Multifunctional scaffolds with improved antimicrobial properties and

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Biological and Medical Applications of Materials and Interfaces

Multifunctional scaffolds with improved antimicrobial properties and osteogenicity based on piezoelectric electrospun fibers decorated with bioactive composite microcapsules Alexander S. Timin, Albert R Muslimov, Mikhail V Zyuzin, Oleksii O Peltek, Timofey E Karpov, Igor S Sergeev, Anna I Dotsenko, Alexander A Goncharenko, Nikita Yolshin, Artem Sinelnik, Bärbel Krause, Tilo Baumbach, Maria A. Surmeneva, Roman V Chernozem, Gleb B. Sukhorukov, and Roman A. Surmenev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09810 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018

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ACS Applied Materials & Interfaces

Multifunctional Scaffolds with Improved Antimicrobial Properties and Osteogenicity Based on Piezoelectric Electrospun Fibers Decorated with Bioactive Composite Microcapsules Alexander S. Timin1,2*, Albert R. Muslimov1, Mikhail V. Zyuzin3, Oleksii O. Peltek4, Timofey E. Karpov4, Igor S. Sergeev4, Anna I. Dotsenko1, Alexander A. Goncharenko4, Nikita Yolshin5, Artem Sinelnik6, Bärbel Krause7, Tilo Baumbach7,8, Maria A. Surmeneva2, Roman V. Chernozem2, Gleb B. Sukhorukov4,9, Roman A. Surmenev2* 1

First I. P. Pavlov State Medical University of St. Petersburg, Lev Tolstoy str., 6/8, 197022,

Saint-Petersburg, Russian Federation 2

Physical Materials Science and Composite Materials Centre, National Research Tomsk

Polytechnic University, Lenin Avenue, 30, 634050, Tomsk, Russian Federation 3

Department of Nanophotonics and Metamaterials, ITMO University, St. Petersburg 197101,

Russian Federation 4

Peter The Great St. Petersburg Polytechnic University, Polytechnicheskaya, 29, 195251, St.

Petersburg, Russian Federation 5

Research Institute of Influenza, Ministry of Healthcare of the Russian Federation, prof. Popova

str., 15/17, 197376, Saint-Petersburg, Russian Federation 6

Department of Dielectric and Semiconductor Photonics, ITMO University, St. Petersburg

197101, Russian Federation 7

Institute for Photon Science and Synchrotron Radiation, Karlsruhe Institute of Technology,

76344 Eggenstein-Leopoldshafen, Germany 8

Laboratory for Applications of Synchrotron Radiation (LAS), Karlsruhe Institute of Technology

(KIT), 76049 Karlsruhe, Germany 1 ACS Paragon Plus Environment

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School of Engineering and Materials Science, Queen Mary University of London, Mile End

Road, London E1 4NS, United Kingdom Correspondence to Associate Prof. Dr. Roman Surmenev and Dr. Alexander Timin at [email protected] and [email protected], [email protected].

Keywords: Polymer scaffolds, polyelectrolyte and hybrid microcapsules, sol-gel coating, cell adhesion, antibacterial properties, osteogenic differentiation

Abstract The incorporation of bioactive compounds onto polymer fibrous scaffolds with further control of drug release kinetics is essential to improve the functionality of scaffolds for personalized drug therapy and regenerative medicine. In this study, the polymer and hybrid microcapsules were prepared and used as drug carriers, which are further deposited onto polymer microfiber scaffolds [polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB) and PHB doping with the conductive polyaniline (PANi) of 2 wt% (PHB-PANi)]. The number of immobilized microcapsules decreased with the increase in their ζ-potential due to electrostatic repulsion with the negatively charged fiber surface, depending on the polymer used for the scaffold’s fabrication. Additionally, the immobilization of the capsules in dynamic mechanical conditions at a frequency of 10 Hz resulted in an increase in the number of the capsules on the fibers with the increase in the scaffold piezoelectric response in the order PCL< PHB < PHB-PANi, depending on the chemical composition of the capsules. The immobilization of microcapsules loaded with different bioactive molecules onto the scaffold surface enabled multimodal triggering by physical (ultrasound, laser radiation) and biological (enzymatic treatment) stimuli, 2 ACS Paragon Plus Environment

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providing controllable release of the cargo from scaffolds. Importantly, the microcapsules immobilized onto the surface of the scaffolds did not influence the cell growth, viability and cell proliferation on the scaffolds. Moreover, the attachment of human mesenchymal stem cells (hMSCs) on the scaffolds revealed that the PHB and PHB-PANi scaffolds promoted adhesion of hMSCs compared to that of the PCL scaffolds. Two bioactive compounds, antibiotic ceftriaxone sodium (CS) and osteogenic factor dexamethasone (DEXA), were chosen to load the microcapsules and demonstrate the antimicrobial properties and osteogenesis of the scaffolds. The modified scaffolds had prolonged release of CS or DEXA, which provided an improved antimicrobial effect, as well as enhanced osteogenic differentiation and mineralization of the scaffolds modified with capsules compared with that of individual scaffolds soaked in CS solution or incubated in an osteogenic medium. Thus, the immobilization of microcapsules provides a simple, convenient way to incorporate bioactive compounds onto polymer scaffolds, which makes these multimodal materials suitable for personalized drug therapy and bone tissue engineering.

Abbreviations: Polycaprolactone – PCL Poly(3-hydroxybutyrate) – PHB PANi – Polyaniline Poly(3-hydroxybutyrate) doping of the conductive PANi of 2 % wt – PHB-PANi Non-degradable poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) capsules with 3 bilayers of polyelectrolytes – PAH caps

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Degradable dextran sulfate/poly(L-arginine) capsules with 3 bilayers of polyelectrolytes – PARG caps Degradable hybrid silica capsules – SiO2 caps Non-degradable poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) capsules functionalized with gold nanorods – PAH/Au caps Fluorescein isothiocyanate – FITC-BSA BSA conjugated with tetramethylrhodamine – TRITC-BSA Dextran conjugated with Alexa Fluor 647 – AF647-DEX Dexamethasone – DEXA Ceftriaxone sodium – CS PCL scaffold decorated with PARG caps – PCL@PARG caps PCL scaffold decorated with PAH caps – PCL@PAH caps PCL scaffold decorated with Au/PAH caps – PCL@Au/PAH caps PCL scaffold decorated with SiO2 caps – PCL@SiO2 caps PHB scaffold decorated with PARG caps – PHB@PARG caps PHB scaffold decorated with PAH caps – PHB@PAH caps PHB scaffold decorated with Au/PAH caps – PHB@Au/PAH caps PHB scaffold decorated with SiO2 caps – PHB@SiO2 caps PHB-PANi scaffold decorated with PARG caps – PHB-PANi@PARG caps PHB-PANi scaffold decorated with PAH caps – PHB-PANi@PAH caps PHB-PANi scaffold decorated with Au/PAH caps – PHB-PANi@Au/PAH caps PHB-PANi scaffold decorated with SiO2 caps – PHB-PANi@SiO2 caps PCL scaffold decorated with CS-loaded SiO2 caps – PCL@CS/SiO2 caps


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PHB scaffold decorated with CS-loaded SiO2 caps – PHB@CS/SiO2 caps PHB-PANi scaffold decorated with CS-loaded SiO2 caps – PHB-PANi@CS/SiO2 caps PCL scaffold decorated with DEXA-loaded SiO2 caps – PCL@DEXA/SiO2 caps PHB scaffold decorated with DEXA-loaded SiO2 caps – PHB@DEXA/SiO2 caps PHB-PANi scaffold decorated with DEXA-loaded SiO2 caps – PHB-PANi@DEXA/SiO2 caps

1.

Introduction Tissue engineering is a promising approach in regenerative medicine that can provide

new methods for organ restoration and overall enhancement of medical treatment.1 As an interdisciplinary field, tissue engineering relies on advances that related fields of science can provide. There are three general tissue engineering tools that are necessary for the successful tissue regeneration, also known as the tissue engineering triad, i.e., cells to provide the tissue growth, scaffolds to strengthen the organ and set its shape, and, finally, signaling molecules to modify cell differentiation. First, mesenchymal stem cells (MSCs) are currently used in the field of tissue engineering,2 not only due to the ability of MSCs to differentiate in the adipogenic and osteogenic directions,3 but also due to the their ability to act as a modulator of the stroma of newly formed tissue. MSCs can realize a supporting function in the tissue that artificially forms de novo. In particular, MSCs can provide the necessary additives, such as the vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMP-2), for improved osteogenesis.4 Scaffolds are the next important factor for successful tissue regeneration. These fibrous networks are providing sites for the cell adhesion as well as for mechanical support.5



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Scaffolds must be adequately porous for cell growth and must possess a three-dimensional structure that allows sufficient nutrient and metabolite transport. The scaffolds materials should be compatible with cells and endogenous tissue and should also contribute to the cell adhesion, proliferation, and directed differentiation. The mechanical properties of scaffolds should match the mechanical properties of a forming tissue to support the cell growth until full tissue recovery is attained. Moreover, these biomaterials should be biodegradable.1 Polyesters are the main biodegradable polymers, which have received increasing attention, revealing a wide range of properties from rigid brittle to flexible plastics.6 The best-known biodegradable polymers are polycaprolactone (PCL) and poly(3-hydroxybutyrate) (PHB). PCL possesses suitable mechanical and biodegradable properties for the development of functional hybrid scaffolds. PCL shows good results as a platform to repair damaged/diseased human tissue as well as in controlled drug release.7 At the same time, PHB is simultaneously biodegradable and has piezoelectric polymers with good biocompatibility in vitro and in vivo for wound dressing,8 cardiac repair,9 bone10 and nerve tissue.11 Electrical stimuli can evoke desirable cellular responses, especially from cells belonging to electrically excitable tissues.12 A recent study has shown that the most significant effect on fibroblasts has been revealed in cases of scaffolds with the largest piezoelectric constants, such as non-biodegradable polyvinylidene fluoride.13 Biodegradable PHB has insufficient piezoelectric charge constant values compared with that of polyvinylidene fluoride (PVDF). However, the piezoelectric performance of PHB can be successfully improved via the addition of a conductive biocompatible polymer, such as polyaniline (PANi)14 in the structure of PHB.15 In vitro MSCs can be directed to form osteoblasts through the addition of soluble factors. One of these factors is dexamethasone in combination with β-glycerophosphate and ascorbic 6 ACS Paragon Plus Environment

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acid, which can be used as a simple and reliable model factor to increase the osteogenic potential of the test materials.16 Some previous studies showed that the generation and presence of a functional microvascular network design (angiogenesis), which provides oxygen and nutrients to facilitate cell growth, significantly improves the differentiation and functionality of tissues.17 The biocompatible scaffolds can be additionally modified with bioactive molecules and employed not only as the matrix for the cell growth but also as a reservoir for the numerous growth factors and drugs. Such modifications may stimulate the cell differentiation,18 impart scaffolds with additional functions, e.g., antibacterial properties,19 and induce certain cell arrangements.20 At present, the clinically approved materials for scaffolds lack the possibility to stimulate tissue differentiation. Moreover, the possible development of biofilms on the implantable materials is an urgent problem, which can cause tissue regeneration failure.21 To overcome these challenges, the treatment of scaffolds with bioactive compounds is required, which can also have the dose-dependent effect. Thus, these bioactive molecules need to be administered systematically to achieve prolonged drug action and necessary therapeutic concentrations.22 The bioactive compounds are generally introduced in the scaffolds by physical adsorption. The release of physically adsorbed molecules always exhibits an initial burst and cannot last for a long period.23 In addition, drug incorporation in organic/inorganic hybrids and their drug release behavior from the scaffold surface are rarely reported.24-25 Among these works, the scaffolds are usually decorated with nanomaterials (e.g. iron oxide, silver, silica nanoparticles, graphene oxide etc.) possessing only one functionality.26-27 For instance, silica nanoparticles loaded with antibiotics were incorporated into electrospun scaffolds to promote prolonged release profile of antibiotics for antibacterial effect of the scaffolds.25 Calcium phosphate particles loaded with growth factors were also used to modify the surface of the scaffolds in order to induce osteogenic



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differentiation.23 To the best of our knowledge, there are no works, describing the functionalization of a scaffold with carriers containing several drugs. This modification enables multi-functional platform for drug delivery. As an example of such a system, a scaffold can be modified with two types of carriers loaded with antibiotics and growth factors. In this case, loaded antibiotics will prevent implant contamination, whereas growth factors will stimulate fast tissue regeneration. Moreover, it is highly important that these carriers can be tuned to provide the controlled release (prolonged or fast release), resulting in development of novel tailored tissue engineering systems. One interesting strategy to solve the above mentioned challenges is represented by polymer or hybrid microcapsules, which can be used as carriers to deliver bioactive compounds into cells.28 Polymer and hybrid microcapsules have been found to be a unique tool that provides the possibility of in situ deposition of various organic and inorganic materials resulting in the formation of a composite shell with controllable permeability upon chemical and physical external stimuli.19,

29-30

These capsules can be loaded with different cargo, which is protected

from the enzymatic degradation. The walls of the capsule can be made out of different materials, allowing either remote controlled release of whole cargo inside cells31-32 or the prolonged release of cargo.33 The scaffolds modified with polymer and hybrid capsules will make them simultaneously sensitive to physical and chemical (UV light and ultrasound) stimuli, which is of particular significance for the development of novel stimuli-responsive implants that could integrate multiple functionalities such as controllable drug release, improved biocompatibility and biodegradability. This work describes a novel scaffold modification method that uses capsules loaded with bioactive compounds, which induce the osteogenic differentiation of human MSCs (hMSCs).


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Three types of scaffolds, (i) polycaprolactone scaffolds (PCL), (ii) polyhydroxybutyrate scaffolds (PHB) and (iii) polyaniline/polyhydroxybutyrate scaffolds (PHB-PANi), were prepared and modified either with non-degradable or degradable capsules or with silica capsules. The release of cargo after ultrasound and enzymatic treatment from different capsule types attached to the scaffold’s surface was investigated and quantified. Moreover, the remote release of cargo from non-degradable polyelectrolyte capsules functionalized with gold nanorods using a nearinfrared laser was performed. Additionally, the antibacterial activity of the scaffolds modified with capsules loaded with ceftriaxone sodium was demonstrated. 2. Experimental section 2.1.Fabrication of the scaffolds In the present study, nonwoven fibrous scaffolds were fabricated using the conventional electrospinning setup. To prepare pure PCL and PHB scaffolds, polymers were dissolved in chloroform at the concentration of 9 % and 6 %, respectively. The fabrication of the PHB scaffolds with the enhanced piezoelectric performance by means of doping with the conductive PANi of 2 %wt. is described elsewhere.34 A syringe pump was used to feed the solutions through an extension tube capped with blunted 21-gauge needles (inner diameter of 0.51 mm). A 6.5 kV voltage was applied, using a high-voltage power supply. A needle-collector path (8 cm), with a deposition time of 60 min and a fixed injection flow rate of 1.5 mL×h−1, was used during electrospinning. The prepared samples were separated from the collector and used for further experiments and analysis. 2.2.Structural characterization of synthesized scaffolds



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Scanning electron microscopy (SEM) (Quanta 200, FEI, Netherlands) was used to characterize the scaffolds’ morphology. The samples were examined at an accelerating voltage of 10 kV and a magnification of 1000x. An average value of the fiber diameter was determined from approximately 100 random measurements using three images of each scaffold. To investigate the molecular composition, the attenuated total reflectance Fourier transform infrared spectroscopy (FTIR) was conducted using a Nicolet 5700 FT-IR Spectrometer (Thermo Electron Corporation, USA). Each sample was scanned in the range from 400 to 4000 cm-1 with the resolution of 4 cm- 1. The structure and phase composition of the prepared scaffolds were investigated via X-ray diffraction (XRD-6000, Shimadzu, Japan) with Cu Kα radiation (λ = 0.154 nm) in the 2θ range from 5° to 90° with a step size of 0.02 °/2θ at 40 kV and 30 mA. The database of ICDD PDF 4+ was used to identify phases. To investigate the surface chemical composition, X-ray photoelectron spectroscopy (XPS) with a Phoibos 150 analyzer, an unmonochromated XR-50 Mg Kα X-ray source (hν=1253.6 eV) and an FG 20 flood gun for compensation of the recharge effect from the SPECS (Germany) were used. The obtained spectra were

analyzed

using

the

CASA-XPS

software35

(v.2.3.17PR1.1;

product

of CasaXPS Software Ltd., USA). The topography of the fiber surface was studied using atomic force microscopy (AFM) in a tapping mode. The lateral and vertical resolutions of the method are 1 nm and 0.1 nm, respectively. The wavelength (λ) of the laser was 650 nm. Investigation of the surface potential distribution on a fiber surface was studied by Kelvin probe force microscopy (KPFM). The measurements were carried out by means of a NanoScanTech (Dolgoprudny, Russia) Certus Standard V atomic-force microscope under normal conditions. Antimony-doped single crystal silicon probes, namely, Tips Nano NSG30/Au probes, were employed. The preparation and examination of the surfaces was done in air. To measure the


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roughness of the investigated samples, an atomic force microscope Ntegra Aura (NT-MDT, Russia) was used. The measurements were performed in a tapping mode with Si-cantilevers (NSG01). The water contact angle was measured to investigate the wetting behaviour of the scaffolds. The analysis was assessed using static contact angle measurements and performed using drop shape analysis (DSA25, Krüss GmbH, Germany). Five droplets (volume - 5 μL) were seeded on the surfaces of three samples of each studied material with the size of 1×1 cm2, and the resulting average contact angle was calculated with the standard deviation. 2.3.Synthesis and characterization of the polymer and hybrid capsules Four different types of capsules were synthesized as previously reported:36-38 (i) nondegradable poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) capsules with 3 bilayers of polyelectrolytes (PAH caps), (ii) degradable dextran sulfate/poly(L-arginine) capsules with 3 bilayers of polyelectrolytes (PARG caps), (iii) degradable hybrid silica capsules (SiO2 caps), and (iv) non-degradable poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) capsules functionalized with gold nanorods (PAH/Au caps).32 Different bioactive compounds were loaded into the cavity of the capsules according to the previously published protocols:28 (i) BSA conjugated with fluorescein isothiocyanate (FITC-BSA), (ii) BSA conjugated with tetramethylrhodamine (TRITC-BSA), (iii) dextran conjugated with Alexa Fluor 647 (AF647DEX), (iv) dexamethasone (DEXA), and (v) ceftriaxone sodium (CS). The capsules were characterized by optical microscopy, scanning electron microscopy and transmission electron microscopy. Details and the full data set are given in the Supporting Information. 2.4. Surface modification of scaffolds with capsules



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All synthesized types of capsules were immobilized onto the surfaces of the different scaffolds separately. The capsules were added to the previously prepared scaffolds, shaken for 1 hour and rinsed twice with MilliQ water. The stability of the attached capsules onto the scaffold surface was checked with the water jet method and rotation method. The details and the full data set are given in the Supporting Information. The schematic illustration of the preparation procedure of PCL/PHB and PHB-PANi scaffolds with their further modification with either polymer or hybrid capsules is presented in Figure 1.

Figure 1. Schematic illustration of the preparation procedure of PCL/PHB and PHB-PANi scaffolds, as well as synthesis of polymer and hybrid capsules. Scaffolds were prepared with the conventional electrospinning setup. Polymer capsules were synthesized using Layer-by-Layer


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technique. To prepare hybrid capsules sol-gel synthesis was involved. Finally, capsules were immobilized onto the surface of scaffolds. 2.5. Release studies The release of the cargo from PAH, PARG, SiO2 capsules loaded with FITC-BSA was investigated after ultrasound, pH and enzyme treatment for different time periods, whereas remote opening of PAH/Au capsules loaded with DEX-AF647 was probed with a near-infrared (NIR) laser. The amount of released cargo from the PAH, PARG and SiO2 caps was measured with a CLARIOstar ® (BMG LABTECH, Germany) multifunctional reader (excitation/emission = 490/525 nm). Additionally, scaffolds with immobilized capsules before and after treatments were visualized with confocal microscopy (Leica TCS SP8, Germany). Finally, the amount of released cargo was plotted versus time of treatment. For the remote laser opening, a laser writing system was used. The defined volume of the scaffold with attached non-degradable PAH/Au capsules was irradiated with the TiF_100 laser Avesta_Project Russia (central wavelength 790 nm). Afterwards, laser-treated scaffolds were observed under the confocal microscope. To prove the prolonged release of CS and DEXA from the biodegradable silica hybrid capsules, the SiO2 capsules were loaded with the model compounds (CS or DEXA) and shaken for several days at room temperature. The absorbance of supernatant was checked each day using a multifunctional reader. The amount of the released compounds was then plotted versus time of incubation. The details and the full data set are given in the Supporting Information. 2.6. Cell culture Human mesenchymal stem cells (hMSCs) were obtained from the bone marrow of healthy donors and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with penicillin/streptomycin, fetal bovine serum and L-glutamine. Confirmation of cell type was 13 ACS Paragon Plus Environment


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performed using flow cytometry (FACS Aria, BD, USA) with a multicolor antibody panel in accordance with the consensus criteria of the International Society of Cellular Therapy.39 The analysis shows that the cell population expressed low levels ( 95%) of typical hMSC markers, e.g., CD105, CD90 and CD73. The details and the full data set are given in the Supporting Information. 2.7. Toxicity studies Human mesenchymal stem cells (hMSCs) were seeded onto PCL, PHB and PHB-PANi scaffolds modified with different PAH, PARG, SiO2 capsules placed into a 96-well plate with each well filled with Vmedium=100 µL and left for 24 h. Then, the cells were washed once with PBS, and a new cell growth medium containing 10% vol. Alamar Blue was added to each well for 4 h at 37°C and 5% CO2. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a CLARIOstar ® (BMG LABTECH, Germany) multifunctional reader. The viability of the hMSCs was assumed to be proportional to the recorded fluorescence intensity. Additionally, cell nuclei were stained with 4′,6-diamidin-2-phenylindole (DAPI) and visualized with a confocal microscope (Leica TCS SP8, Germany, diode laser 405 nm as an excitation source, LP filter 420 nm). All toxicity experiments were repeated 3 times. The details and the full data set are given in the Supporting Information. 2.8. Proliferation studies Proliferation of hMSCs on scaffolds with immobilized capsules was measured on days 1, 3 and 7. The cells were seeded onto PCL, PHB and PHB-PANi scaffolds modified with PAH, PARG and SiO2 capsules previously placed into a 96-well plate. On the 1st, 3rd and 7th day, the


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cells were stained with Calcein AM (Invitrogen) and visualized with a confocal microscope (Leica TCS SP8, Germany, argon laser 488 nm as an excitation source, BP filter 505-530). To quantify the number of cells on the scaffolds, the calibration curve representing the number of attached cells onto scaffolds was obtained using the Alamar Blue reagent. For this, a known number of cells were seeded into a 96-well plate and left overnight in the incubator at 37°C and 5% CO2. The next day, the old growth medium was replaced with a growth medium containing 10% vol. Alamar Blue and hMSCs were incubated for 4 h at 37°C and 5% CO2. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using the CLARIOstar ® (BMG LABTECH, Germany) multifunctional reader. Knowing that the number of living cells is proportional to the recorded fluorescence intensity, the calibration curve (fluorescence intensity versus number of cells) was plotted. Using the calibration curve, the number of cells attached on scaffolds hMSCs on days 1, 3 and 7 was estimated and plotted for all samples. The proliferation experiments were repeated 3 times. The details and the full data set are given in the Supporting Information. 2.9. Antibacterial studies Antimicrobial activity in vitro was evaluated by agar disc-diffusion assay.40 The bacterial strain, E. coli ATCC25922, was used in this study. First, 200 µL of bacterial suspension (1.5 x 108 CFU/ml) was spread on brain heart (BH) agar plates, and then, scaffold discs were gently placed on the surfaces of the agar plates. The scaffolds soaked in ceftriaxone sodium (CS) solution (4 mg/mL) was used as positive controls. The plates were incubated in the dark for 24 h at 37°C, and the zone of inhibition (ZOI) around each specimen was measured with a digital caliper. The experiments were carried out for 15 days. BH agar plates were replaced every 2



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days, and the ZOIs were measured until they disappeared. Each assay condition was repeated three times. The details and the full data set are given in the Supporting Information. 2.10.

Osteogenic Differentiation Human mesenchymal stem cells (hMSCs) were seeded onto PCL, PHB and PHB-PANi

scaffolds modified with SiO2 caps loaded with dexamethasone (DEXA) previously placed into 24-well plates with each well filled with Vmedium= 500 µL. The cells were left for 14 and 21 days at 37°C and 5% CO2. The cell growth medium was changed each third day. Afterwards, osteogenic differentiation was examined by Alizarin S Red staining, calcein staining and the alkaline phosphatase test. All measurements were repeated 3 times, and the full data sets are given in the Supporting Information. 2.11.

Statistics

Statistical analyses were performed using Prism5 software (Graph Pad, La Jolla, CA, USA). All values were plotted as averages, including standard deviations of the means. The Student’s t-test and ANOVA were used to determine the significant differences between multiple sets of experimental data.

3. Results and discussion 3.1 Characterization of scaffolds PCL, PHB and PANi polymers were chosen to prepare biodegradable electrospun fibrous scaffolds as a delivery platform for capsules. The morphology, phase and molecular composition were studied using SEM, XRD and FTIR techniques and are presented in detail in the Supporting Information. The comparison with previously fabricated scaffolds based on the same polymers revealed no difference in porosity, fiber diameter, molecular and phase composition.34, 41 16 ACS Paragon Plus Environment

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However, the chemical composition of the scaffold surfaces has still not been fully studied. Figure 2A represents survey XPS spectra of the PCL, PHB and PHB-PANi scaffolds. Only the C 1s and O 1s chemical states were observed on the surface of the scaffolds. The N 1s peak on the surface of PHB-PANi scaffolds was not registered from PANi,42 therefore, PANi is located inside the PHB fibers, since the XPS depth sensitivity is 2-3 nm. It is known that PCL and PHB possess different molecular structures but have the same chemical groups (C-C(H), C-O and C=O) on the surface,43-44 which was observed for the PCL, PHB and PHB-PANi scaffolds (Figure 2B).



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Figure 2. A) Survey XPS spectra of pristine PCL, PHB and PHB-PANi scaffolds. B) The highresolution XPS spectra of the C 1s and O1s chemical states from the surface of pristine PCL, PHB and PHB-PANi scaffolds. 3.1.Scaffold development via decoration of polymer and hybrid microcapsules onto electrospun fibers There are many reports on the functionalization of the electrospun fiber surface by various drug carriers, including micelles, and silica nanoparticles.23,

45-46

In this study, we

focused on the use of polymer (non-biodegradable PAH caps; biodegradable PARG caps) and hybrid (SiO2 caps) microcapsules for the functionalization of electrospun microfibers. The polymer capsules (PAH caps and PARG caps) were synthesized through the layer-by-layer (LbL) technique, while the sol-gel method was used for preparation of hybrid silica-coated capsules (SiO2 caps). The morphology of polymer and hybrid capsules is shown in Figure 3A – C. As is evident, the SiO2 caps are, in contrast to the PARG and PAH caps, fully covered by a dense layer of an inorganic nanostructure (silica shell), which endows them with enhanced mechanical strength. The capsules can serve as a unique tool with triggered drug release upon external and internal stimuli.19 It is worth mentioning that we can combine several stimuliresponsive mechanisms into one intelligent capsule that can be beneficial in some drug delivery situations, offering an effective strategy to enhance the therapeutic efficacy of many remedies. The physicochemical characterization of PCL, PHB and PHB-PANi scaffolds was performed using SEM, XRD, FTIR spectroscopy and is presented in the Supporting Information (Figure S1).



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Figure 3. SEM images of capsules and scaffolds modified with polymer and hybrid microcapsules: A) PARG caps = (DEXS/PARG)3 capsules, 3 corresponds to the number of bilayers of polyelectrolytes. B) SiO2 caps = (DEXS/PARG)3/SiO2 capsules. C) PAH caps =


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(PSS/PAH)3 capsules. D) PCL scaffolds with immobilized PARG caps. E) PCL scaffolds with immobilized SiO2 caps. F) PCL scaffolds with immobilized PAH caps. G) PHB scaffolds with immobilized PARG caps. H) PHB scaffolds with immobilized SiO2 caps. I) PHB scaffolds with immobilized PAH caps. J) PHB-PANi scaffolds with immobilized PARG caps. K) PHB-PANi scaffolds with immobilized SiO2 caps. L) PHB-PANi scaffolds with immobilized PAH caps. The surface properties of the scaffolds such as surface roughness, hydrophilicity etc. play a crucial role in cell adhesion and proliferation.47 Therefore, the AFM analysis was performed to characterize the surface roughness of investigated materials. Also, the contact angels were measured to determine the wettability of all the tested scaffolds. The AFM images of the scaffold surfaces and the analysis of root mean square roughness are shown in Figure 4 and Supporting Information, Figure S12. As seen in Figure 4B, the surface of PCL was smoother than that of PHB and PHB-PANi. The immobilization of capsules slightly increased the surface roughness of the scaffolds. When measuring an average water contact angle (CA), modification of the scaffolds surface with capsules shows the decrease in the average CA, which indicates an increased hydrophilicity (Supporting Information, Figure S12). We further discuss the influence of surface properties of the scaffolds on the cell adhesion and proliferation.



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Figure 4. Characterization of the surface roughness using AFM: A) 2D images of AFM analysis for PCL and PCL@SiO2 caps samples. B) Quantification of the surface roughness of electrospun scaffolds with and without immobilized capsules (Data are presented as mean ± standard deviation. n = 8. ∗ represents p < 0.05 and ∗∗ represents p < 0.005). Several strategies can be employed to modify scaffold surfaces with nano/microparticles that include specific binding of particles to the scaffold, gelatin entrapment and nonspecific binding.48 In the present study, capsules were immobilized onto the scaffolds surface via nonspecific interaction, which is determined by molecular-scale interactions: electrostatic, van der Waals, and hydrophobic interactions.48 From the Figure 5A, it can be seen that the microcapsules (PARG caps) are unevenly distributed onto the scaffold’s fibers. The average ζpotential of the prepared capsules has been previously characterized and is negative: -15 mV for SiO2 caps, -23 mV for PAH caps and -35 mV for PARG caps.49 At the same time, according to the literature, an average value of the fibrous surface potential of PCL scaffolds is -10 mV due to the orientation of its carbonyl groups on the surface,50 observed in the high-resolution XPS spectrum (Figure 2). According to the KPFM measurements, PHB scaffolds also have negative 22 ACS Paragon Plus Environment

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surface potential, which is -7 mV (Supporting Information, Figure S2). Due to small negative charge values of the scaffold surfaces, we assume that the electrostatic repulsion is not leading to adsorption mechanism. Instead, the van der Waals interactions between the scaffold surface and capsules are considered as dominant.51 Since PARG capsules possess the highest ζ-potential, we can conclude that in this case the electrostatic repulsion contributes more significantly that results in a lower number of PARG capsules attachment onto the scaffold surface compared to PAH and SiO2 capsules (Figure 5B). It should be noted that the largest number of capsules of all types was observed on the PHB-PANi scaffolds (Figure 5B), which can be explained taking into account their piezoelectric properties. The piezoelectric properties of the PHB-PANi scaffolds can result in the changes of the surface charge, which can result in the higher number of adsorbed capsules.52 Notably, the interactions between the microcapsules and the scaffold’s surface was sufficiently strong to survive the mechanical impacts. To test this, the water jet and rotation methods were tested to study the stability of the attached microcapsules on the scaffolds (see Supporting Information). The obtained data revealed that in all cases of the abovementioned impacts, only 10-15% of the microcapsules desorbed from the scaffolds.



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Figure 5. CLSM images of scaffolds modified with capsules: A) (upper row, from left to right) PCL, PHB and PHB-PANi scaffolds modified with (DEXS/PARG)3 capsules loaded with FITCBSA, (bottom row, from left to right) PCL, PHB and PHB-PANi scaffolds modified with (DEXS/PARG)3 capsules loaded with FITC-BSA and TRITC-BSA separately. Scale bars 24 ACS Paragon Plus Environment

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correspond to 10 µm. B) Amount of capsules attached onto the surface of the scaffold. (The results were presented as the average value ± standard deviation, n = 3 independent experiments; ∗ represents p < 0.05). 3.2.Comparative triggering by ultrasound, enzymes and laser radiation As mentioned above, different methods were used for cargo incorporation into electrospun microfibers.53 Despite the sustained drug release, the polymer scaffolds possess a limited release rate, relying on the passive diffusion of drugs from the electrospun fibers. The use of stimuli-responsive microcapsules enables on-demand release of encapsulated drugs because they can sense and respond to signals and variations in the surrounding environment, leading to microstructure changes and drug release.19 To assess the drug responsive-triggered release after successful immobilization of capsules onto electrospun fibers, the external and internal stimuli were performed. These stimuli include the following: (1) physical stimuli, such as ultrasound and laser radiation, and (2) biological stimulus, such as enzyme (trypsin). A well-known mechanism of rapid capsule breakage is ultrasound, which is mostly effective in the case of the capsule shell modified with the inorganic materials.54 As can be seen from Figure 6, the hybrid silica capsules were more sensitive to this external trigger than were simple polymer capsules. After the first 30 sec of treatment, more than 50% of the cargo was released from the hybrid capsules. Confocal images of the silica capsules showed that the SiO2 caps were broken into small fragments even after the first seconds of ultrasound treatment (Figure 6A). At the same time, simple polymer capsules were slightly deformed after exposure to ultrasound. An explanation for the higher sensitivity of hybrid capsules to the ultrasound could be that the silica shell increases the density gradient across the interface between the shell and



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surrounding water, what can result in the higher absorption of acoustic energy.55 The release curves (Figure 6B) reveal the fast response of the hybrid capsules to the ultrasound. This response enables the possibility of creating high local concentrations of the delivered drugs, which are necessary to achieve therapeutic effects. At the same time, the systematic drug concentrations remain low, which reduces the toxic influence of delivered molecules on the whole body. The polymer capsules showed almost no release after ultrasound treatment, suggesting that this type of capsule possesses a certain stiffness, which helps to survive acoustic impacts.56 It is worth mentioning that all types of scaffolds were not affected with ultrasound treatment.


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Figure 6. CLSM micrographs of scaffolds modified with capsules before and after ultrasound and enzymatic treatment: A) (upper row, from left to right) PCL scaffold modified with SiO2 caps after 0, 20 and 60 sec of ultrasound treatment, (bottom row, from left to right) PCL scaffold modified with PAH caps after 0, 20 and 60 sec of ultrasound treatment. B) Percentage of released FITC-BSA from scaffolds modified with polymer and hybrid capsules after ultrasound treatment. C) (upper row, from left to right) PCL scaffold modified with PARG caps after 0, 2 and 24 h of enzymatic treatment, (bottom row, from left to right) PCL scaffold modified with PAH caps after 0, 2 and 24 h of enzymatic treatment. D) Percentage of released FITC-BSA from scaffolds modified with polymer and hybrid capsules after enzymatic treatment versus time of treatment. (The results were presented as the average value ± standard deviation). Trypsin (2 mg/ml, pH 7.4) was used as a model compound to perform the enzymatic degradation of capsules attached to the scaffolds. For enzymatic degradation, the samples were placed into the incubator at 37°C, 5% CO2 for 24 h. As expected, capsules made of natural polyelectrolytes (PARG caps) were more sensitive to the enzymatic treatment (Figure 6C). Similar processes of biodegradation usually occur inside cells, in particular, inside the lysosomal compartments, where capsules are collected after internalization.28, 57 From Figure 6D, one can see the cargo release curves from different capsules immobilized onto scaffolds after enzymatic treatment. In comparison to the treatment with ultrasound, the enzymatic degradation takes more time. The data for the degradable hybrid capsules show that 50% of drug was released from the capsules after 12 hours of incubation. Non-degradable PAH caps did not show significant release of cargo after 24 h of trypsin-treatment. The slight increase in released fluorescence intensity from the PAH caps after 24 h can be explained as the leakage of capsules over time. As before, the enzymes did not show any visible effect on the scaffolds’ morphology. With such dosage



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forms, it is possible to deliver drugs for a prolonged period of time, which can be very useful, especially, in the case of antibiotics administration, with the purpose to prevent contamination of cell tissues. The variation of the capsule types immobilized onto the scaffold surface allows to prepare a smart multifunctional platform, which can be designed with the possibility to integrate several drugs and their further controllable release under different stimuli. Indeed, the ability to immobilize multiple cargo molecules onto the scaffold surface and release them on demand in a tailored order is an unique feature. To address it, FITC-BSA loaded SiO2 caps (that are responsive to ultrasound) and TRITC-BSA loaded PARG caps (that are responsive to enzyme treatment) were simultaneously immobilized onto the same scaffold surface (Figure 7). As illustrated in Figure 7A – C, only SiO2 caps are broken upon ultrasound radiation, demonstrating burst cargo release (FITC-BSA), while PARG caps retained their contents (TRITC-BSA) without significant release of cargo. In other case, when scaffolds modified with SiO2 caps and PARG caps were treated by trypsin (biological stimulus), the degradation of PARG caps is observed and release of TRITC-BSA is detected (Figure 7D – F). Although SiO2 caps are a bit leaked, but the cumulative release of TRITC-BSA from PARG caps is higher in comparison with the leakage of FITC-BSA from SiO2 caps. Thus, integration of several different types of capsules (each loaded with a specific cargo and released upon a unique stimulus) onto the same scaffold allows to prepare a platform to achieve the release of the capsules’ content in response to different stimuli.


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Figure 7. Dual triggering release of FITC-BSA and TRITC-BSA from the scaffolds modified with PARG and SiO2 caps under external (ultrasound) and internal (enzyme treatment) stimuli: A) illustration of SiO2 caps destruction under ultrasound with further cargo release. B) CLSM micrographs of PCL scaffold modified with PARG and SiO2 caps after 0, 20 and 60 sec of ultrasound treatment. C) Percentage of the released FITC-BSA/TRITC-BSA from the scaffolds modified with polymer and hybrid capsules after ultrasound treatment. D) Illustration of PARG



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caps degradation in the presence of trypsin with further cargo release. E) PHB scaffold modified with PARG and SiO2 caps after 0, 2 and 24 h of enzymatic treatment. D) Percentage of the released FITC-BSA/TRITC-BSA from the scaffolds modified with polymer and hybrid capsules after enzymatic treatment versus treatment time. (The results were presented as the average value ± standard deviation, *p < 0.05). As mentioned above, delivery of therapeutically effective drug concentrations is often limited by the high toxicity of these drugs for the whole body. Thus, finding noninvasive methods to deliver toxic compounds remains a challenging task. Moreover, it is important not only to deliver but also to control the release of drugs to create high local concentrations of these compounds in the damaged cells. Biocompatible scaffolds modified with near-infrared (NIR) light-sensitive capsules can be potentially good candidates for the aforementioned challenges. As an example, vaccination can be mentioned, where the triggered-only release of bioactive compounds is of high interest.58-59 Among the various light sources, NIR light (800-1200 nm) is the best for biomedical applications due to its high transparency in living tissues with negligible damage.60-61 Remote light activation allows noninvasive opening of the capsules at desired time points or when a specific target site is reached. To demonstrate the remote-controlled release of cargo, non-degradable capsules functionalized with gold nanorods (maximum of absorbance at the wavelength 800 nm) and loaded with AF647-DEXS were used. Successful incorporation of Au nanorods into the capsule’s wall can be seen from transmission electron microscopy images (Figure 8 B1, B2). It can be seen that the nanorods are evenly distributed in the capsule’s wall without large aggregations, suggesting that the maximum absorption peak of Au nanorods remains at 800 nm even after attachment to the capsule’s wall, since the interaction of nanorods located in close


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proximity to each other results in a redshift of the surface plasmon resonance band.62 PCL, PHB and PHB-PANi scaffolds with defined volumes (100x100x10 µm) were illuminated with a laser operating at 790 nm (within the biological window) with a power of 80 mW. The laser beam randomly heated the capsules inside the chosen volume, inducing the rupture of capsules due to the absorption of NIR light by Au nanorods and converting it into heat. Figure 8D (left) shows a confocal microscopy image of capsules before opening. Here, one can see PAH/Au capsules attached to the fiber of scaffold. However, after laser scanning (Figure 8D right), the changes in the shape of the capsules can be observed (Figure S9). The interactions between the laser beam and gold nanoparticles through thermal processes are able to deform/disassemble polyelectrolyte capsules and, thus, change their permeability. Interestingly, that the organic fibers of the scaffold were not affected with the laser beam at the used power, suggests that only Au-functionalized capsules were sensitive to the laser beam and the NIR irradiation, which is located in the biologically “friendly” window (700-1000 nm). Thereby, using NIR-responsive polymer capsules for their remote activation inside cells to reach high local drug concentrations is feasible.



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Figure 8. Sketch of polyelectrolyte (PSS/PAH)3 capsules functionalized with Au nanorods immobilized onto PCL scaffolds before and after NIR laser irradiation: A) TEM image of Au nanorods. The scale bar corresponds to 80 nm. B1, B2) TEM image of (PSS/PAH)3 capsule functionalized with Au nanorods. C) CLSM micrograph of (PSS/PAH)3 capsules modified with Au nanorods and loaded with TRICT-BSA. D) CLSM micrograph of (PSS/PAH)3 capsules modified with Au nanorods, loaded with TRICT-BSA and immobilized on a PCL scaffold before (left) and after (right) irradiation. Thus, in the present work various mechanisms of cargo release (external and internal) were demonstrated. The main advantages of external triggering (ultrasound or laser irradiation) are non-invasiveness, the absence of ionizing radiations, the cost effectiveness and simple regulation 32 ACS Paragon Plus Environment

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of tissue penetration depth by tuning the frequency, cycles and exposure time. Ultrasound therapy has been effectively used for diverse biomedical applications such as therapy in musculoskeletal disorders,55 e.g. knee osteoarthritis,63 inflammation of the attachment of the plantar fascia in the course of calcaneal spurs64 and etc. However, the ultrasound/laser impact above biological limits can have negative effect on the physiological activity of the cells (e.g. decrease in metabolite cell production, cell morphology, and detachment of the cells).65 Therefore, it is highly important to control the initial parameters of external exposure. 3.3. Cell adhesion, viability and proliferation during cultivation on the scaffolds decorated with polymer and hybrid microcapsules After the functionalization of scaffolds with capsules of different types, the adhesion, viability and proliferation of hMSCs onto the scaffold surfaces were evaluated. For comparison, experiments with scaffolds without capsule modifications were also performed. The cell viability and cell density were evaluated after 24 h of incubation. DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining was used to visualize the adherence of the hMSCs to the scaffolds. To check whether the cells internalize the capsules previously attached to the scaffold surface, confocal microscopy was use.66 The cell cytoskeleton and the nucleus were fluorescently stained, and the co-localization of the red fluorescently labeled capsules within the cellular compartments was evaluated with z-stack images of hMSCs, as shown in Figure 9. It can be noted that the cells are able to internalize the capsules attached to the scaffolds, as verified with the orthogonal view from different planes of the confocal images. An indicator of the capsule internalization was the red signal coming from the labeled capsules surrounded with a green signal coming from the stained cytoskeleton.



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Figure 9. CLSM micrographs of hMSCs adhered on different scaffolds with immobilized PAH caps. Cell nuclei were stained with DAPI (blue), cytoskeleton with AF488 phalloidin (green). The third column is the orthogonal view from different planes (x/y, x/z or y/z) of the stained cells.


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Figure 10 shows the cell viability measured with the Alamar Blue assay and the cell density after 24 h of culture. According to the obtained data, the microcapsules did not affect the cell viability and adhesion of hMSCs. The obtained data suggest nontoxic effects of capsules, which corresponds to the previous studies.28 Thus, the incubation of hMSCs with scaffolds modified with microcapsules at concentrations relevant for the practical application did not result in a reduced viability and adhesion of cells. The hMSCs attached onto PHB, PHB-PANi before and after immobilization of capsules showed higher viability than did those attached onto PCL before and after capsule functionalization (80% of viability). This result is related with less cell adhesion, which showed hMSCs on the PCL scaffold (approximately 40% less adherent cells as for PHB and PHB-PANi scaffolds).



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Figure 10. CLSM micrographs of hMSCs attached and grown on different scaffolds modified with polymer and hybrid capsules: A) Nuclei of hMSCs were stained with DAPI. B) Cell viability (expressed in %) of hMSCs after 24 hours incubation with scaffolds modified with capsules determined by Alamar Blue assay. C) Cell density on all scaffolds modified with capsules 24 h after seeding. (The results were presented as the average value ± standard deviation, n = 10. ∗ represents p < 0.05 and ∗∗ represents p < 0.005).

The proliferation of hMSCs on scaffolds modified with different capsules was checked on days 1, 3 and 7 (Figure 11). According to the obtained results, the cell density increased with increasing incubation time, indicating that hMSCs are able to proliferate onto the scaffold surfaces. As before, the least cells were found on the PCL scaffolds (approximately 35% fewer cells on the 1st and 3rd day and 20% fewer on the 7th day in comparison to those of other scaffold types). The hMSCs proliferated approximately at the same rate on the PHB and PHB-PANi scaffolds (data not shown). As before, the capsules did not affect the hMSCs proliferation. This result is again in agreement with the previous studies.67 Interestingly, since the scaffolds have 3D structures, the hMSCs obviously have more space to grow. Thus, even at day 7 the proliferation of cells was not suppressed by contact inhibition.68



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Figure 11. Proliferation of hMSCs, adhered on different scaffolds modified with polymer and hybrid capsules: A) CLSM images of cells on scaffolds. hMSCs were stained with calcein AM (green), capsules were loaded with TRITC-BSA (red) after 1, 3 and 7 days of incubation. B) Density of hMSCs cells attached on different scaffolds modified with polymer and hybrid capsules after 1, 3 and 7 days of incubation. (The results were presented as the average value ± standard deviation, *p < 0.05).

The reduced hMSCs adhesion and proliferation on the PCL scaffolds can be associated with smaller values of surface roughness of PCL fibers and weaker reversible adsorption of proteins compared to PHB and PHB-PANi scaffolds, which have almost the same roughness. As reported earlier, cells are very sensitive to the surface roughness. Many studies showed that increased surface roughness results in an increased number of human bone marrow cells adhered to the scaffold surface.69 Additionally, a higher roughness in case of PHB/ PHB-PANi scaffolds promotes better protein adsorption (Supporting Information, Figure S3) and, therefore, supports increased cell attachment. Interestingly, the average water CA decreased after immobilization of capsules onto the surface of the scaffolds (~ 17 %). This did not affect cell adhesion and proliferation, which remained approximately constant. Although, hydrophilicity of the scaffolds with capsules is slightly increased, but the contribution of hydrophilic capsules immobilized onto the surface of the fibers is not enough to significantly change the cell adhesion and proliferation due to uneven distribution of the micrometric capsules. In the previous works for scaffolds modification, the hydrophilic nanoparticles were usually used to cover the whole surface of the scaffolds, which led to the increased hydrophilicity.70



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3.4. Antimicrobial evaluation of scaffolds decorated with SiO2 caps containing CS Previously, it has been demonstrated that hMSCs adhere and proliferate on scaffolds with and without capsules, indicating that capsules, adsorbed on the surface of scaffolds, did not affect cell functions. Among all the tested capsules in this study, the hybrid SiO2 caps were used for the further encapsulation of commercially available model antibiotic ceftriaxone sodium (CS) and dexamethasone (DEXA), used as a low-molecular-weight osteoinductive factor. SiO2 caps have been chosen as the most suitable carriers for incorporation of low-molecular-weight compounds, such as CS and DEX.49 To examine the antimicrobial effect of the scaffolds, hybrid SiO2 caps loaded with CS were immobilized onto the scaffold surface. Scaffolds modified with hybrid SiO2 caps loaded with DEXA were used to induce osteogenesis. Since bacterial contaminations are the major source of implant rejection and, thus, the failure of tissue regeneration, an antimicrobial agent (CS) was chosen to probe the antibacterial properties of scaffolds functionalized with bioactive hybrid capsules. To keep the concentration of CS per scaffold surface constant, Ncaps ~ 6 x 106 of SiO2 caps were added per tested sample (this value was chosen as the maximum adsorption capacity of the capsules for the PCL scaffold, Figure 5B). The loading amount of CS in SiO2 caps per scaffold was 2 mg per 0.32 cm2 of scaffold for every type of tested scaffold (PCL, PHB and PHB-PANi). The release profile of CS from SiO2 caps in PBS (pH 7.4) at 37 oC and the effect of CS concentration on the viability of hMSCs are shown in Figure 12A, B. After incubation for 16 days (384 hours), the cumulative release of CS from the capsules was 85 ± 5%. The viability data indicate the low cytotoxicity of CS for wide concentrations. The same tendency in the release profile of CS was observed when the experiment was performed in the presence of hMSCs seeded on the scaffold surface in the cell culture medium (Supporting Information, Figure S12). It should be noted that all the


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scaffolds loaded with CS exhibited comparable cell viability to the controls (scaffolds without SiO2 caps containing CS). Thus, scaffolds modified with SiO2 caps can sustain release of CS in the presence of cells and CS in our case has no significant cytotoxicity on the hMSCs. Next, the agar disc-diffusion test was performed to evaluate the antibacterial activity of scaffolds modified with hybrid SiO2 caps containing CS (2 mg per 0.32 cm2) against E. coli ATCC25922. E. Coli was used as a standard microbial organism model with well-known sensitivity to ceftriaxone (CS). On the second day of incubation with E. coli, it was found that the zones of inhibition (ZOIs) were approximately the same for all tested samples (scaffolds@CS/SiO2 caps and scaffolds/CS), excluding those of the negative controls (PCL, PHB and PHB-PANi without CS treatment). Nonetheless, the antibacterial activities as measured by the ZOI values of the scaffolds modified with SiO2 caps containing CS were comparable with the ZOI values for the scaffolds soaked in CS solution during the first 4 days of incubation. However, after 4 days of incubation, the ZOIs of scaffolds soaked in CS solution disappeared, while the scaffolds modified with SiO2 caps containing CS showed further antimicrobial activity (Figure 12C, Figure S13). The changes in the ZOI values for different scaffolds over time are shown in Figure 12D. Based on the analysis of the ZOI values, the scaffolds modified with SiO2 caps containing CS exhibited prolonged antimicrobial effects, indicating the vital role of capsules with antimicrobial agents for enhancing the duration of antibacterial activities of the scaffolds.



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Figure 12. The antibacterial effect of SiO2 capsules loaded with ceftriaxone (CS) immobilized on different scaffolds: A) Cell viability (expressed in %) of hMSCs after 24 hours incubation 42 ACS Paragon Plus Environment

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with scaffolds modified with SiO2 capsules loaded with CS determined by Alamar Blue assay. B. Release of CS from SiO2 capsules during 15 h of incubation. C) Antimicrobial activity in vitro was evaluated by agar disc-diffusion assay using E. coli. Samples (0.32 cm2) were placed on to the bacterial inoculated brain heart (BH) agar plates. The plates were incubated under dark conditions for 15 days at 37°C (pictures shown on days 2, 4 and 8). The BH agar plates were replaced every 2 days, and zones of inhibition were measured until they disappeared. Scaffold soaked CS (4 mg/mL) was used as the positive control, whereas PCL, PHB and PHB-PANi were used as the negative controls. Each experiment was repeated 3 times. D) Measured zone of inhibitions versus time of incubation. (The results were presented as the average value ± standard deviation).

3.5. Evaluation of osteogenic differentiation The potential to differentiate between the varieties of connective tissue cell types, such as osteoblasts, adipocytes and chondrocytes, can be considered as one of the main functional characteristic of hMSCs.10 Osteoblasts can be induced to produce extracellular calcium deposits. This process is called mineralization, which is a key factor for bone regeneration. Therefore, we examined the osteogenic differentiation of hMSCs on scaffolds modified with SiO2 caps loaded with DEXA. As with the antibacterial studies, Ncaps ~ 6 x 106 of SiO2 caps were used for immobilization on the surface of each tested sample. The DEXA content in scaffolds @SiO2 caps was calculated to be 0.0235 mg per 0.32 cm2. The release of DEXA from the scaffolds modified with SiO2 caps (PCL@SiO2 caps, PHB@SiO2 caps and PHB-PANi@SiO2 caps) was checked. The obtained data suggest an initial burst release followed by a continuous release of DEXA during 24 days of incubation (Figure S14). 43 ACS Paragon Plus Environment


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The mineralized matrix formation was examined by Alizarin red S (ARS) staining and the extent of calcium deposition was quantified by extracting the dye from the scaffold structure (Figure 13A, C). Additionally, the evaluation of mineralization was performed by staining the samples with calcein, followed by CLSM analysis, which identifies positively stained calceincalcium complexes (Figure 13B). As shown in Figure 13A, after 14 and 21 days of cultivation, the positive control (PCL, PHB and PHB-PANi scaffolds incubated in osteogenic medium) and DEXA-loaded scaffolds (PCL@DEXA/SiO2 caps, PHB@DEXA/SiO2 caps and PHB-PANi@ DEXA/SiO2 caps) showed positive staining signals, while the negative controls (PCL, PHB and PHB-PANi scaffolds incubated in growth medium) barely showed any visible signal. The staining signal coming from scaffolds modified with SiO2 caps loaded with DEXA was higher than that of the positive control. The CLSM images confirmed the presence of calcium deposition on the scaffolds via formation of green fluorescent complexes of calcium with calcein (Figure 13B, Figure S15). A quantitative analysis of ARS staining was performed by eluting ARS from the stained cells and detecting the absorbance at 405 nm normalized to the DNA amount (Figure 13C). The scaffolds with immobilized SiO2 caps containing DEXA displayed an increased amount of calcium deposits compared with that of the positive control (PCL, PHB and PHB-PANi scaffolds incubated in osteogenic medium). Thus, the prolonged release of DEXA from the scaffolds modified with SiO2 caps induces the matrix mineralization more effectively than that of the osteogenic medium. Also, it should be noted that PHB and PHB-PANi possess higher matrix mineralization abilities than those of PCL.


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Figure 13. Evaluation of osteogenic differentiation of hMSCs: A) Digital images of different samples after staining with Alizarin Red S (ARS). Scaffolds incubated in osteogenic medium (OM) without capsules were used as the positive controls, scaffolds incubated in cell growth medium (GM) without capsules were used as the negative control. B) CLSM images of the calcium minerals deposited by hMSCs after 14, 21 days of incubation. Cell nuclei were stained with DAPI (blue), cytoskeleton with AF 633 phalloidin (green), calcium minerals with calcein



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(green). C) The production of a mineralized matrix determined by quantifying the amount of Alizarin Red S that stained the mineralized matrix. D) ALP activity of hMSCs grown on different scaffolds modified with DEXA/SiO2 capsules cultured for 14 and 21 days. (The results were presented as the average value ± standard deviation, *p < 0.05).

In addition, an ALP activity assay was carried out as an early stage marker to investigate the osteogenic differentiation activity of hMSCs seeded on the scaffolds. As shown in Figure 13D, the ALP activities of hMSCs cultured on the scaffolds with immobilized SiO2 caps containing DEXA were enhanced in comparison to those of the positive controls (PCL, PHB and PHB-PANi scaffolds incubated in osteogenic medium) during 14 and 21 days incubation, which is in good agreement with the previous results. The results of the ALP activity assay relate to the effect of DEXA released from the SiO2 caps attached on the scaffold surface. Again, PHB and PHB-PANi scaffolds showed more improved ALP activities than the PCL scaffold did. The ALP activity assay and calcium deposition results clearly indicated that the scaffolds with immobilized DEXA/SiO2 caps showed enhanced osteogenic differentiation of hMSCs. The release of DEXA from the scaffolds modified with SiO2 caps occurs in a sustainable manner, which can stimulate the enhanced osteogenic differentiation of hMSCs. Moreover, this fact can be explained with the internalization of DEXA-loaded capsules with stem cells (as shown in Figure 9).67 Based on the obtained results, we can conclude that several factors are involved in enhanced osteogenic differentiation of hMSCs on scaffolds. Firstly, the appropriate surface properties of PHB and PHB-PANi scaffolds (e.g. surface roughness, fiber diameter, scaffold matrix, protein adsorption etc.) favoured higher cell adhesion compared to PCL, and


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subsequently increased osteogenic differentiation. Secondly, a high biocompatibility of PHB and PHB-PANi scaffolds promotes cell proliferation, which also supports osteogenic differentiation. Thirdly, sustained release of DEXA from SiO2 caps and capsule internalization within hMSCs synergistically results in the enhanced osteogenic differentiation. The degradable or non-degradable polymer capsules, as well as hybrid capsules can be used in different cases. When using biodegradable (PARG) capsules, the prolonged release of bioactive compounds can be achieved via the enzymatic degradation (internal triggering, Figure 6). While capsules functionalized with silica coating or gold nanoparticles are sensitive to the ultrasound and laser irradiation, respectively (external triggering). External physical impacts induce fast burst like release of a high dose of cargo (Figures 6 – 8). Thus, the main advantage of our developed platform is that different mechanisms of triggering can be applied to the particular type of capsules. Due to the improved surface properties of PHB and PHB-PANi (e.g. roughness, protein adsorption etc.), the number of attached cells was enhanced with better proliferation ability compared to PCL. As a drawback for our drug carrier-scaffold systems, an additional step in capsules synthesis is required. Hybrid capsules require additional 3 hours for silica deposition at a basic pH, as well as PAH/Au capsules require deposition of additional Au layer that not always can be beneficial for the encapsulated cargo. Conclusions In this work, the polymer and hybrid microcapsules were immobilized onto the surface of polymer electrospun microfibers with the purpose to develop drug-loaded fibrous scaffolds possessing controlled drug incorporation and its further release. Even though the scaffolds were based on different polymers, a decrease in the microcapsules number on the fiber surface with the increase in their negative ζ-potential was observed, which is likely due to the electrostatic 47 ACS Paragon Plus Environment


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repulsion with the negatively charged fibers surface. Additionally, dependent on the nature of the capsules, immobilization of the capsules in a dynamic mechanical system at a frequency of 10 Hz resulted in an increase in the number of microcapsules on the fibers with the increase in their piezoelectric response, i.e., from PCL (non-piezoelectric) to PHB-PANi, which possess significantly higher piezoelectric performance than that of pure PHB. The microcapsuledecorated scaffolds demonstrated controllable responsive release of cargo upon physical (ultrasound, laser radiation) and biological (enzyme treatment) stimuli, which was due to the unique physicochemical properties of the polymer and hybrid microcapsules. Therefore, such modified scaffolds with triggered release of the bioactive agents might be effectively used to enhance the bioactive properties of these agents. At the same time, the immobilization of microcapsules did not influence the cell growth, viability and proliferation on the scaffolds. Additionally, the cell attachment study revealed that the PHB and PHB-PANi scaffolds possess enhanced adhesion of hMSCs compared to that of the PCL scaffold. In the example of ceftriaxone and dexamethasone encapsulated inside the capsules, antimicrobial properties and osteogenesis of the scaffolds were found, which were hindered after the immobilization of capsules onto the electrospun microfibers. The capsules loaded by ceftriaxone showed a consistent release profile that provides the mid-term antimicrobial (for 16 days) effect of scaffolds against gram-negative bacteria growth, as tested for E. coli. More importantly, the dexamethasone-loaded capsule-decorated composite scaffolds presented an enhanced osteogenic differentiation and mineralization compared with that of individual scaffolds incubated in an osteogenic medium. Thus, this study provides a fast and convenient method to obtain microcarriers-incorporated composite scaffolds, which can be considered as advantageous for drug loading and controllable and prolonged drug release. This strategy to combine drug-loading


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and controlled release of drugs in one system may facilitate a promising multifunctional platform with different mechanisms of triggering for different fields of modern medicine, including personalized drug therapy and bone tissue engineering. Acknowledgements

The preparation of capsules and their characterization were supported by the Russian Science Foundation, No. 17-73-10023 (A.S.T.). Additionally, this work was partly supported by the grant of the Russian Foundation for Basic Research, No. 18-015-00100 (3.2 and 3.3 parts of Results and Discussion), Ministry of Education and Science of RF, No. 3.1500.2017/4.6 and State Order NAUKA (No. 11.1233.2017/4.6;11.7293.2017/8.9). M. V. Z acknowledges the President’s Scholarship SP-1576.2018.4 and M. A. S. acknowledges the President’s Scholarship МК6287.2018.8. We also thank Mr. Mikhail Zhukov for the performed AFM measurements and Ms. Ekaterina Chudinova for her assistance in SEM characterization of the prepared samples and microcapsules synthesis.

Supporting information Other experimental results, describing the scaffold preparation and characterization using AFM, SEM, XRD, contact angel etc.; synthesis of nano- and micromaterials; procedure of microcapsule loading with cargo; capsule immobilization onto scaffold surface; release studies; cell studies (e.g. adhesion, toxicity and proliferation); antibacterial analysis; procedure for analysis of osteogenic differentiation are available in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Author contributions



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A.S.T. initiated and performed this work and manuscript. A.R.M. conceived and designed the basic idea and structures. A.R.M., O.O.P., T.E.K., I.S.S., A.I.D., N.Y. performed all biological experiments. M.V.Z., A.S. and A.A.G. performed the synthesis and characterization of capsules and release studies. M.A.S., R.V.C. synthesized scaffolds and performed their characterization. T. B., B.K. provided the XPS analysis. G.B.S. and R.A.S. analyzed the results. A.S.T, M.V.Z., A.R.M., R.V.C., and R.A.S. co-wrote the manuscript. All authors contributed to discussion and reviewed the manuscript. Notes The authors declare no competing financial interests. References (1) O'brien, F. J. Biomaterials & Scaffolds for Tissue Engineering. Mater. today 2011, 14 (3), 88-95. (2) Kim, H.; Kim, H. W.; Suh, H. Sustained Release of Ascorbate-2-phosphate and Dexamethasone from Porous PLGA Scaffolds for Bone Tissue Engineering Using Mesenchymal Stem Cells. Biomaterials 2003, 24 (25), 4671-4679. (3) Martins, A.; Duarte, A. R. C.; Faria, S.; Marques, A. P.; Reis, R. L.; Neves, N. M. Osteogenic Induction of hBMSCs by Electrospun Scaffolds with Dexamethasone Release Functionality. Biomaterials 2010, 31 (22), 5875-5885. (4) Ren, Q.; Cai, M.; Zhang, K.; Ren, W.; Su, Z.; Yang, T.; Sun, T.; Wang, J. Effects of Bone Morphogenetic Protein-2 (BMP-2) and Vascular Endothelial Growth Factor (VEGF) Release from Polylactide-poly (ethylene glycol)-polylactide (PELA) Microcapsule-based Scaffolds on Bone. Braz. J. Med. Biol. Res. 2018, 51 (2).


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graphical abstract 617x513mm (150 x 150 DPI)

ACS Paragon Plus Environment

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Multifunctional scaffolds with improved antimicrobial properties and

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