Patterning Multi-Nanostructured Poly(l-lactic acid) Fibrous Matrices to

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Patterning Multi-Nanostructured Poly(L-lactic acid) Fibrous Matrices to Manipulate Biomolecules Distribution and Functions Wenwu Xiao, Qingtao Li, Huimin He, Wenxiu Li, Xiaodong Cao, and Hua Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18423 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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ACS Applied Materials & Interfaces

Patterning Multi-Nanostructured Poly (L-lactic acid) Fibrous Matrices to Manipulate Biomolecules Distribution and Functions Wenwu Xiao1,2,#, Qingtao Li 1,2,3,#, Huimin He 1,2, Wenxiu Li 1,2, Xiaodong Cao 1,2,4,*, Hua Dong 1,2,*

1.

Department of Biomedical Engineering, School of Materials Science and Engineering,

South China University of Technology, Guangzhou, 510006, China 2.

National Engineering Research Center for Tissue Restoration and Reconstruction

(NERC-TRR), Guangzhou, 510006, China 3.

School of Medicine, South China University of Technology, Guangzhou, 510006, China

4.

Guangdong Province Key Laboratory of Biomedical Engineering, South China

University of Technology, Guangzhou, 510641, China

*Corresponding authors: E-mail: [email protected] (X. Cao), [email protected] (H. Dong)

ACS Paragon Plus Environment

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ABSTRACT

Precise manipulation of biomolecules distribution and functions via biomolecule-matrix interaction is very important and challenging for tissue engineering and regenerative medicine. As a well-known biomimetic matrix, electrospun fibers often lack the unique spatial complexity compared to their natural counterparts in vivo and thus cannot deliver fully the regulatory cues to biomolecules. In this paper, we report a facile and reliable method to fabricate micro- and nanostructured poly (l-lactic acid) (PLLA) fibrous matrices with spatial complexity by combination of advanced electrospinning and agarose hydrogel stamp based micropatterning. Specifically, advanced electrospinning are used to construct multi-nanostructures of fibrous matrices whilst solvent-loaded agarose hydrogel stamps are used to create microstructures. Compared with other methods, our method shows extreme simplicity and flexibility originated from mono-/multispinneret conversion and limitless micropatterns of agarose hydrogel stamps. Three types of PLLA fibrous matrices including patterned nano-Ag/PLLA hybrid fibers, patterned bicompartment polyethylene terephthalate (PET)/PLLA fibers and patterned hollow PLLA fibers are fabricated and their capability to manipulate biomolecules distribution and functions, i.e., bacteria distribution and antibacterial performance, cell patterning and adhesion/spreading behaviors, protein adsorption and delivery, are demonstrated in detail. The method described in our paper provides a powerful tool to restore spatial complexity in biomimetic matrices and would have promising applications in biomedical engineering field.

KEYWORDS: side-by-side electrospinning; coaxial electrospinning; agarose hydrogel stamps; antibacteria effects; cell patterning; protein delivery


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1. INTRODUCTION Precise manipulation of biomolecules distribution and functions via biomolecule-matrix interaction is one of the most challenging issues in tissue engineering and regenerative medicine 1-3

. Thanks to the great progress in biomaterial science and technology, biomimetic matrices that

partially recapture some key physicochemical characteristics of the in vivo microenvironment have been developed successfully4. However, restricted by the fabrication processes, such matrices normally lack the unique spatial complexity, compared to their natural counterparts which deliver regulatory cues for biomolecules distribution and functions. Although 3D printing can yield spatially complex matrices, it cannot meet all the requirements due to the low printing resolution (typically >200 µm) and limited printable biomaterials5. As a result, numerous efforts are still being made to explore novel and reliable fabrication methods that can restore spatial complexity in matrices. Among these efforts, electrospun fibers are often used as prototype materials, due to their similarity to extracellular matrix (ECM) and low fabrication cost

6-13

.

Generally, the spatial complexity of electrospun fibers is realized by subtle design of the micro/nano-structures. In recent years, the majority of the advances in fabricating diverse nanostructured fibrous matrices are achieved via multi-spinneret configurations, which allows the spatially ordered combination of different materials at nanoscale level. For example, coreshell and hollow fibers can be prepared using coaxial electrospinning14-16 whilst bi-/multicompartment fibers are harvested through side-by-side electrospinning17. In contrast to nanostructure, microstructured fibrous matrices are usually obtained via either introducing collectors with microscale layouts18-21, or incorporating magnetic nanoparticles into the fibrous matrices and simultaneously imposing a magnetic field22, or employing near-field


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electrospinning23-24. Besides, another alternative strategy to generate microstructured fibrous matrices lies in the post-modification based on microelectromechanical system (MEMS) or inject-printing25-26. Despite of the relatively independent development in nano- and microstructured fibrous matrices, there are few literatures describing the simultaneous control within these two scales, which in our opinion would enhance dramatically the spatial complexity and thus provide more accurate cues for biomolecules distribution and functions. In this paper, we propose a facile and robust method to fabricate micro- and nano-structured fibrous matrices with spatial complexity by combination of advanced electrospinning and agarose hydrogel stamp based micropatterning. Specifically, advanced electrospinning such as conventional electrospinning, side-by-side electrospinning and coaxial electrospinning are used to construct multi-nanostructures of fibrous matrices. The as-formed fibrous matrices are then contacted with solvent-loaded agarose hydrogel stamps to efficiently remove part of the matrix materials in a very short time (ca. 10~30 seconds) and thus create microstructures (or namely, micropatterns). The merits of our method are the extreme simplicity and flexibility rooted in mono-/multi-spinneret conversion and infinite micropatterns of agarose hydrogel stamps. Herein poly(l-lactic acid) (PLLA), a linear aliphatic polyester with excellent biocompatibility and biodegradability, is chosen as a proof-of-concept material to prepare three types of PLLA fibrous matrices including patterned nano-Ag/PLLA hybrid fibers, patterned bicompartment PET/PLLA fibers and patterned hollow PLLA fibers (Scheme 1). Moreover, we further demonstrate their capability to manipulate biomolecules distribution and functions, i.e., bacteria distribution and antibacterial performance, cell patterning and adhesion/spreading behaviors, protein adsorption and delivery.


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Scheme 1. Patterning multi-nanostructured PLLA fibrous matrices via solvent-loaded agarose hydrogel stamps: (a) electrospinning multi-nanostructured fibrous matrices (i.e., nano-Ag/PLLA hybrid fibers using conventional electrospinning, bicompartment PET/PLLA fibers using sideby-side electrospinning, hollow PLLA fibers using coaxial electrospinning); (b) fabricating agarose hydrogel stamps via soft lithography; (c) microcontacting multi-nanostructured fibrous matrices with solvent-loaded agarose hydrogel stamps; (d) immersing coaxial electrospun fibrous matrices into n-hexane solution to extract inner castor oil; (e) patterned multi-nanostructured fibrous matrices and refreshable agarose hydrogel stamps.


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2. EXPERIMENTAL SECTION 2.1 Materials and Reagents Poly (L-lactic acid) (PLLA, Mv=50000) was purchased from Polymtek Company (China) and polyethylene

terephthalate

(PET)

was

obtained

from

Yibao

Company

(China).

Hexafluoroisopropanol (HFIP), Rhodamine B, fluoranthene, bovine serum albumin (BSA) and castor oil were bought from Aladdin (China). Silver nanoparticles (20 nm) and agarose were purchased from Suzhou Tanfeng graphene Technology Company and Huankai Company, respectively. Bone mesenchymal stem cells (BMSCs) were obtained from Cyagen Biosciences (China) and cultured in Dulbecco’s Modified Eagle’s Medium (PAA) supplemented with 10% fetal bovine serum (Life Technologies, Gibco, USA) and 1% penicillin/streptomycin. Escherichia coli (E.coli) were bought from China Center for Type Culture Collection. Calcein AM, propidium iodide (PI) were purchased from Tongren Company and fluorescein diacetate (FDA), Triton X-100 were obtained from Aladdin Company. Phalloidin-FITC probe and DAPI were bought from AAT Bioquest Company. Polydimethylsiloxane (PDMS) (Sylgard 184) was purchased from Dow Corning Company (USA) and negative photoresist (NR21-20000P) was bought from Futurrex (USA). Bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) was obtained from Solarbio Company (China). All the materials and reagents were used as received without further purification. 2.2 Fabrication of PDMS masters and agarose hydrogel stamps PDMS masters were prepared via standard soft lithography techniques27. Briefly, a mixture of PDMS and curing agent with 10:1 (w/w) ratio was poured onto a resist-patterned silicon wafer, degassed by vacuum oven, cured on a hotplate at 80 oC and then peeled off from the silicon


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wafer. Agarose hydrogel stamps were fabricated by heating aqueous agarose solution (7.5 wt %) at 80 oC, casting onto PDMS masters for 30 min, peeling off the solidified agarose hydrogel. The as-prepared agarose hydrogel stamps were subsequently soaked in 2-2-2-trifluoroethanol (TFE) solution overnight. 2.3 Fabrication and characterization of nano-Ag/PLLA hybrid fibrous matrices, bicompartment PET/PLLA fibrous matrices and hollow PLLA fibrous matrices 0.9 g of PLLA was dissolved in 6 ml of HFIP solution to prepare ca.15 wt % of PLLA solution. 0.12 g, 0.3 g or 0.6 g of silver nanoparticles were then added into the solution and stirred overnight. A simple setup with a needle tip (mounted on a syringe) and a glass slide (1 cm×1 cm) surrounded by grounded copper square ring was employed to electrospun nanoAg/PLLA hybrid fibrous matrices. The distance between the syringe needle and collector was ca.12 cm and the applied voltage was set as 12-18 kV. The PLLA solution was delivered at a constant flow rate of 1.0 mL/h and the collecting time was 1 min. 15 wt % of PLLA and 20 wt % of PET dissolved in HFIP was used to prepare bicompartment PET/PLLA fibers, which were achieved by a dual-spinneret with the same diameter of 0.4 mm. The collecting distance was similar to simple electrospinning and the applied voltage was 9-11 kV. The flow rates of PLLA and PET were 0.5 mL/h and 0.35 mL/h. In order to fabricate hollow PLLA fibers, coaxial electrospinning was carried out with pure castor oil as the core solution and 15 wt % of PLLA dissolved in HFIP as shell solution. The inner and outer diameters of the metal coaxial nozzle were 0.5 mm and 1.5 mm, respectively. The distance between the syringe needle and collector was ca. 12 cm, the applied voltage was set


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as 15-18 kV, the core flow rate was 0.2 mL/h and the shell flow rate was 0.6 mL/h. The asformed fibers were then soaked in n-hexane overnight to remove the castor oil in the inner layer. Surface morphologies of the multi-nanostructured fibrous matrices were characterized by scanning electronic microscopy (SEM, Zeiss, Germany) and optical microscopy (Qlympus, Japan). Transmission electron microscopy (TEM, JEM-1200F, JEOL, Tokyo, Japan) was used to confirm the bicompartments in PET/PLLA fibers. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra (Nexus Por Euro, USA) were collected to identify various components before and after treatment with agarose hydrogel stamps. Fluorescence images were observed using confocal laser scanning microscope (CLSM, Leica, Germany). 2.4 Cell and bacterial culture Patterned bicompartment PET/PLLA fibrous matrices were first fixed by 2 wt % of agarose solution to prevent floating during cell culture, then sterilized using 75% of ethanol for 30 min, followed by rinsing with sterilized PBS at least 3 times. After seeding BMSCs (density: 1×105 cells per well) for two days, the unattached cells were gently rinsed away with PBS. Thereafter, 200 µl of PBS solution containing 1‰ (v/v) of calcein AM and 1‰ (v/v) of PI was added per well and incubated for 30 min at 37 oC. For cytoskeleton staining, the samples were fixed using 4% formaldehyde, then 0.1% Triton X-100, phalloidin-FITC probe, and DAPI were injected successively. The rinsed samples were characterized by CLSM. E.coli bacterial colonies of the strains were stored at 4 oC and cultured at 37 oC in a flask containing nutrient agarose medium for 12 h on an orbital shaker at 150 rpm. The seeding density of E.coli is 2×108 per well. After cultured for 6 h, the unattached E.coli were washed 3


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times by PBS and then 200 µL of live/dead dye solution containing 2% of FDA and 1% of PI was added and incubated for 20 min. 2.5 Protein adsorption and release The patterned hollow PLLA fibers were soaked into BSA-FITC solution for 6 h on an orbital shaker at 50 rpm, followed by rinsing with PBS at least 3 times. The protein adsorption amount was identified by CLSM. Protein release was monitored according to a method described in a previous study28. In brief, 3 mg of the patterned fibrous matrices with the length of 500 µm and 1200 µm were placed in 3 mL of PBS buffer containing BSA. Protein release was performed in an incubator shaker at 37 oC for 24 h. At appropriate intervals, 1 ml of the supernatant was removed and replenished with an identical volume of fresh buffer. BSA concentration in the supernatant was determined using UV absorption at a wavelength of ∼205 nm (Tu-1901, China). Each sample was assayed in triplicate. 2.6 Statistical Analysis All fluorescence images were analyzed with Image J software. A one-way ANOVA followed by a Tukey test for means comparison was performed to assess the level of significance by employing the SPSS 19.0 statistics software. Results are expressed as the mean ± standard error, and p < 0.05 was designated as statistically significant.

3. RESULTS AND DISCUSSIONS


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3.1 Patterning nano-Ag/PLLA hybrid fibrous matrices to manipulate live/dead E.coli bacteria distribution and antibacterial area In our study, the first nanostructured PLLA fibrous matrices were fabricated by incorporating Ag nanoparticles into PLLA fibers (see TEM image of Ag nanoparticles in Fig. S1, Supporting information). Fig. 1a and Fig. S2 (Supporting information) show the SEM and TEM images of electrospun nano-Ag/PLLA hybrid fibrous matrices with various Ag contents. As can be seen, nano-Ag/PLLA hybrid fibers with low Ag content (0-2 wt %) exhibit homogeneous morphology and smooth surface, indicating the uniform distribution of Ag nanoparticles inside fibers. In contrast, when the Ag content exceeds 5 wt %, small clots can be observed along the fibers due to the agglomeration of Ag nanoparticles. Besides, the presence of Ag in nanoAg/PLLA hybrid fibers was also confirmed using EDS and the data are illustrated in Fig. 1b. Obviously, there are no characteristic peaks of Ag in pure PLLA fibers. With the increased Ag contents from 2 wt % to 10 wt %, the characteristic peaks of Ag become more and more evident, further proving the successful addition of Ag nanoparticles into PLLA fibers. After contacting with TFE-loaded agarose hydrogel stamps, high-quality micropatterns can be constructed on nano-Ag/PLLA hybrid fibrous matrices regardless of the Ag content. Fig. 1c shows the optical images of patterned nano-Ag/PLLA hybrid fibrous matrices containing 2 wt % of Ag nanoparticles (see optical images of patterned nano-Ag/PLLA hybrid fibrous matrices containing 5 wt % and 10 wt % of Ag nanoparticles in Fig. S3, supporting information). It is clear that no residual fibers exist in the contacted area whilst the fibers in the uncontacted area still remain good configuration, implying effective and precise removal of nano-Ag/PLLA mats by TFEloaded agarose hydrogel stamps.


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Figure 1. Fabrication and characterization of patterned nano-Ag/PLLA hybrid fibrous matrices: (a) SEM images of pure PLLA and nano-Ag/PLLA hybrid fibers with Ag content in the range of 2-10 wt %; (b) EDS profiles of pure PLLA fibers and nano-Ag/PLLA hybrid fibers; (c) optical pictures of patterned nano-Ag/PLLA hybrid fibrous matrices (2 wt % Ag). Considering the broad-spectrum biocidal properties of Ag+ and/or Ag29, the antibacterial performance of patterned nano-Ag/PLLA hybrid fibrous matrices against E.coli bacteria was investigated. Our preliminary results reveal that the live/dead E.coli distribution and antibacterial area can be manipulated by the Ag content and patterns of the fibrous matrices (Fig. 2a). Herein the role of Ag content is identified through the live/dead staining of E.coli cultured on patterned nano-Ag/PLLA hybrid fibrous matrices with various Ag contents (Fig. 2b). Most of E.coli are alive on the whole surface of patterned pure PLLA fibrous matrices, indicative of no antibacterial effects for both pure PLLA and bare glass substrates. As the Ag content increases,


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the amount of dead E.coli on the fiber surface rises dramatically, owing to the antibacterial properties of Ag nanoparticles. Interestingly, the amount of dead E.coli on bare glass surface without fibers is also dependent on the Ag content of fibers, which can be attributed to the trace amount of Ag or Ag+ released from surrounding fibrous matrices during culture process. Fig. 2c lists the live/dead E.coli ratio as a function of the Ag content. The live/dead E.coli ratio is the highest (ca. 4.8-4.9) for the patterned pure PLLA fibrous matrices (0 wt % Ag) and spatially shows no significant difference. In terms of patterned nano-Ag/PLLA hybrid fibrous matrices with 2 wt % of Ag, the live/dead E.coli ratio changes a little on glass surface but declines sharply on fiber surface, resulting in their significant difference (p < 0.0001). However, when the Ag content continues growing to 5 wt % and 10 wt %, such a ratio starts to decrease on bare glass while remains steady on fiber surfaces, and finally shows no significant difference all over the fibrous matrices. Nevertheless, the huge difference of live/dead E.coli ratios between bare glass and fibers containing 2 wt % of Ag implies the possibility of spatially controlling live/dead E.coli distribution and antibacterial area by the patterns of fibrous matrices. To further confirm this, nano-Ag/PLLA hybrid fibrous matrices with diamond, stripe, hexagon and cross patterns were used for E.coli culture and the corresponding fluorescence images show the obvious boundaries of live/dead E.coli bacteria which perfectly match these patterns (Fig. 2d). The precise and arbitrary manipulation of live/dead E.coli distribution and antibacterial area via patterned nano-Ag/PLLA hybrid fibrous matrices provides a new and promising tool to control the area of bacterial biofilms, benefitting the study on their formation mechanisms and relevant infection effects30.


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Figure 2. Manipulation of live/dead E.coli bacteria distribution and antibacterial area via patterned nano-Ag/PLLA hybrid fibrous matrices: (a) schematic illustration on controlling the distribution of live/dead E.coli bacteria; (b) live/dead staining of E.coli bacteria cultured on patterned nano-Ag/PLLA hybrid fibrous matrices for 6h; (c) live/dead E.coli bacteria ratio between bare glass and nano-Ag/PLLA fiber surfaces (**, p < 0.01; ****, p < 0.0001); (d) arbitrary manipulation of antibacterial area by micropatterns on nano-Ag/PLLA hybrid fibrous matrices. 3.2 Patterning bicompartment PET/PLLA fibrous matrices to manipulate BMSCs distribution and adhesion/spreading behaviors


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Figure 3. Fabrication and characterization of patterned bicompartment PET/PLLA fibrous matrices: (a) SEM (left) and TEM (right) images of bicomponent PET/PLLA fibers fabricated by side-by-side electrospinning; (b) optical (top) and fluorescence (bottom) images of patterned bicompartment PET/PLLA fibrous matrices (fluorescence dye was added into PLLA only); (c) selective removal of PLLA component from bicompartment PET/PLLA fibrous matrices using agarose hydrogel stamp (fluorescence dyes were added in both PET and PLLA components, i.e., blue for PET component and red for PLLA component): (c1) merged fluorescence image of PET and PLLA components before patterning, (c2) merged fluorescence image of PET and PLLA components after patterning, (c3) individual fluorescence image of PET component after patterning; (c4) individual fluorescence image of PET component on agarose hydrogel stamp surface after patterning; (c5) individual fluorescence image of PLLA component after patterning; (c6) individual fluorescence image of PLLA component on agarose hydrogel stamp surface after patterning; (d) FTIR spectra of PET, PLLA, PET/PLLA and PET/PLLA (treated by solventloaded agarose hydrogel stamp).


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Bicompartment PET/PLLA fibers were fabricated via side-by-side electrospinning. SEM and TEM images in Fig. 3a show that each fiber consists of two compartments aligned parallel to each other, in good agreement with the fiber architecture reported in the literature31. The fibers were then treated with TFE-loaded agarose hydrogel stamps. Different from pure PLLA and nano-Ag/PLLA hybrid fibers mentioned in section 3.1 where the fibers in the contacted area are removed thoroughly, fibers can still be seen in the contacted area for bicompartment PET/PLLA fibers although patterns are well defined (Fig. 3b). To distinguish the observation more clearly, Rhodamine B (red fluorescence) was mixed into PLLA. The resulting bicompartment PET/PLLA fibers show no fluorescence in the contacted area after patterning process. This phenomenon was further confirmed by adding another dye (fluoranthene, blue fluorescence) into PET compartment. As illustrated in Fig. 3c, the original bicompartment PET/PLLA fibers is rose (Fig. 3c1), an overlapping color of red (PLLA) and blue (PET). Patterning these fibers leads to the disappearance of red fluorescence but reservation of blue fluorescence in the contacted area (Fig. 3c2). Individual monitoring of PET shows negligible change of blue fluorescence intensity in patterned bicompartment PET/PLLA fibers (Fig. 3c3) as well as the absence of blue fluorescence on agarose hydrogel stamp surface (Fig. 3c4). On the contrary, individual monitoring of PLLA proves that red fluorescence vanishes in the contacted area (Fig. 3c5) and correspondingly complementary pattern is observable on agarose hydrogel stamp surface (Fig. 3c6). In addition to optical measurement, ATR-FTIR profiles were collected to explore the change in fiber mats after contacting with agarose hydrogel stamp. The characteristic peaks of PET (730 cm-1: vibration of benzene ring, 1730 cm-1: C=O stretching, 3055 cm-1: C-H stretching of benzene ring) and PLLA (1757 cm-1: C=O stretching, 1181 cm-1: symmetric stretching of CO-C, 1087 cm-1: C-O stretching) can be found distinctly in bicompartment PET/PLLA fibers.


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However, the treated bicompartment PET/PLLA fibers only show PET characteristic peaks (Fig. 3d). All these data suggest that TFE can only dissolve PLLA rather than PET. As a consequence, PLLA in the bicompartment PET/PLLA fibers can be removed selectively by TFE-loaded agarose hydrogel stamp, causing local generation of monocompartment PET fibers in the contacted area. In another word, patterning bicompartment PET/PLLA fibrous matrices would result in an ordered assembly of two different fibers. It should be pointed out that this patterning strategy is also applicable to core-shell PET/PLLA hybrid fibrous matrices with PLLA as outer layer and PET as inner layer (Fig. S4, supporting information). Due to their morphological and dimensional similarity to native ECM fibers and fibril bundles, electrospun fibrous matrices have been well recognized as a robust biomimetic platform to study cell–cell and cell–matrix interactions, and ultimately promote tissue repair and regeneration32-34. Aware of the essence of spatially complex ECM materials in regulating cellular activities and functions in vivo, it is desirable to develop the capabilities of fabricating patterned ECM-like surfaces in vitro and studying cell responses. Fig. 4 shows BMSC behaviors after culturing on patterned bicompartment PET/PLLA fibrous matrices. As we know, PLLA is hydrophobic whilst PET is hydrophilic (Fig. S5, supporting information). BMSCs are inclined to adhere to PET instead of PLLA, indicative of cell patterning effects (Fig. 4a). To better correlate cell adhesion and spreading with material patterns, BMSCs were cultured on patterned bicompartment PET/PLLA fibrous matrices with three shapes (circle, diamond or hexagon) and dimensions (100, 200, 400 µm). It can be seen from Fig. 4b that BMSCs are more likely to adhere onto the PET fiber surface (or namely, the area contacted by agarose hydrogel stamp) in all cases after 1 day culture. Upon closer observation, one may notice that the preferential adhesion and spreading of BMSCs is more remarkable when the pattern size increases from 100 to 400 µm. In


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our opinion, one possible reason is that larger pattern promotes cell communication and thus cell proliferation. Similar phenomena were also reported by Jia et al who studied cell adhesion to patterned fibronectin on polycaprolactone (PCL) fibers

32

. Even culturing for longer time (for

example 2 day), cell patterning is still very clear (Fig 4c). Although further work is needed to understand the intrinsic mechanism for pattern-induced cell responses, our results demonstrate that cellular distribution and functions can be regulated by patterning bicompartment PET/PLLA fibrous matrices.


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Figure 4. Manipulation of BMSC distribution and adhesion/spreading behaviors on patterned bicompartment PET/PLLA fibrous matrices: (a) schematic illustration on manipulating BMSC distribution; (b) fluorescence images of BMSC cultured on patterned bicompartment PET/PLLA fibrous matrices with three shapes (circle, diamond or hexagon) and dimensions (the diameter of circle, the side length of diamond and hexagon were 100, 200 and 400 µm, respectively); (c) fluorescence images of BMSC cultured on bicompartment PET/PLLA fibrous matrices with concentric circles and circle dots patterns. 3.3 Patterning hollow PLLA fibrous matrices to manipulate protein adsorption and delivery Core-shell PLLA fibers were initially collected via coaxial electrospinning with castor oil and PLLA as core and shell materials respectively. To ensure the core-shell structure, it is critical to control the flow rates of core and shell solutions during the electrospinning process (Figure S6, supporting information). After extracting castor oil by hexane, hollow PLLA fibers can be acquired. SEM images (Fig. 5a) show that the as-prepared PLLA fibers are quite smooth and uniform, and hollow structure can be observed easily from the cross sectional view. Fig. 5b compares the FTIR spectra of castor oil, PLLA, core-shell PLLA fibers with castor oil inside and hollow PLLA fibers. The characteristic peaks of castor oil (3500 cm-1: O-H stretching, 30002800 cm-1: C-H stretching and 1650 cm-1: C=O stretching) and PLLA (1757 cm-1: C=O stretching, 1181 cm-1: symmetric stretching of C-O-C, 1087 cm-1: C-O stretching) can be both found in core-shell PLLA fibers, indicating the presence of castor oil in the core-shell fibers before extraction. In contrast, the vanishing peaks of castor oil in hollow PLLA fibers prove the complete removal of castor oil after extraction.


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Biomolecule and drug delivery via carriers benefits from both the protection of encapsulated biomolecules or drugs from hazardous environment and the sustained release for a desired period35. An efficient carrier should meet at least two conditions, i.e., high loading capacity and adjustable release rate. In view of the high specific surface area, hollow fibers are very promising carriers for biomolecule or drug delivery. Unfortunately, their release rate is hard to control. One possible reason is the lack of a strategy to precisely manipulate the length of hollow fibers. In our opinion, this problem can be solved by patterning aligned hollow PLLA fibers via agarose hydrogel stamps, as shown in Fig. 5c. Herein BSA, as a model protein or drug, was used to evaluate the loading capacity and release rate of patterned hollow PLLA fibers. Fig. 6a shows the fluorescence images of solid and hollow fibers after treating with various concentrations of FITC-labelled BSA (FITC-BSA). It is obvious that the fluorescence intensity (or namely, the loading capacity of FITC-BSA) of these two kinds of fibers increases as the concentration of FITC-BSA rises from 0.25 mg/mL to 1.0 mg/mL. In comparison to solid fibers, hollow fibers exhibit much higher loading capacity despite of the same fiber diameter and length (Fig. 6b). Further quantitative analysis shows that the loading efficienties of hollow fibers and solid fibers are 5.6% and 3.4% respectively (average fiber length: 1.2 mm). The BSA release rate was examined by taking hollow fibers with the length of 500 and 1200 µm as examples. The release curves in Fig. 6c display that a burst release of BSA occurs both for hollow fibers of 500 and 1200 µm long during the first 4 h. However, an obvious steady release can be observed for hollow fibers of 1200 µm long whilst burst release still continues for hollow fibers of 500 µm long until all the BSA molecules come off from the fiber surface. This is possibly due to the longer diffusion distance of BSA from the inner surface of longer hollow fibers. Anyway, the close relationship between BSA release rate and fiber length indicates the feasibility to regulate


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the release rates of encapsulated biomolecules or drugs by controlling the hollow fiber length which can be easily realized through the patterning of aligned hollow fibers.

Figure 5. Fabrication and characterization of patterned hollow PLLA fibrous matrices: (a) SEM images of hollow PLLA fibers prepared by coaxial electrospinning; (b) FTIR spectra of castor oil, PLLA, the coaxial fibers before and after extraction; (c) optical images of patterned hollow PLLA fibrous matrices. Scale bar: 200 µm (top) and 100 µm (bottom).


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Figure 6. Manipulation of protein adsorption and release via patterned hollow PLLA fibrous matrices: (a) fluorescence images of solid and hollow PLLA fibers after immersing in FITCBSA solution with different concentrations; (b) the quantitative data on BSA adsorption amount on hollow and solid PLLA fibers; (c) BSA release curves of hollow PLLA fibers with the length of 500 µm and 1200 µm. The measurements were conducted after immersing 3 mg of hollow PLLA fibers in 1 mg/mL of BSA solution for 6 h.

4. CONCLUSION In summary, this paper reports the fabrication of patterned PLLA fibrous matrices with multi-nanostructures by integrating advanced electrospinning and agarose hydrogel stamp based patterning, and the manipulation of biomolecules distribution and functions using these fibrous


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matrices. Three independent yet interconnected work are conducted, i.e., patterning nanoAg/PLLA hybrid fibrous matrices to manipulate live/dead E.coli bacteria distribution and antibacterial area; patterning bicompartment PET/PLLA fibrous matrices to manipulate BMSCs distribution and adhesion/spreading behaviors; patterning hollow PLLA fibrous matrices to manipulate protein adsorption and delivery. Our results reveal that biomolecules distribution (live/dead E.coli bacteria distribution, BMSCs patterning, protein adsorption) and functions (antibacterial performance, BMSCs adhesion and spreading, protein delivery) can be controlled effectively by micropatterning these multi-nanostructured fibrous matrices. We believe the methodology and even the PLLA fibrous matrices exploited in our paper would have promising applications in biomedical engineering field.

ASSOCIATED CONTENT Supporting Information. TEM image of Ag nanoparticles and the particle size distribution, TEM images of nanoAg/PLLA hybrid fibers with Ag content in the range of 0-10 wt%, photo images of patterned nano-Ag/PLLA hybrid fibrous matrices with the Ag contents of 5 wt % and 10 wt %, the images of patterned core-shell PET/PLLA hybrid fibrous matrices (PLLA as outer layer and PET as inner layer) fabricated via coaxial electrospinning, and the contact angle of PET and PLLA films. These files are available free of charge.

AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected], Xiaodong Cao. *E-mail: [email protected], Hua Dong Author Contributions Xiao and Li contribute equally to this work. ACKNOWLEDGMENT This work was financially sponsored by the National Natural Science Foundation of China (Grant No. 51373056, 51372085, 21574045), Natural Science Foundation of Guangdong Province (2016A030311010) and Fundamental Research Funds for the Central Universities (2017ZD014).

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TOC figure


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Scheme 1. Patterning multi-nanostructured PLLA fibrous matrices via solvent-loaded agarose hydrogel stamps: (a) electrospinning multi-nanostructured fibrous matrices (i.e., nano-Ag/PLLA hybrid fibers using conventional electrospinning, bicompartment PET/PLLA fibers using side-by-side electrospinning, hollow PLLA fibers using coaxial electrospinning); (b) fabricating agarose hydrogel stamps via soft lithography; (c) microcontacting multi-nanostructured fibrous matrices with solvent-loaded agarose hydrogel stamps; (d) immersing coaxial electrospun fibrous matrices into n-hexane solution to extract inner castor oil; (e) patterned multi-nanostructured fibrous matrices and refreshable agarose hydrogel stamps. 262x260mm (300 x 300 DPI)



Figure 1. Fabrication and characterization of patterned nano-Ag/PLLA hybrid fibrous matrices: (a) SEM images of pure PLLA and nano-Ag/PLLA hybrid fibers with Ag content in the range of 2-10 wt %; (b) EDS profiles of pure PLLA fibers and nano-Ag/PLLA hybrid fibers; (c) optical pictures of patterned nano-Ag/PLLA hybrid fibrous matrices (2 wt % Ag). 311x188mm (150 x 150 DPI)


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Figure 2. Manipulation of live/dead E.coli bacteria distribution and antibacterial area via patterned nanoAg/PLLA hybrid fibrous matrices: (a) schematic illustration on controlling the distribution of live/dead E.coli bacteria; (b) live/dead staining of E.coli bacteria cultured on patterned nano-Ag/PLLA hybrid fibrous matrices for 6h; (c) live/dead E.coli bacteria ratio between bare glass and nano-Ag/PLLA fiber surfaces (**, p < 0.01; ****, p < 0.0001); (d) arbitrary manipulation of antibacterial area by micropatterns on nanoAg/PLLA hybrid fibrous matrices. 142x134mm (300 x 300 DPI)



Figure 3. Fabrication and characterization of patterned bicompartment PET/PLLA fibrous matrices: (a) SEM (left) and TEM (right) images of bicomponent PET/PLLA fibers fabricated by side-by-side electrospinning; (b) optical (top) and fluorescence (bottom) images of patterned bicompartment PET/PLLA fibrous matrices (fluorescence dye was added into PLLA only); (c) selective removal of PLLA component from bicompartment PET/PLLA fibrous matrices using agarose hydrogel stamp (fluorescence dyes were added in both PET and PLLA components, i.e., blue for PET component and red for PLLA component): (c1) merged fluorescence image of PET and PLLA components before patterning, (c2) merged fluorescence image of PET and PLLA components after patterning, (c3) individual fluorescence image of PET component after patterning; (c4) individual fluorescence image of PET component on agarose hydrogel stamp surface after patterning; (c5) individual fluorescence image of PLLA component after patterning; (c6) individual fluorescence image of PLLA component on agarose hydrogel stamp surface after patterning; (d) FTIR spectra of PET, PLLA, PET/PLLA and PET/PLLA (treated by solvent-loaded agarose hydrogel stamp). 99x66mm (300 x 300 DPI)


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Figure 4. Manipulation of BMSC distribution and adhesion/spreading behaviors on patterned bicompartment PET/PLLA fibrous matrices: (a) schematic illustration on manipulating BMSC distribution; (b) fluorescence images of BMSC cultured on patterned bicompartment PET/PLLA fibrous matrices with three shapes (circle, diamond or hexagon) and dimensions (the diameter of circle, the side length of diamond and hexagon were 100, 200 and 400 µm, respectively); (c) fluorescence images of BMSC cultured on bicompartment PET/PLLA fibrous matrices with concentric circles and circle dots patterns. 198x314mm (300 x 300 DPI)



Figure 5. Fabrication and characterization of patterned hollow PLLA fibrous matrices: (a) SEM images of hollow PLLA fibers prepared by coaxial electrospinning; (b) FTIR spectra of castor oil, PLLA, the coaxial fibers before and after extraction; (c) optical images of patterned hollow PLLA fibrous matrices. Scale bar: 200 m (top) and 100 m (bottom). 125x108mm (300 x 300 DPI)


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Figure 6. Manipulation of protein adsorption and release via patterned hollow PLLA fibrous matrices: (a) fluorescence images of solid and hollow PLLA fibers after immersing in FITC-BSA solution with different concentrations; (b) the quantitative data on BSA adsorption amount on hollow and solid PLLA fibers; (c) BSA release curves of hollow PLLA fibers with the length of 500 µm and 1200 µm. The measurements were conducted after immersing 3 mg of hollow PLLA fibers in 1 mg/mL of BSA solution for 6 h. 103x75mm (300 x 300 DPI)



76x42mm (300 x 300 DPI)


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