Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal

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Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal Wound Healing Feiyan Gao, Wenfeng Li, Jia Deng, Jinlan Kan, Tingwang Guo, Bochu Wang, and Shilei Hao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01725 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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ACS Applied Materials & Interfaces

Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal Wound Healing Feiyan Gao, †, Wenfeng Li, †, Jia Deng, § Jinlan Kan, † Tingwang Guo, §Bochu Wang, †, *

†

and Shilei Hao †, *

Key Laboratory of Biorheological Science and Technology, Ministry of Education, College

of Bioengineering, Chongqing University, Chongqing 400030, China. § College

of Environment and Resources, Chongqing Technology and Business University,

Chongqing, 400067, China.

* Correspondence:

Bochu Wang and Shilei Hao Bioengineering College, Chongqing University, No. 174, Shapingba Main Street, Chongqing, China, 400030. Tel./Fax: +86-23-65102507 E-mail: [email protected] (B. Wang); [email protected] (S. Hao)

KEYWORDS: human hair keratin proteins, recombinant keratin 37, recombinant keratin 81, wound healing, nanoparticles

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ABSTRACT In recent years, the favourable enhanced wound healing properties and excellent biocompatibility of keratin derived from human hair have attracted considerable attention. Recombinant keratin proteins can be produced by recombinant DNA technology and have higher purity than extracted keratin. However, the wound healing properties of recombinant keratin proteins remain unclear. Herein, two recombinant trichocyte keratins including human type I hair keratin 37 and human type II hair keratin 81 were expressed using a bacterial expression system, and recombinant keratin nanoparticles (RKNPs) were prepared via an ultrasonic dispersion method. The molecular weight, purity, and physicochemical properties of the recombinant keratin proteins and nanoparticles were assessed using gel electrophoresis, circular dichroism, mass spectrometry, and SEM analyses. The RKNPs significantly enhanced cell proliferation and migration in vitro, and the treatment of dermal wounds in vivo with RKNPs resulted in improved wound healing associated with improved epithelialization, vascularization and collagen deposition and remodelling. In addition, the in vivo biocompatibility test revealed no systemic toxicity. Overall, this work demonstrates that RKNPs are a promising candidate for enhanced wound healing, and this study opens up new prospects for the development of keratin biomaterials.

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1. INTRODUCTION Wound healing has significant economic and social implications and has received considerable attention globally. 1-2 Accelerating wound healing after skin damage and restoring tissue integrity and function remain central concerns for patients and doctors. 3

One approach for enhancing wound healing is the use of biomaterials, which can

promote cell adhesion and proliferation by creating a suitable microenvironment to improve the outcome of the wound healing process.

4-5

Many biomaterials, including

collagen, cellulose, chitosan, gelatine, silk, hyaluronic acids, and alginate, have been employed for wound healing. 1, 6-8 Among these, keratin-based biomaterials are considered excellent biomaterials for wound healing because of their intrinsic cell growth and proliferation ability, biocompatibility, biodegradability, and natural abundance.

9-12

Keratin, an important

subset of fibrous proteins, belongs to the broad category of insoluble proteins that are mainly expressed in the epithelium and hair and constitute the largest subgroup of intermediate filaments (IFs). 9, 13-15 Traditional keratin biomaterials have been extracted from human hair, feather and wool and used for medical applications.

11, 16-17

Keratin

regulates cell growth through binding to the adaptor protein in wounded stratified epithelia. 18 In addition, keratin strongly enhances collagen deposit, fibroblast adhesion and keratinocyte migration because of the abundance of peptide sequences of the LDV and RGD.

11, 19

However, the purity of extracted keratin is poor due to the complex

components of hair, wool and feather. For example, keratin extracted from human hair is enriched in keratin proteins, keratin-associated proteins, and IF proteins.

20

In

addition, there are numerous methods for human hair keratin extraction and isolation, which may influence the characteristics of keratin extraction, such as the composing of amino acid and molecular weights.21-23 Recombinant expression is an important way to improve the purity and quality of keratin proteins. 24-25 Recombinant keratin proteins have been employed to investigate the structural features of keratin IFs, and in vitro assembly of the recombinant keratin proteins into Ifs can be observed.

26-28

Furthermore, we have used recombinant

trichocytic keratins for haemostasis for the first time. 29 Keratin biomaterials extracted from natural resources have yielded impressive results in haemostatic, wound healing, nerve regeneration, bone repair, and drug transport applications. 3


10, 12, 30

Therefore,


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research on the biomedical applications (for example, wound healing) of recombinant keratin proteins is essential for the development of keratin biomaterials. The aim of the present study was to investigate the wound healing ability of recombinant human hair keratin proteins. High conservation of the sequence and structure of human IF has been reported in previous studies.

31-32

The structure of IF

proteins is highly conserved and consists of 4 α-helical coiled-coil domains separated by non-helical linker regions, suggesting that they could have evolved from a primordial gene. However, there are 11 acidic type I family and 6 neutral-basic type II family trichocytic keratin proteins, and there is an obvious relationship between sequence homology at the protein level and the arrangement of keratin genes in each of the type I and type II clusters.

13, 33

Therefore, two recombinant trichocyte keratins

including human type I hair keratin 37 and human type II hair keratin 81 were expressed using bacterial expression system in this study. Nanoparticles have recently emerged as a promising nanomaterial for wound healing that can achieve rapid closure of deep wounds due to the nanobridging effect between the nanoparticles and tissue matrix. 3, 34-35

Therefore, recombinant keratin nanoparticles (RKNPs) were prepared via the

ultrasonic dispersion method to evaluate their wound healing effect.

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The detailed

procedure is listed in Figure 1. To provide a comprehensive understanding of the wound healing application of recombinant human hair keratin proteins, the physicochemical properties of recombinant human hair keratin proteins and RKNPs were investigated, and the in vitro cell bioactivity and in vivo full-thickness wound healing properties of RKNPs were evaluated. 2. MATERIALS AND METHODS 2.1. Cloning of Recombinant Keratin Proteins. Gene sequence data of human hair keratin 37 (8688) and 81 (3877) obtained from GenBank (Table S1). A pET-28a (+) plasmid was used for cloning. The human hair keratin gene (K37 or K81) was amplified by PCR and then cloned into the NdeI and XhoI sites in pET-28a (+) by enzyme digestion and ligation following the manufacturer’s instructions to obtain the plasmids pET-28a (+) – K37 and pET-28a (+) – K81. pET-28a (+) – K37 and pET-28a (+) – K81 were then amplified in E. coli TOP 10 cells and confirmed by plasmid extraction and sequencing. 4


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2.2. Expression and Purification of Recombinant Keratin Proteins. Recombinant Keratin 37 (RK37) and Recombinant Keratin 81 (RK81) were expressed as described in our previous study.

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The plasmids pET-28a (+) – K37 and pET-28a

(+) – K81 were separately used to transform E. coli BL21 (DE3). The transformed cells were inoculated in 10 mL of Luria–Bertani (LB) medium and incubated overnight at 37 °C, 250 rpm with 50 μg/mL kanamycin. The cells were subsequently diluted 1:100 in LB media and induced at an optical density of 0.6–0.8 using IPTG at a final concentration of 0.5 mM. The cells were harvested 4 h after the induction by centrifugation at 10,000 rpm for 15 min. Purification of RK37 and RK81 from total bacterial proteins was achieved in several steps following previous studies with some modifications. 29, 36-38 The cells were resuspend with buffer A (pH 8.0) containing 50 mM Tris, 20 mM β-mercaptoethanol 150 mM NaCl and 5 mM EDTA. The solution were ultrasonic disrupted and centrifuged at 10,000 rpm for 1 h. The precipitates was collected and resuspend with buffer B (adding 1 M urea and 0.5 % Triton X-100 into buffer A). The sample was then centrifuged at 10,000 rpm for 1 h. Finally, the insoluble fraction were collected and dissolved into buffer C (50 mM Tris pH 8.0, 2.5 M NaCl, 8 M urea, 5 mM EDTA, 20 mM β-mercaptoethanol), which was then centrifuged at 10,000 rpm for 1 h at 4 °C. The supernatant was collected and dialyzed against 8 M urea and 20 mM β-mercaptoethanol. The solution was collected and stored for further analysis. 2.3. Characterization of Recombinant Keratin Proteins. 2.3.1.

Gel Electrophoresis Analysis. RK37 or RK81 was dissolved in buffer A

and then mixed with 4 μL of 5X SDS loading buffer and 0.6 M β-mercaptoethanol. The samples (20 μL) were heated at 100 °C for 10 min prior to gel electrophoresis in 10-12 % gradient Tris-HCL gels at 120 V for 90 min. The gels were subsequently washed three times with deionized water and stained for 30 min with Coomassie Brilliant Blue G-250. 2.3.2.

Circular Dichroism Analysis. Circular dichroism (CD) analysis of RK37

and RK81 was performed using a JascoJ715 spectropolarimeter (Tokyo, Japan) at room temperature, and a circular quartz cell with a pathlength of 0.1 cm was used for dilute

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samples. Spectra were scanned from 190 to 300 nm at a rate of 100 nm/min. All tests were carried out three times. 2.3.3.

UV-Vis Spectra Analysis. The UV-Vis spectra of keratin RK37 and

RK81 were acquired using a spectrophotometer (UV-2450; Shimadzu, Kyoto, Japan). Briefly, RK37 or RK81 was dissolved in 8 M urea buffer. The buffer was used as the blank, and spectral data were recorded over a wavelength range of 200 to 400 nm. 2.3.4.

FT-IR Analysis. Infrared spectra of RK37 and RK81 were recorded in a

spectral range ranging from 400 to 4000 cm-1. The recombinant keratin samples were prepared by freeze-drying, and the dried protein samples were ground with KBr at a ratio of 1:100 and pressed into a thin disk for testing. 2.3.5.

Mass Spectrometry Analysis. The molecular weights of the recombinant

keratin were confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (SHIMADZU Corporation, MALDI-7090, Japan). A 2-μL aliquot of 1 mg/mL RK37 or RK81 was mixed with 2 μL of sinapic acid matrix (10 mg/mL) (Sigma) and then dissolved in a milli-Q purified water/acetonitrile mixture (50/50, v/v) for MALDI -TOF analysis. The acceleration voltage was 20 kV, and the spectrum was recorded in a linear mode by averaging 100 laser irradiations at a power setting of 120 system units. 2.4. Preparation of RKNPs. Recombinant K37 nanoparticles (RKNP37) and recombinant K81 nanoparticles (RKNP81) were prepared using an ultrasonic dispersion method based on our previous study.

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Briefly, RK37 or RK81 was

dissolved at different concentrations (0.1 %, 0.15 % and 0.2 %, w/v) in 8 M urea (pH 9.0), and the keratin solution was then injected into DI water (pH 3.0, adjusted with dilute solutions of HCl) under sonication via a needle using a syringe pump with a speed of 0.25 mL/min. The ultrasonic treatment was performed by an ultrasonic cell disruption system (JY92-II, Scientz, China), and the power of the ultrasonic cell disrupter was 400 W. The resulting RKNPs were lyophilized overnight by a freeze dryer (230, Modulyod, USA) and stored. Keratin extract nanoparticles (KNPs) were also prepared using the above method for the evaluation of in vivo wound healing; keratin extracts (0.15 %, w/v) were dissolved in ultrapure water and injected into DI water (pH 3.0) under sonication. 10 6


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2.5. Characterizations of RKNPs. RKNP suspensions with different concentrations were sonicated in DI water (pH 3.0, adjusted with dilute solutions of HCl). The particle size and zeta potential of the RKNPs were measured by photon correlation spectroscopy using a nano ZS90 Zetasizer (Malvern Instruments, UK). Furthermore, the surface morphology of RKNP37 and RKNP81 was observed using a scanning electron microscope (SEM). The nanoparticle suspensions were spread on a piece of aluminium foil and dried at room temperature. The dried nanoparticles were then coated with gold metal under vacuum and examined (Hitachi, S-3400N, Japan). 2.6. Cell Experiments. 2.6.1.

Culture Conditions. HaCaT keratinocytes purchased from ATCC and

grown at 37 °C in 5 % CO2 were used for in vitro cell experiments. The cells were cultured in DMEM with 10 % FBS supplemented with 1 % penicillin-streptomycin and sub-cultured every 4 days. 2.6.2.

Cell Proliferation Assay. The effect of RKNPs on HaCaT cell

proliferation was determined by the MTT assay. 39 HaCaT cells were seeded in 96-well plates at a density of 2 ×104 cells/well and incubated for 24 h. RKNPs sterilized via gamma ray-irradiation at a dose of 25 kGy were added to the wells at different concentrations. Cells cultured without RKNPs were used as a control. After 24 h of incubation, MTT was added to each well and further incubated for 4 h at 37 °C. Cell viability was measured at 490 nm using a microplate reader (ELx808, BioTek, USA), and the activity of untreated cells was set to 100 % for calculations. In addition, cell viability was assessed using a LIVE/DEAD viability/cytotoxicity assay kit (YEASEN biotechnology, Shanghai, China). Double staining of calcein-AM and ethidium homodimer-1 was performed to analyse cell viability and cell death, respectively. The fluorescent images of each well under a green channel and red channel were captured on a fluorescence microscope (XDS-2FL, Optika, Italy). The experiments were conducted triplicate. 2.6.3.

Cell Scratch Assay. HaCaT cells (2 × 106 cells/well) were seeded in 24-

well plates and grown until reaching surface monolayer confluence. Using a sterile 200μL pipette tip, a cell-free area was created by scraping the monolayer.

40-41

The cells

were washed twice with PBS, and the culture medium was changed to medium 7



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containing RKNPs (0.25 mg/mL). Cells without RKNPs were used as the control. Pictures of the scratches were captured by a microscope (UOP photoelectric technology, DSZ2000X, China) after different incubation times (0, 12, 24, and 36 h). Photographs were taken accurately at the same location to record the process of scratch repair. The experiment was repeated three times, and a representative picture was selected. Furthermore, the wound area in each picture was determined by measuring the cell-free area using ImageJ image analysis software. The scratch closure rate was calculated to express the migration rate according to equation (1): Scratch closure rate = [(At0-At) / At0] × 100 %

(1)

where At0 is the scratch area at the initial time and At is the corresponding scratch area after incubation with RKNPs for different times. 2.7. In Vivo Wound Healing. 2.7.1.

Rat Wound Model. Healthy male SD rats weighing at least 200 g were

chosen for the in vivo wound healing study. The rats were housed at 24–26 °C with free access to food and water under a controlled 12/12 light/dark cycle. The animal experimental protocol was approved by the Animal Ethical and Experimental Committee of the Third Military Medical University (Chongqing, China). The fullthickness wound model was established as follows: the rats were anaesthetized by intraperitoneal administration of 10 % chloral hydrate, and 2 cm × 2 cm open-excision full-thickness wounds were created on the dorsal surface of the rats. Subsequently, the dorsal wounds were covered with 0.500 mg of RKNP37, RKNP81 or KNPs and fixed with Tegaderm film (3M, St Paul, MN). A wound covered with Tegaderm film was used as the control. After surgery, each rat was placed in an individual cage. Wound treatments and Tegaderm dressing changes were conducted every three days until wound healing or sacrifice. In addition, the wounds were gently cleaned with gauze and washed with sterilized saline to thoroughly remove residue at every wound dressing change. 42 Wounds were photographed periodically with a digital camera, and the size was measured using ImageJ software to calculate the wound area closure percentage using equation (2): 8


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Wound area closure (%) = [A0 – A] /A0 × 100 %

(2)

where A0 is the original wound area and A is the actual wound area at the time interval. 2.7.2.

Histological Observation. The reconstructed skin tissue samples were

fixed in 10 % formalin, embedded in paraffin. Subsequently, the samples were cut from the paraffin-embedded blocks and stained with haematoxylin & eosin (H&E) and Masson's trichrome for routine histological analysis and collagen deposition analysis, respectively. 2.8. Biocompatibility Test. Twenty-four male SD rats (approximately 250 g) were subcutaneously implanted with RKNPs or KNPs (20 mg) for 7, 14, 21 and 28 days to assess their biocompatibility and biodegradability. Briefly, the skin was surfacesterilized with iodine after anaesthesia of the animal as described above, the skin was sheared, and a tissue bag was formed by blunt dissection. The sterilized RKNP37, RKNP81 and KNPs were compressed into disks and then placed in a subcutaneous pocket via a 5-mm incision. Photographs were taken to assess the biodegradability of the RKNPs and KNPs at the selected intervals. The major tissue organs of the rats (including heart, liver, spleen, lung and kidney) were fixed in 4 % paraformaldehyde (PFA) for histopathological examination. Furthermore, blood samples were obtained by heart puncture at different days after surgery, which were used to evaluate the proinflammatory responses. Serum was stored at -80 ° C until the inflammatory cytokine interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were detected using an enzyme-linked immunosorbent assay kit (Quantikine, R&D Systems, Minneapolis, USA). The evaluation was carried out according to the kit’s protocols. 2.9. Statistical Analyses. All experiments were performed at least in triplicate unless stated otherwise. Values are expressed as the means ± standard deviations. The significance of the data was analysed using a two-tailed Student’s t-test and ANOVA with a Dunnet post hoc test where appropriate. p< 0.05 indicated a significant difference. All analyses were performed using GraphPad Prism software (Version 6.0). 9



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3. RESULTS AND DISCUSSION 3.1. Recombinant Keratin Expression and Purification. RK37 and RK81 were expressed and purified after expression using an E. coli expression system. The yields of RK37 (type I) and RK81 (type II) were 20 mg/L and 12 mg/L, respectively. Each protein was further purified in buffer, and purified full-length proteins were isolated from inclusion bodies. In addition, the recombinant keratin proteins were analysed by SDS-PAGE after purification. The various stages of the protein purification process were documented (Figure 2A), and the molecular weights of RK37 and RK81 were approximately 49 kDa and 55 kDa, respectively. Furthermore, the single band in the gel indicated that pure recombinant keratins were obtained by prokaryotic expression in this study. Gel electrophoresis of keratin extracts usually reveals multiple bands, demonstrating a complex of multiple proteins in the extract.

11, 23

Therefore, the

presence of a single band upon gel electrophoresis suggested greater purity of the recombinant keratin proteins compared with the keratin extracts. 3.2. Characterizations of Recombinant Keratins. CD analysis of RK37 and RK81 was performed to elucidate the secondary structures of the proteins in solution. Negative minimum absorption bands were observed at 210 nm and 222 nm in the CD spectra of RK37, and the CD spectra of RK81 displayed double minima near 208 and 218 nm (Figure 2B). The overall shapes were reminiscent of the CD spectra of α-helices, although the minima were slightly shifted. 43-44 UV-Vis absorption measurement is a simple way to explore structure formation. The UV-Vis absorption spectra of RK37 and RK81 was also analysed in this study (Figure 2C), and the results showed the maximum absorption wavelength of RK37 and RK81 was approximately 260 nm. A previous study reported that the UV-Vis spectra of a keratin hydrolysate solution showed a wide peak in the range of 250-280 nm.

45

Furthermore, RK37 and RK81 were dissolved in 8 M urea for UV-Vis analysis in this study, and the characteristic band showed a blue shift in the presence of urea. 46 The FTIR spectra of RK37 and RK81 in the region 400–4000 cm−1 are given in Figure 2D, and the spectra of recombinant keratin proteins exhibited several characteristic bands at 1650 cm-1 (primary amide vibrations), 1541 and 1260 cm

-1

(secondary and tertiary amide vibrations), 3292 cm-1 (C-H and O-H vibrations), and 10


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2926 cm-1 (-CH2 vibrations). These major band data agree with published data for extracted keratins. Mass spectrometry was performed to verify the molecular weights of the RK37 and RK81 expressed in the present study. As shown in Figure 2E and 2F, a strong peak was observed at 55.08 kDa or 50.32 kDa in the spectra of RK81 and RK37, respectively, in agreement with the results of SDS-PAGE analysis. 3.3. Preparation and Characterizations of RKNPs. The RKNPs were prepared using the ultrasonic dispersion method in this study based on the dissolution of recombinant keratin proteins in DI water (pH 3.0). Inclusion body proteins were obtained via the bacterial expression system, since trichocyte keratin proteins are insoluble in nature. 29 Therefore, the recombinant keratin proteins were dissolved in urea solution (8 M), and the agglomeration was observed when the recombinant keratin solution was pumped into DI water at pH 3.0 adjusted with dilute solutions of HCl. In addition, ultrasonication was used to uniformly disperse the particles at the nano scale. 47 The procedure for the fabrication of RKNPs is illustrated in Figure 1. The morphology of the RKNPs was characterized using SEM. As shown in Figure 3A-D, both RKNP37 and RKNP81 with 0.15 % keratin were spherical in shape, and the size of the nanoparticles ranged from approximately 200 nm to 500 nm. The mean size of RKNP37 (Figure 3E) and RKNP81 (Figure 3F) was 307.80 and 305.86 nm, respectively. In addition, the effect of different keratin concentrations on the size and zeta potential values of the keratin nanoparticles was studied (Table 1). The mean size of the nanoparticles increased with increasing keratin concentration. The mean particle size of RKNPs ranged from 269.56 ± 13.90 nm to 503.70 ± 16.50 nm, and the range of the PDI was 0.20 to 0.29. Furthermore, the zeta potential of RKNPs under pH 3.0 ranged from 13.91 ± 0.21 to 21.90 ± 0.13 mV with increasing keratin concentration. The zeta potential adopted positive values when the pH of the formulation was below the isoelectric point, and the isoelectric point values of RK37 and RK81 were 4.84 and 5.36, respectively. Moreover, the CD spectra of RKNP37 and RKNP81 were analysed (Figure S1), and an α-helical structure was observed in the spectra, indicating that the secondary structure of the proteins did not change after nanoparticle preparation. 11



3.4. Cell Experiments. The effects of RKNP37 and RKNP81 on cell proliferation rates and viability were evaluated in this study. In the live/dead viability/cytotoxicity assay, the percentages of viable cells (green fluorescence) increased with increasing RKNP concentration from 0.25 to 1.00 mg/mL (Figure 4A), indicating a cell proliferation effect of both RKNP37 and RKNP81, although a few dead cells were found (red staining). Viability was further investigated with the MTT assay (Figure 4B). RKNP37 and RKNP81 significantly increased HaCaT cell proliferation compared to blank medium, and the cell growth was dependent on the concentration of RKNPs. Furthermore, RKNP81 displayed a stronger cell proliferation ability than RKNP37 when the concentration of RKNPs was higher than 0.75 mg/mL. In vitro scratch wound healing assays were performed out in a single layer of HaCaT cells with a 200-L pipette tip to observe the effects of RKNPs on the healing process. As shown in Figure 4C and 4D, RKNP37 and RKNP81 induced significant cell migration compared to the control group within 36 h, as observed by increased closure of the scratch area. Moreover, RKNP81 promoted more cell migration than RKNP37 at 36 h. Cell proliferation and migration at the wound edge co-occurred during wound healing. 48 The attachment and proliferation of fibroblast and keratinocyte cells in keratin extracts have been reported,

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and keratin extracts have also displayed

favourable ability in cell migration. 50 This study showed that RKNPs can also regulate cell proliferation and migration, which is beneficial for accelerated healing of skin wounds. Furthermore, RKNP81 showed stronger cell proliferation and migration regulation ability compared to RKNP37. 3.5. In Vivo Wound Healing Evaluation. The wound healing efficiency of the RKNPs and KNPs was investigated in vivo, and the wounds were photographed on days 0, 7, 14, 21 and 28. (Figure 5A). The wounds treated with RKNP37, RKNP81 and KNPs showed faster closure compared to the control group after 7 days of treatment, and the wound size reductions were 49.66 %, 56.4 % and 40.33 % for the wounds covered by RKNP37, RKNP81 and KNPs, respectively (Figure 5B). By contrast, there was a 29.7 % reduction of the wound area in the control group. On day 14, the control group displayed 69.5 % wound closure, whereas RKNP37, RKNP81 and KNPs showed wound closures of 81.0 %, 85.5 % and 74.26 %, respectively. These results indicated that both KNPs and RKNPs can accelerate wound healing significantly within 2 weeks, and RKNPs 12


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displayed stronger wound healing ability than KNPs due to the high purity of the former, suggesting that the keratin proteins in extracts play a key role in the wound healing process. In addition, RKNP81 treatment led to a significantly enhanced therapeutic effect compared with RKNP37, revealing a stronger wound healing effect of RKNP81. The wound healing process was further evaluated through histological examination. The histological evaluation of dermal wound healing by H&E staining and Masson's trichrome staining on days 7, 14, 21 and 28 are shown in Figure 6A. At day 7, an increase in inflammatory cell number and cell infiltration was observed in the tissue section of the control group. By contrast, a reduction of inflammation was observed in the RKNP37 and RKNP81 treatments, and the proliferative phase began with the presence of activated fibroblasts (black arrows). Fibroblasts play a key role in tissue remodelling during wound healing, and early migration of fibroblasts could promote ECM synthesis and tissue regeneration. The morphology and epidermal thickness were also characterized, as shown in Figure 6B (dashed white line outlines). After day 14, the epidermis was thinner in wounds treated with RKNP37 and RKNP81 than in those treated with KNPs and the control group. Furthermore, the RKNP81 treatment displayed a thinner epidermal thickness than the RKNP37 treatment at day 28 and was almost identical to the normal skin epidermal thickness. Additionally, the RKNP37 and RKNP81 groups showed a greater number of skin appendages after day 21 (Figure 6C), similar to normal skin tissue. Finally, more blood vessels (black asterisks) were observed in the RKNP37 and RKNP81 groups compared to the KNPs and control groups at day 14 (Figure 6D). The regeneration of blood vessels is critical for the formation of vascular networks and to provide nutrients and oxygen to cells during the wound healing process. Collagen is the principal component of the skin and can restore the structural integrity of damaged tissues in wound healing. Masson’s trichrome staining was carried out at day 28 to assess the collagen deposition in the wound region after different treatments. As shown in Figure 6A, more collagen deposition was found in the RKNP37 and RKNP81 groups compared to the control group. The wounds treated with RKNPs exhibited a stronger healing capacity compared to those treated with KNPs, which could be attributed to the higher purity of the 13



recombinant keratin. Since keratin extracts derived from human hair contain keratin proteins, keratin-associated proteins and IF proteins, these results indicate that keratin proteins play a critical role in wound healing and that an increase in keratin content in nanoparticles can provide a better regenerative effect on wound healing. In addition, the RKNP81 treatments had a much higher rate of wound closure than the RKNP37 treatments during the early healing stage, which can be attributed to the stronger cell proliferation and migration abilities of RKNP81 compared with RKNP37, as supported by the faster epithelialization, vascularization and collagen deposition and remodelling in the histological examination of the RKNP81 group. 3.6. Biocompatibility Test. The in vivo biocompatibility of RKNP37, RKNP 81 and KNPs was assessed in SD rats through subcutaneous implantation. No animals died after the end of the experiment, and no abnormal manifestations were observed, including wound infections and behaviours of moving difficulty. The in vivo degradation of RKNPs and KNPs is shown in Figure 7. A gradual reduction of implanted RKNPs and KNPs can be observed with time, and the shape of the disks began to change after day 7 of implantation, although no erythema or oedema was observed. In addition, RKNP37 and RKNP81 were degraded completely at day 28, while a few KNPs could still be found at this time point. The systemic toxicity of RKNPs and KNPs were evaluated by H&E staining of the heart, lungs, liver, kidneys and spleen of rats. As shown in Figure 8A, no significant organ damage was observed in the RKNP and KNP groups. Moreover, there is no significant histopathological differences in main organs between the normal and test rats. Furthermore, the proinflammatory response after RKNP37, RKNP81 and KNP implantation was evaluated by measuring serum levels of the inflammatory cytokines IL-6 and TNF-α (Figure 8B). The results showed that the RKNPs and KNPs caused a minimal or no inflammatory response. In addition, there was no significant increase in cytokine levels compared to control group, which suggested that RKNPs does not cause undesirable tissue inflammatory or immunotoxic reactions, and no systemic toxicity was found after implantation.

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As natural biomaterials, keratins have attracted much attention in biomedical applications due to their intrinsic biocompatibility and biodegradability. Interestingly, favourable biocompatibility and biodegradation of RKNPs were observed in this study. RKNPs did not induce systemic toxicity or tissue inflammatory or immune responses. Furthermore, the in vivo degradation rate of RKNPs was faster than that of KNPs, largely due to the high purity of the recombinant protein. 4. CONCLUSION In conclusion, this study is the first to develop RKNPs for wound healing. Different types of trichocyte keratin proteins (K37 and K81) were expressed using a bacterial expression system and prepared as nanoparticles by an ultrasonic dispersion method. RKNPs displayed favourable effects on cell migration and proliferation, and the RKNPs promoted enhanced wound healing by improving epithelialization, vascularization and collagen deposition and remodelling. In addition, RKNPs also exhibited favourable biocompatibility and biodegradation, and it can be concluded that RKNPs represent potential candidates for wound-healing and tissue-engineering applications. ■ ASSOCIATED CONTENT Supporting Information Gene sequence data of human hair keratin 37 and 81 (Table S1) and CD spectra of RKNP 37 and RKNP 81. ACKNOWLEDGMENTS The authors acknowledge financial assistance provided by the National Natural Science Foundation of China (Project no. 31600770 and 81501484), the Chongqing Research Program of Basic Research and Frontier Technology (Project no. cstc2018jcyjAX0836), and the Fundamental Research Funds for the Central Universities (Project no. 2018CDQYSG0007). Notes The authors declare no competing financial interest.

15



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Figure legends Figure 1. Scheme of the bacterial expression of human hair keratin proteins and nanoparticle preparation. Figure 2. Physicochemical properties of recombinant keratin proteins. Expression and purification of RK37 and RK81 (A). Lane M: protein molecular weight marker, lane 1: inclusion body isolated in buffer A, lane 2: insoluble fraction isolated in buffer B, lane 3: supernatant fraction in buffer C, lane 4: proteins recovered after dialysis. The CD 18


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spectra (B), UV-Vis spectra (C), FT-IR spectra (D), and mass spectra (E and F) of RK37 and RK81 are shown. Figure 3. Morphology and particle size distribution of RKNPs. SEM images of RKNP37 (A and B) and RKNP81 (C and D) with different magnifications indicated a spherical shape. The mean sizes of the corresponding RKNP37 (E) and RKNP81 (F) were 307.80 and 305.86 nm, respectively. Figure 4. Effects of RKNPs on the proliferation and migration of HaCaT cells. (A) Fluorescent images of HaCaT cells treated with the live/dead cell assay kit after 24 h of incubation with various concentrations of RKNPs. (B) Proliferation studies of the effects of various concentrations of RKNPs on HaCaT cells. (C) Microscope images of HaCaT cells after scratch and treatment with RKNPs for different times. (D) Quantification of the scratch closure rate. *p < 0.05, **p < 0.01 relative to the control group. Figure 5. (A) Healing progression of full-thickness cutaneous wounds treated with RKNP37, RKNP81 and KNPs. (B) Rate of wound closure on days 0, 7, 14, 21 and 28. *p < 0.05, **p < 0.01 relative to the control group. Figure 6. (A) Representative images of sections of wounded skin stained by H&E and Masson's trichrome after treatment with RKNP37, RKNP81 and KNPs. H&E staining shows the border of the epidermal layer (dashed white line outlines), blood vessel (black asterisks) formation, and the presence of activated fibroblasts (black arrows) and skin appendages (red arrows). The thickness of the epidermal layer (B), skin appendages (C) and blood vessel area (D) per square millimetre of healed wounds were quantified after H&E staining. Figure 7. Representative images of in vivo degradation of compressed RKNP37, RKNP81 and KNPs disks after subcutaneous implantation in the rat. Figure 8. (A) H&E staining of the main organs (heart, lung, kidney, spleen and liver) after subcutaneous implantation of RKNPs and KNPs. The variation of the relative IL-6 (B) and TNF-α (C) levels was quantified in all groups.

Table 1 19



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Effect of different concentrations of recombinant keratin proteins on the characteristics of nanoparticles (mean ± S. D., n = 3).

Recombinant

Size

Zeta potential

Concentration Keratin RK37

RK81

nm

PDI

mV

0.10 %

271.75±19.20

0.72±0.02

14.05±0.27

0.15 %

307.80±20.60

0.61±0.08

21.90±0.13

0.20 %

503.70±16.50

0.90±0.05

20.40±0.40

0.10 %

269.56±13.90

0.65±0.12

13.91±0.21

0.15 %

305.86±21.50

0.59±0.04

21.20±0.35

0.20 %

500.22±18.20

0.89±0.03

19.80±0.23

20


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Figure 1. Scheme of the bacterial expression of human hair keratin proteins and nanoparticle preparation. 91x57mm (300 x 300 DPI)



Figure 2. Physicochemical properties of recombinant keratin proteins. Expression and purification of RK37 and RK81 (A). Lane M: protein molecular weight marker, lane 1: inclusion body isolated in buffer A, lane 2: insoluble fraction isolated in buffer B, lane 3: supernatant fraction in buffer C, lane 4: proteins recovered after dialysis. The CD spectra (B), UV-Vis spectra (C), FT-IR spectra (D), and mass spectra (E and F) of RK37 and RK81 are shown. 213x119mm (300 x 300 DPI)


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Figure 3. Morphology and particle size distribution of RKNPs. SEM images of RKNP37 (A and B) and RKNP81 (C and D) with different magnifications indicated a spherical shape. The mean sizes of the corresponding RKNP37 (E) and RKNP81 (F) were 307.80 and 305.86 nm, respectively. 319x156mm (300 x 300 DPI)



Figure 4. Effects of RKNPs on the proliferation and migration of HaCaT cells. (A) Fluorescent images of HaCaT cells treated with the live/dead cell assay kit after 24 h of incubation with various concentrations of RKNPs. (B) Proliferation studies of the effects of various concentrations of RKNPs on HaCaT cells. (C) Microscope images of HaCaT cells after scratch and treatment with RKNPs for different times. (D) Quantification of the scratch closure rate. *p < 0.05, **p < 0.01 relative to the control group.


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Figure 5. (A) Healing progression of full-thickness cutaneous wounds treated with RKNP37, RKNP81 and KNPs. (B) Rate of wound closure on days 0, 7, 14, 21 and 28. *p < 0.05, **p < 0.01 relative to the control group.



Figure 6. (A) Representative images of sections of wounded skin stained by H&E and Masson's trichrome after treatment with RKNP37, RKNP81 and KNPs. H&E staining shows the border of the epidermal layer (dashed white line outlines), blood vessel (black asterisks) formation, and the presence of activated fibroblasts (black arrows) and skin appendages (red arrows). The thickness of the epidermal layer (B), skin appendages (C) and blood vessel area (D) per square millimetre of healed wounds were quantified after H&E staining.


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Figure 7. Representative images of in vivo degradation of compressed RKNP37, RKNP81 and KNPs disks after subcutaneous implantation in the rat.



Figure 8. (A) H&E staining of the main organs (heart, lung, kidney, spleen and liver) after subcutaneous implantation of RKNPs and KNPs. The variation of the relative IL-6 (B) and TNF-α (C) levels was quantified in all groups.


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TOC 91x57mm (300 x 300 DPI)


Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal

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