400 ppm) in L+D2 group. (F) SEM images of MRSA morphologies interacting with HHMs and HHMs-Lyso in D2 and L+D2 groups (scale bars = 200 nm) and corresponding schemes of antibacterial mechanism. (G) Schematic illustration of photothermal therapy of HHMs-Lyso for MRSA-infected wound in vivo. (H) Quantitative assessment of bacterial counts in corresponding wounds by spread plate method at 1 and 2 days (n = 6). (I) Percentage of neutrophils (versus all cells) in wounds by corresponding H&E staining (n = 6). (J) Quantitative analysis of


72

ACS Nano

Page 73 of 78 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

corresponding MRSA-infected wounds over time (12 days, n = 6). Error bars indicate means ± standard deviations: *P < 0.05, **P < 0.01, ***P < 0.001 (t test). NS, not significant (P > 0.05).


73

ACS Nano 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

Page 74 of 78


74

Page 75 of 78 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

ACS Nano

Figure 4. Tissue repair and related mechanism. (A) Quantitative analysis of corresponding wounds over time (12 days, n = 4). (B) Quantitative detection of collagen deposition by corresponding Masson's trichrome staining (n = 6). (C) Quantitative detection of collagen deposition by corresponding Sirius red staining (n = 6). (D) Volcano plot of the iTRAQ-based proteomic results of 3940 differently expressed proteins (P < 0.05), including 231 downregulated proteins (cutoff value of 0.83-fold for expressed variation) and 243 upregulated proteins (cutoff value of 1.2-fold for expressed variation) following HHMs treatment. (E) Heatmap analysis of these differential proteins (231 downregulated proteins and 243 upregulated proteins) corresponding to volcano plot (n = 3). (F) GO classification (biological process, molecular function, and cellular component) of all differential proteins and enriched GO terms in top 20. (G) KEGG annotations of identified proteins underlying pathways and processes, and enriched KEGG pathways in the top 5 terms. (H) PPI network of these proteins in "protein digestion and absorption" pathway. Error bars indicate means ± standard deviations: *P < 0.05, **P < 0.01, ***P < 0.001 (t test).


75

ACS Nano 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

Page 76 of 78

Figure 5. Related mechanism of tissue repair. (A) Relative expression of proteins in the PPI network in proteomics (n = 3). (B) Quantitative Col14a1 protein enrichment by PRM


76

Page 77 of 78 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

ACS Nano

technology (n = 3), and corresponding enrichment of three peptide sequences (NLVVDDETATSLR, IGILITDGK, and VTVTPVYTVGEGVSVSAPGK). (C) Schematic illustration of potential mechanism involved in tissue repair after HHMs treatment. Error bars indicate means ± standard deviations: *P < 0.05, **P < 0.01, ***P < 0.001 (t test).


77

ACS Nano 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

Page 78 of 78

For Table of Contents Only


78

Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant

Recommend Documents