Antibacterial porous microcarriers with pathological state responsive

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Antibacterial porous microcarriers with pathological state responsive switch for wound healing Canwen Chen, Yuxiao Liu, Lingyu Sun, Guopu Chen, Xiuwen Wu, Jianan Ren, and Yuanjin Zhao ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00134 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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

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

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

ACS Applied Bio Materials

Antibacterial porous microcarriers with pathological state responsive switch for wound healing Canwen Chen1, Yuxiao Liu2, Lingyu Sun2, Guopu Chen1, Xiuwen Wu*,1, Jianan Ren*,1, Yuanjin Zhao*,1,2 1

Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University,

Nanjing 210002, China 2

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, Nanjing 210096, China.

KEYWORDS: inverse opal; drug delivery; wound healing; microcarrier; antibacterial

ABSTRACT: In this paper, novel antibacterial porous microcarriers with a pathological state responsive switch were developed to promote wound healing. The porous microcarrier includes a n inverse opal scaffold generated by a temperature-responsive hydrogel that deforms according to different temperatures, and a biodegradable sodium alginate hydrogel used as a vehicle to load drugs. Thus, the microcarrier is endowed with the ability of releasing loaded drugs when the temperature changes in the wound site due to an inflammation reaction. Without inflammation, the microcarriers will be locked and the release of drug molecules will not continue, which prevents the abuse of drugs and further improves the safety of the therapeutic treatment. The designed microcarriers could eliminate inflammation significantly and promote the formation of granulation-tissue, angiogenesis and collagen deposition in the wound site. These features indicate that these microcarriers with a pathological state responsive switch are ideal for promoting wound healing,ACS and will have great potential in biomedical applications. Paragon Plus Environment 1

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

Page 2 of 23

INTRODUCTION Microbial infection can severely hinder the healing process, leading to necrosis at the wound site, sepsis, and even death.1-5 Systemic, topical or oral administration of antibiotics are frequently prescribed to patients suffering from infectious wounds.6-11 However, most of these traditional treatments are limited by their fluctuant drug concentration in blood, side effects and even antibiotic-resistance after repeated infection and inflammation. To overcome these defects, alternative treatments of bacterial infection, such as impregnated gauzes and antibacterial ointments, have been used.12-18 Among them, microparticle-based drug delivery microcarriers have promise owing to their effective, stable, safe and monitorable characteristics in drug delivery.19-24 Various biodegradable and biocompatible polymers have been used to make microcarriers, including gelatin, collagen, polycaprolactone and poly (2-hydroxyethyl methacrylate).25-28 Such particles can increase the loading of drugs, prolong the expiration date of active molecules, and greatly reduce the side effects.29-32 Although the use of these microcarriers have been used to promote wound healing, their static and unintelligent drug release cannot adapt to the changeable pathological state of a wound site, which leads to a poor therapeutic effect, drug waste, and limited application. Thus, drug microcarriers with a pathological-state responsive switch during the release process are still strongly anticipated. In this paper, we developed an intelligent drug-delivery microcarrier with a pathologicalstate responsive switch, by combining an inverse opal scaffold generated by a temperatureresponsive hydrogel and biodegradable sodium alginate hydrogel used to encapsulate drugs. Inverse

opal particles

possess

unparalleled

features

of

uniform,

three-dimensional

interconnecting pores and specific optical characteristics, which makes them ideal scaffolds in drug delivery and tissue engineering.33-37 In addition, vancomycin, the most important treatment for methicillin-resistant Staphylococcus aureus (MRSA) infections,38, 39 can be loaded into the ACS Paragon inverse opal pores and encapsulated by Plus a Environment second biocompatible gel. Therefore, the 2

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


combination of inverse opal scaffolds and vancomycin could be conducive to the development of microcarriers for promoting wound healing. Additionally, since the inverse opal scaffold is generated by a temperature-responsive hydrogel, the process of releasing the loaded drugs from the microcarriers can be activated once the temperature changes in the wound sites due to the inflammation reaction.40 Inflammation is the switch in this drug delivery system because the carriers releases the drug at the infected wound site as long as inflammation occurs. Alternatively, the microcarriers will be locked and drug release will not continue when inflammation (i.e. a temperature increase) is not present, which avoids drug waste and further improves the safety of the therapeutic treatment. These features indicate that inverse opal microcarriers with a smart switch triggered by a pathological state are ideal for promoting wound healing and have great potential for further biomedical applications.

EXPERIMENTAL SECTIO Materials and Methods: SiO2 nanoparticles were purchased from Nanjing Nanorainbow Biotechnology Co., Ltd. N-Isopropylacrylamide (NIPAM, 97%), Calcium chloride, Fluorescein vancomycin, 2-hydroxy-2-methylpropiophenone photoinitiator (97%), N-methylol acrylamide (97%) were all purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Hydrofluoric acid and N-Methylolacrylamide were purchased from Aladdin Industrial Corporation (Shanghai, China). Sodium alginate was purchased from Alfa Aesar China Ltd (Heysham, Lancs). All other reagents were of the best grade available and used as received. Generation of template colloidal crystal beads: The silica colloidal crystal beads (SCCBs) were prepared by the droplet template method. The silica nanoparticles solution and silicon oil were loaded into the microfluidic device. In consequence, the silica nanoparticles solution was wrapped in silicone oil to form droplets in the microfluidic channel. The droplets were collected in a box which filled with 500 CS silicon oil. Next, the boxes were placed at 80°C in an oven which ACS Paragon Plus Environment

3


provided a high temperature to evaporate of water in the droplets. After curing overnight, the silicon oil in the boxes and SCCBs were removed by washing with hexane. Finally, the SCCBs that removed silicon oil were placed at 750°C for 4h. The concentration of the silica nanoparticles solution loaded into the microfluidic was 18% (w/v). The parameters of oil and dispersed phase were set as 0.4mL/h and 10mL/h, respectively. Fabrication of PNIPAM hydrogel inverse opal particles: Pregel solution for the generation of thermosensitive inverse opal particles included N-isopropyl acrylamide and N-methylol acrylamide (with different w/t), 2-hydroxy-2-methylpropiophenone (1.5 % v/v). The mass ratio of N-isopropylacrylamide to N-methylolacrylamide was 9:1 and its LCST is close to 37°C. The SCCB is immersed in the pregel solution. They were then exposed to UV light (365 nm, 100 W, 1 minute) in a near zero environment to polymerize the pregel solution. The prepared SCCB-containing bulk was sequentially immersed in a buffer solution for 8 hours. After the hydrogel bulk was stirred into pieces by a stirrer, the hybrid beads were filtered from the buffer solution, which is a key step for furthering separating hydrogel and hybrid beads. Finally, the thermally responsive inverse opal particles were obtained by removing the SCCB by immersion in sodium hydroxide (10%, v/v) for 4 hours. Drug encapsulation and controlled release experiment: Sodium alginate pre-gel solution with different concentration of 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 3.0% (w/v) were previously prepared and mixed with 2.5mg/ml FITC-vancomycin. The inverse opal particles were immersed in the mixture for more than 2h. Then 2.0% (w/v) calcium chloride was added into the pre-gel solution contending FITC-vancomycin and particles to realize the gelatin of calcium alginate hydrogel. The prepared hydrogel containing the vancomycin loading particles were immersed in the buffer solution for 60 minutes in succession. After stirring the hydrogel into pieces by a stirrer, the particles were filtered from the buffer solution. For each ACS (w/v) ParagonofPlus Environment concentration from 0.75% to 3.0% calcium alginate hydrogel, the samples were 4

Page 4 of 23

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


suspended in 1.5 ml buffer consisted of PBS (pH 7.4 ± 0.05). These samples were incubated for more than twenty days at room temperature with shaking (200 rpm). Fluorescent photographs of drug-loaded inverse opal particles were taken by a microscope with high resolution CCD (OLYMPUS DP73) (OLYMPUS BX53) and processed by Cell Standard software to characterize the fluorescence intensity by the obtained green gray value. Fluorescent cross-sectional images were obtained using a confocal microscope (OLYMPUS FV500-IX81). Antibacterial test in vitro: Gram-positive S. aureus was selected as a representative bacterium in the antibacterial test because S. aureus is the most common pathogen and drug-resistant bacteria in the clinic and the drug vancomycin is a specific powerful weapon for anti-Gram-positive. Bacterial suspensions were prepared by the addition of S. aureus isolates until the turbidity based on the McFarland standard was about 0.5. Subsequently, the bacterial isolate was cultured on mannitol agar at 37 °C. Simultaneously, the PNIPAM inverse opal particles loaded with 0.25%, 1%, 3% sodium alginate and vancomycin, respectively, were put into the 96-well palate. Then the 200 ul of 0.5 MCF bacterial liquid of staphylococcus aureus was added into it. After that, the 96-well plate was placed in 37°C incubator. Then we took a small amount of uniform bacterial liquid and dilute 100 times and spread on 1.5% LB agar plate every half hour and calculated the number of colonies. Preparation of Acute Wound Infection Model: Sixteen adult male Sprague-Dawley rats (body weight: 220 g–250 g) were used for this experiment. The animals live at a temperature of 24 ° C and a humidity of 55%, and were allowed free access to food and water. All the animal care and experimental protocols were reviewed and approved by Animal Investigation Ethics Committee of Jinling Hospital. All rats were fasted overnight and received general anaesthesia through intraperitoneal injections of 10% chloral hydrate at 0.5 mL/100 g. Infectious wounds were then created by ACS Paragon Plus Environment

5


Page 6 of 23

removing round skin with 1 cm diameter from the back of these animals and injecting 0.1 mL of bacterial suspension into the defect area. All rats were randomly divided into four groups. After that, different interventions PBS solution, PNIPAM hydrogel inverse opal particles, vancomycin, and PNIPAM hydrogel inverse opal particles with vancomycin encapsulated by 0.25% sodium alginate hydrogel were treated with each group, respectively. There was no dead during the experiment. After one week, the rats were sacrificed and granulation tissues over the wound were excised and cut into two pieces. In addition, two pieces of granulation tissue were stored in 10% neutral formaldehyde for immunohistochemistry and histological analysis, as well as immunofluorescence staining. Effectiveness of PNIPAM hydrogel inverse opal particles for wound healing: Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, Immunohistochemistry and Immunofluorescence staining: The samples of granulation tissue were taken out of the formaldehyde, followed by dehydration and then embedded in paraffin. A microtome was used to prepare serial sections in accordance with standard protocols. Sections with a thickness of 5 μm were treated for hematoxylin and eosin staining (H&E), as well as immunohistochemical evaluation. Immunohistochemical sections were stained with IL-6 and TNF-α. Besides, the other sections were reacted with primary antibodies CD31 (KEYGEN, KGYM0118–7) and α-smooth muscle actin (α-SMA) (KEYGEN, KGYT5053-6) overnight at 4 °C for neovascularization.

RESULTS and DISCUSSION In a typical experiment, silica colloidal crystal beads (SCCBs) were used as a template for generation of temperature-responsive inverse opal scaffolds. The particles were fabricated by the process shown in Figure 1. Microfluidic devices were used to generate droplet templates where silica nanoparticles could self assemble to form beads. In an evaporationinduced crystallization process, the silica nanoparticles were tightly packed and formed a ACS Paragon Plus Environment

6

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


hexagonally arranged, ordered microstructures in the droplets. The resultant SCCBs had a round shape and highly ordered, regularly arranged nanostructure (Figure 2d and Figure S1d-e). Additionally, the interconnected nanovoids of the silica nanoparticles provide space for pregel infiltration. To achieve this, the SCCBs were immersed in the PNIPAM pregel solution. After being exposed to ultraviolet irradiation, the pregel solidified and the inside of the SCCBs was efficiently filled with the PNIPAM hydrogel (Figure 2e). The inverse opal particles were obtained after removing the SCCB template (Figure S1f). Consequently, the Poly(Nisopropylacrylamide) (PNIAPM) inverse opal particles showed highly uniform, interconnected round pores because of the ordered arrangement template, which aids i n t h e loading and release of drug. In addition, because of the effect of Bragg diffraction, these particles were vividly colored (Figure 2f). According to the Bragg equation： λ = 1.633dnaverage

(1)

where λ is the wavelength of the reflected light, d is the center-to-center distance between the nearest nanoparticles or nanopores and naverage refers to the mean refractive index of the materials, the main peak position λ can be estimated. Thus, the structural colors and specific reflection peaks of the particles could shift synchronously with changing λ or d. (Figure S1a-c).

ACS Paragon Plus Environment

7


Figure 1. Schematic illustration of the fabrication process and the application of the inverse opal particles with responsive switch triggered by pathological state.

Figure 2. The reflection images of three kinds of particles: (a) the template SCCBs; (b) the PNIPAM hybrid SCCBs; (c) the inverse opal particles. The SEM images of three kinds of particles:(d) surface microstructure of template SCCBs; (e) inner microstructure of the PNIPAM hybrid SCCBs; (f) the surface microstructure of inverse opal particles. The scale bars are 200 μm in a-c, and 500 nm in d-f.

PNIPAM hydrogel was chosen to generate the inverse opal scaffold because of its temperature responsiven e s s to enable a smart response to the pathological state of the wound site, where the body temperature would increase because of an inflammatory reaction caused by the invasion of bacteria. It was empolyed as a model hydrogel and was demonstrated as a proofof-concept of the thermo-responsive drug delivery system. In addition, for further research, through some chemical modifications, the PNIPAM hydrogel would be imparted with biodegradable abilities, which could be more versatile for biomedical applications.41 PNIPAM hydrogel with a volume phase transition temperature (VPTT) around 37°C would shrink and extrude the encapsulated drug under the high-temperature environment of an infectious wound. To confirm this feature, fluorescein isothiocyanate-vancomycin (FITC-Van) was employed as a model drug to investigate the smart drug delivery system. Sodium alginate hydrogels with different concentrations were used to encapsulate FITC-Van into the PNIPAM scaffold. ACS Paragon According to confocal laser scanning images,Plus theEnvironment drug completely and evenly filled the inverse 8

Page 8 of 23

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


opal scaffold (Figure 3a and Figure S2a). To verify the performance of PNIPAM drug carriers, the release kinetics of three groups were recorded and compared. The microcarriers and their surrounding environment were exposed to different temperatures, with room temperature considered the control group (Figure 3b). The temperatures of the experimental groups were 37°C and 45°C (above VPTT), respectively, as shown in Figure 3c and 3d. In addition, different temperature exposure cycles of 15, 30, 45 and 60 min were also used to investigate the release situation. The drug could be released for at least 20 days and up to 30 days without temperature stimulation, mimicking a normal, non-infected wound (Figure 3b and Figure S2b). Compared with the control group, the release of vancomycin increased in the other two groups because the PNIPAM hydrogel porous inverse opal scaffold particles shrank above the VPTT, extruding the encapsulated drugs from inside alginate hydrogel through the external force from NIPAM hydrogel scaffold. I t w a s d e m o n s t r a t e d t h a t t h e P NIPAM gel inverse opal particles has a shrinkage when temperature is above VPTT, which led to the almost invisibility of the porous microstructure (Figure S3). Additionally, the amount of drug released negatively correlated with the concentration of sodium alginate, regardless of temperature exposure cycle (15 min), as the lower concentration hydrogels are more sensitive to scaffold contraction owing to their more porous internal structure. As a result, the relative high temperatures could accelerate the drug release by triggering the shrinking of the PNIPAM hydrogel scaffold. In practice, approximately 50% of the vancomycin in the low concentration calcium alginate was released within two temperature cycles, while three temperature cycles were needed to release half of the vancomycin in a 2% hydrogel (Figure 3c and 3d). This phenomenon of various released amounts in different hydrogel concentrations could meet the demands for diverse delivery systems according to different clinical needs. Notably, with the i n c r e a s i n g time of e v e r y temperature cycle, the release amount of the first temperature cycle would also increase (Figure S4), which could be ascribed to the more sufficient time provided for the ACS Paragon Plus Environment

9


Page 10 of 23

particles to shrink and extrude the encapsulated drugs. In conclusion, the amount of drug released from the microcarriers will increase, promoting wound healing when inflammation persists or is repeating. Alternatively, the microcarriers will be locked and the release of drug molecules will not continue when there is no appropriate inflammatory reaction. These features help to avoid the abuse of antibiotics and further improve the safety of the therapeutic treatment.

Figure 3. (a) LSCM images of the FITC-Vancomycin loaded PNIPAM hydrogel inverse opal particles. Optical slices 1-9 (parallel to horizontal) are indicative of the images taken in the zdirection from the top of the beads to the bottom; (b) Cumulative release curves of the FITCdextran from the PNIPAM hydrogel inverse opal particles with different concentrations (w/v) of encapsulated calcium alginate hydrogel at room temperature; (c-d) Release of the FITC-dextran from the particles under different temperature stimuli and different cycles: (c) one temperature cycle with 15 mins at 37°C; (d) one temperature cycle with 15 mins at 45°C. ACS Paragon Plus Environment

10

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


We conducted antibacterial tests under simulated inflammatory conditions in vitro to further observe the antibacterial efficiency of microcarriers under inflammation for promoting wound healing. Three representative sodium alginate gel concentrations, 0.25%, 1% and 3%, were chosen. PNIPAM hydrogel inverse opal particles with vancomycin encapsulated by different concentrations of sodium alginate (the experimental groups) and without vancomycin (the control group) were added to a 96-well plate. 0.5 McFarland (MCF) S. aureus bacterial liquid was added to the wells and the plate was incubated at 37°C to mimic an inflammatory environment. Compared with the control group, the smart microcarriers all showed significant bactericidal action with the release of antibiotic triggered by the relatively high temperature. Additionally, the bacteria were all killed in approximately 1.5 hours when the sodium alginate hydrogel was 0.25%. The bacteria were all killed in approximately 3 hours when the sodium alginate hydrogel was 1% or 3% (Figure S5). The faster elimination of bacteria with the 0.25% sodium alginate hydrogel that was more sensitive to the shrinkage of temperature-responsive inverse opal scaffold because of its relatively weak strength. The reason that sodium alginate hydrogel with low concentration is more sensitive to the shrinkage of temperature-responsive inverse opal scaffold is that their high content of water in hydrogel and their weak strength between intermolecular hydrogen bonds could make them be affected by external force more easily.42,43 However, this effect w as ineffective when the strength of hydrogel increased. Thus, a slight difference was observed between the groups of 1% and 3% sodium alginate.


11


Page 12 of 23

Figure 4. Images of wounds on Day 0, Day 3, Day 5, Day 7 from different groups: (i)Day 0, (ii) Day 3, (iii) Day 5, (iv) Day 7; (a) the PBS group, (b) the PNIPAM hydrogel inverse opal particles group, (c) the vancomycin group, (d) the inverse opal particles with vancomycin group. Representative H&E stained histologic longitudinal sections of different groups: (e) the PBS group, (f) the PNIPAM hydrogel inverse opal particles group, (g) the vancomycin group, (h) the inverse opal particles with vancomycin group. The scale bars are 800μm.

We also conducted animal experiments to evaluate the effects of the microcarriers for wound healing. The specific particles for the tests in this part were selected based on the previous studies. All the research protocols were approved by Animal Investigation Ethics Committee of Jinling Hospital and followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). To evaluate the temperature elevation of the infected wounds on the rats in the experimental group, we measured the temperature of rats suffering from infected wound or clean wound. It was demonstrated that the temperature of rats suffering clean wound and infected wound was around 35.8°C and 37.6 °C, respectively (Figure S8). There was an obvious temperature elevation in rats suffering from infected wounds. Wounddeficient rats were randomly divided into four groups. These rats were treated with the following treatments: phosphate buffer saline (PBS) solution (control group), PNIPAM hydrogel inverse opal particles, vancomycin, and PNIPAM hydrogel inverse opal particles with vancomycin encapsulated by 0.25% sodium alginate hydrogel. During the course of the experiment, the rats survived and were sensitive to the external stimulus. The recovery of infectious wounds in each group were recorded 0, 3, 5, and 7 days after operation (Figure 4ad) . (Figure 4a-d). H&E staining of the regenerated tissue was used to histologically analyze ACS Paragon Plus Environment

12

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


the wound recovery of each group (Figure 4e-h). It was found that inverse opal particles with vancomycin group had the largest thickness of the granulation tissue and wound closure rate, followed by the vancomycin group, then the PNIPAM hydrogel inverse opal particles group, and the lowest was the PBS group (Figure S6 and S7). PBS could not prevent further damage in the wound sites owing to its liquid nature. In the PNIPAM hydrogel inverse opal particles group, the hydrogel particles played a protective role but the effect was not significant. As a result, the thickness of the granulation tissue in this group was also relatively thin. However, the regenerated granulation tissue in the two groups with vancomycin was perfect because infected wounds heal quickly via the efficient bactericidal action of vancomycin. In addition, compared with the one-dose vancomycin group, the inverse opal particles with vancomycin group was better because it continuously and intelligently released the drug at the wound, acting as a drug bank and prolonging the time of the drug release. In the process of wound healing, the microcarriers not only can be integrated into the compact scab, but also can be encapsulated into the wound site and contact with inner moist tissue so as to release the drug for promoting wound healing. This feature provides strong support for the microcarriers with the smart drug delivery system as a novel treatment for the wound healing.


13


Figure 5. Neovascularization in different groups: (a) the PBS group; (b) the PNIPAM hydrogel inverse opal particles group; (c) the vancomycin group; (d) the inverse opal particles with vancomycin group. The double staining revealed that CD31 (+) structures were surrounded by α- smooth muscle actin positive cells (indicated with yellow arrows). (e) Quantification of the CD31 labeled structures. The scale bars in a-d are 20μm.

Figure 6. Immunostaining for TNF-α and IL-6 and Mason trichrome staining for collagen in different groups: (a) the Immunostaining for TNF-α; (b) Mason trichrome staining for collagen; (c) the Immunostaining for IL-6; among f-h the (i)-(iv) represent (i) the PBS group, (ii) the PNIPAM hydrogel inverse opal particles group, (iii) the vancomycin group, (iv) the inverse opal particles with vancomycin group, respectively. *P < 0.05，**P < 0.01., The scale bars in a-c are 100μm.

Angiogenesis plays an important role in tissue generation and maturation. However, it is difficult for normal somatic cells, such as vascular cells, to survive in infected wounds because of the severe immune response caused by the high pathogen concentration. Therefore, only by eliminating infection can the cells survive successfully, the vessel and tissue is able to effectively regenerate, which contributes to the wound healing. In the PBS and PNIPAM ACS Paragon Plus Environment

hydrogel inverse opal particles groups, there was little positive staining of vascular structures 14

Page 14 of 23

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


(Figure 5) and little collagen deposition, and the inflammatory factors TNF-α and IL-6 were upregulated, indicating the inflammation was severe (Figure 6). The double staining of CD31 and α- smooth muscle actin was used to assess the vascular structure because they are recognized mark on vascular endothelial cell and smooth muscle cell respectively. The staining of CD31 was green, and α- smooth muscle actin was red. As a result, the vascular development conditions of these two groups were unsatisfactory because there was no means to fight t h e infection (Figure 5a, b). Alternatively, vascular structures were densely distributed in the vancomycin and the inverse opal particles with vancomycin groups (Figure 5c, d), and there was significant amount of collagen and the inflammatory factor TNF-α and IL-6 was downregulated (Figure 6). Thi s phenomenon could b e ascribed to vancomycin eliminating infection in the wound sites. However, compared with the one-dose drug delivery mode in the vancomycin group, the inverse opal particles with vancomycin group had longer and more effective anti-infective impact because of the smart drug delivery. These results were observed statistically (Figure 5e). This also highlights that microcarriers with a smart switch triggered by a pathological state could play a pivotal role in inflammation elimination and tissue engineering.

CONCLUSIONS In summary, we developed microcarriers with a responsive switch that can controllably release drugs according to the pathological state of wound healing. The temperature- responsive PNIPAM hydrogel was employed as inverse opal scaffolds that could be loaded with vancomycin encapsulated in sodium alginate hydrogel. The release of vancomycin from the microcarriers could be controlled by the pathological state in the wound site. In a severe wound infection model, microcarriers with the smart switch significantly promoted the angiogenesis, collagen deposition, and granulation-tissue, followed by alleviating inflammation. Alternatively, the microcarriers do not release drugs if there are no appropriate stimulus of inflammation, which ACS Paragon Plus Environment

avoids the abuse of antibiotics and further improves the safety of the therapeutic treatment. 15


Page 16 of 23

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Reflection images and SEM images of template SCCBs, pNIPAM hybrid SCCBs, inverse opal particles. SEM images of inverse opal particles filled with calcium alginate hydrogel and Confocal images of drug release from composited inverse opal particles. Release behavior of the FITCVancomycin from the composited inverse opal particles under different temperature stimuli and different cycles. Surviving fractions of staphylococcus aureus in different composited inverse opal particles groups. Statistical analysis of the relative connective tissue thickness in the four different interventions in animal model.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; *E-mail: [email protected]; *E-mail: [email protected] Author Contributions W. X. W., R. J. A. and Z. Y. J. conceived and built the experiment, C. C. W. carried out the experiment and analyzed the data. All authors contributed to the preparation of the manuscript.

Funding Sources This study was funded by the National Nature Science Foundation of China (81801971), Key Project of Jiangsu Social Development (BE2016752 and BE2017722), Distinguished Scholars Foundation of Jiangsu Province (JCRCB2016006), Innovation Project of Military Medicine (16CXZ007), and Nanjing Science and Technology Development Project (201803051).


16

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


REFERENCES (1)

Cho, H.; Blatchley, M. R.; Duh, E. J.; Gerecht, S. Acellular and cellular approaches to

improve diabetic wound healing. Adv. Drug Deliv. Rev. 2018, DOI: 10.1016/j.addr.2018.07.019. (2)

Duan, B.; Huang, Y.; Lu, A.; Zhang, L. Recent advances in chitin based materials

constructed via physical methods. Progress in Polymer Science 2018, 82, 1-33. (3)

Li, S.; Dong, S.; Xu, W.; Tu, S.; Yan, L.; Zhao, C.; Ding, J.; Chen, X. Antibacterial

Hydrogels. Advanced science 2018, 5, 1700527. (4)

Gurtner, G. C.; Werner, S.; Barrandon, Y.; Longaker, M. T. Wound repair and

regeneration. Nature 2008, 453, 314-321. (5)

Mofazzal Jahromi, M. A.; Sahandi Zangabad, P.; Moosavi Basri, S. M.; Sahandi Zangabad,

K.; Ghamarypour, A.; Aref, A. R.; Karimi, M.; Hamblin, M. R., Nanomedicine and advanced technologies for burns: Preventing infection and facilitating wound healing. Adv. Drug Deliv. Rev. 2018, 123, 33-64. (6)

Chen, Z.; Wang, Z.; Ren, J.; Qu, X. Enzyme Mimicry for Combating Bacteria and

Biofilms. Accounts of chemical research 2018, 51, 789-799 (7)

Chaloupka, K.; Malam, Y.; Seifalian, A. M. Nanosilver as a new generation of

nanoproduct in biomedical applications. Trends in biotechnology 2010, 28, 580-588 (8) Bei,

Xue, Q.; Liu, X. B.; Lao, Y. H.; Wu, L. P.; Wang, D.; Zuo, Z. Q.; Chen, J. Y.; Hou, J.;

Y. Y.; Wu, X. F.; Leong, K. W.; Xiang, H.; Han, J. Anti-infective biomaterials with surfacedecorated tachyplesin I. Biomaterials 2018, 178, 351-362 (9)

Tatara, A. M.; Kontoyiannis, D. P.; Mikos, A. G. Drug delivery and tissue engineering to

ACS Paragon Plus Environment promote wound healing in the immunocompromised host: Current challenges and future directions. 17


Page 18 of 23

Adv. Drug Deliv. Rev. 2018, 129, 319-329 (10)

Bowler, P. G.; Duerden, B. I.; Armstrong, D. G. Wound microbiology and associated

approaches to wound management. Clinical microbiology reviews 2001, 14, 244-269. (11)

Gao, G.; Jiang, Y. W.; Jia, H. R.; Wu, F. G. Near-infrared light-controllable on-demand

antibiotics release using thermo-sensitive hydrogel-based drug reservoir for combating bacterial infection. Biomaterials 2018, 188, 83-95. (12)

Chen, G. P.; Yu, Y. R.; Wu, X. W.; Wang, G. F.; Ren, J. A.; Zhao, Y. J. Bioinspired

Multifunctional Hybrid Hydrogel Promotes Wound Healing. Advanced Functional Materials 2018, 28, 1801386

(13)

Aderibigbe, B. A.; Buyana, B. Alginate in Wound Dressings. Pharmaceutics 2018, 10,

42.

(14)

Ong, S. Y.; Wu, J.; Moochhala, S. M.; Tan, M. H.; Lu, J. Development of a chitosan-based

wound dressing with improved hemostatic and antimicrobial properties. Biomaterials 2008, 29, 4323-4332

(15)

Chen, C. W.; Liu, Y. X.; Wang, H.; Chen, G. P.; Wu, X. W.; Ren, J. A.; Zhang, H. D.;

Zhao, Y. J. Multifunctional Chitosan Inverse Opal Particles for Wound Healing. ACS nano 2018, 12, 10493- 10500 (16)

Sun, G.; Shen, Y. I.; Harmon, J. W. Engineering Pro-Regenerative Hydrogels for Scarless

Wound Healing. Advanced healthcare materials 2018, 7, e1800016. (17)

ACS Paragon Plus Environment Zhao, X.; Cui, W. G. Disease-triggered hydrogel therapy. Materials Today 2015, 18, 56-

18

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


57. (18)

Zhao, P.; Jin, Z.; Chen, Q.; Yang, T.; Chen, D.; Meng, J.; Lu, X.; Gu, Z.; He, Q. Local

generation of hydrogen for enhanced photothermal therapy. Nature communications 2018, 9, 4241. (19)

Shang, L. R.; Cheng, Y.; Zhao, Y. J. Emerging Droplet Microfluidics. Chemical reviews

2017, 117, 7964-8040. (20)

Zhang, Y. S.; C. Z.; Xia, Y. N. Inverse Opal Scaffolds and Their Biomedical

Applications. Adv. Mater. 2017, 29, 1701115. (21)

Wang H.; Zhao Z.; Liu Y. X.; Shao C. M.; Bian F. K.; Zhao Y. J. Biomimetic enzyme

cascade reaction system in microfluidic electrospray microcapsules. Sci. Adv. 2018, 4, eaat2816. (22)

Zhang, Y.; Fu, Q.; Ge, J. Test-Paper-Like Photonic Crystal Viscometer. Small 2017, 13,

1603351. (23)

Liu, Y. X.; Huang, Q.; Wang, J.; Fu, F. F.; Ren, J. A.; Zhao, Y. J. Microfluidic

generation of egg- derived protein microcarriers for 3D cell culture and drug delivery. Science Bulletin 2017, 62, 1283-1290. (24)

Wu, W.; Dai, Y.; Liu, H.; Cheng, R.; Ni, Q.; Ye, T.; Cui, W. G. Local release of

gemcitabine via in situ UV-crosslinked lipid-strengthened hydrogel for inhibiting osteosarcoma. Drug Delivery 2018, 25, 1642-1651. (25)

Zhang, B.; Cheng, Y.; Wang, H.; Ye, B. F.; Shang, L. R.; Zhao, Y. J.; Gu, Z. Z.

Multifunctional inverse opal particles for drug delivery and monitoring. Nanoscale 2015, 7, 10590-10594 (26)

Liu, Z.; Li, Y.; Li, W.; Xiao, C.; Liu, D.; Dong, C.; Zhang, M.; Makila, E.; Kemell, M.;

Salonen, J.; Hirvonen, J. T.; Zhang, H.; Zhou, D.; Deng, X.; Santos, H. A. Multifunctional ACS Paragon Plus Environment

19


Page 20 of 23

Nanohybrid Based on Porous Silicon Nanoparticles, Gold Nanoparticles, and Acetalated Dextran for Liver Regeneration and Acute Liver Failure Theranostics. Adv. Mater. 2018, 30, e1703393 (27)

Song, Y.; Li, X.; Fan, J. B.; Kang, H.; Zhang, X.; Chen, C.; Liang, X.; Wang, S.

Interfacially Polymerized Particles with Heterostructured Nanopores for Glycopeptide Separation. Adv Mater 2018, 30, e1803299; (28) (29)

Santos, H. A. Materials Science in Finland. Adv. Mater. 2018, 30, e1802557. Li, Y. N.; Yan, D.; Fu, F. F.; Liu, Y. X.; Zhang, B.; Wang, J.; Shang, L. R.; Gu, Z. Z.; Zhao,

Y. J. Composite core-shell microparticles from microfluidics for synergistic drug delivery. Science China Materials 2017, 60, 543-553 (30)

Zhang, Y.; Tu, J.; Wang, D.; Zhu, H.; Maity, S. K.; Qu, X.; Bogaert, B.; Pei, H.; Zhang,

H. Programmable and Multifunctional DNA-Based Materials for Biomedical Applications. Adv Mater 2018, 30, e1703658

(31)

Yu, Y. R.; Chen, G. P.; Guo, J. H.; Liu, Y. X.; Ren, J. A.; Kong, T.; Zhao, Y. J. Vitamin

metal–organic framework-laden microfibers from microfluidics for wound healing. Materials Horizons 2018, 5, 1137-1142 (32)

Sousa, F.; Cruz, A.; Pinto, I. M.; Sarmento, B. Nanoparticles provide long-term stability

of bevacizumab preserving its antiangiogenic activity. Acta biomaterialia 2018, 78, 285-295. (33)

Fu, F. F.; Chen, Z. Y.; Zhao, Z.; Wang H.; Shang, L. R.; Gu, Z. Z.; Zhao, Y. J. Bio-

inspired self-healing structural color hydrogel. PNAS 2017, 23, 5900-5905. (34)

Fu, Q.; Zhu, H.; Ge, J. Electrically Tunable Liquid Photonic Crystals with Large Dielectric

Contrast and Highly Saturated Structural Colors. Advanced Functional Materials 2018, 28, ACS Paragon Plus Environment

20

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


1804628 (35)

Fu, X.; Zhang, Y.; Yuan, H.; Binks, B. P.; Shum, H. C. Controlled Actuation of Liquid

Marbles on a Dielectric. ACS applied materials & interfaces 2018, 10, 34822-34827. (36)

Wang, L.; Cai, P.; Luo, J.; Zhang, F.; Liu, J.; Chen, Y.; Zhu, Z.; Song, Y.; Yang, B.; Liu,

X.; Chen, X.; Wang, S. Engineering subcellular-patterned biointerfaces to regulate the surface wetting of multicellular spheroids. Nano Research 2018, 11, 5704-5715 (37)

Pi, Q.; Maharjan, S.; Yan, X.; Liu, X.; Singh, B.; van Genderen, A. M.; Robledo-Padilla,

F.; Parra-Saldivar, R.; Hu, N.; Jia, W.; Xu, C.; Kang, J.; Hassan, S.; Cheng, H.; Hou, X.; Khademhosseini, A.; Zhang, Y. S. Digitally Tunable Microfluidic Bioprinting of Multilayered Cannular Tissues. Adv Mater 2018, 30, e1706913.

(38)

Bedenic, B.; Car, H.; Slacanac, D.; Sviben, M.; Cacic, M.; Lukic-Grlic, A.; Bencic, A.;

Sijak, D.; Tripkovic, M.; Beader, N. In vitro synergy and postantibiotic effect of colistin combinations with meropenem and vancomycin against Enterobacteriaceae with multiple carbapenem resistance mechanisms. Journal of infection and chemotherapy: official journal of the Japan Society of Chemotherapy 2018, 24, 1016-1019 (39)

Lopez-Iglesias, C.; Barros, J.; Ardao, I.; Monteiro, F. J.; Alvarez-Lorenzo, C.; Gomez-

Amoza, J. L.; Garcia-Gonzalez, C. A. Vancomycin-loaded chitosan aerogel particles for chronic wound applications. Carbohydr. Polym. 2019, 204, 223-231. (40)

Wu, J.; Zheng, Z.; Chong, Y.; Li, X.; Pu, L.; Tang, Q.; Yang, L.; Wang, X.; Wang, F.;

Liang, G. Immune Responsive Release of Tacrolimus to Overcome Organ Transplant Rejection. Adv Mater 2018, 30, e1805018. (41)

Roy, D.; Brooks, W. L. A.; and Brent, S. S. New directions in thermoresponsive ACS Paragon Plus Environment

21


Page 22 of 23

polymers, Chem. Soc. Rev. 2013, 42, 7214.

(42)

Liu, Y.X.; Shao, C. M., Bian, F. K.; Yu, Y. R.; Wang, H.; Zhao, Y. J. Egg components composited

inverse opal particles for synergistic drug delivery ACS Applied Materials & Interfaces, 2018, 10, 1705817064. (43)

Kasapoglu-Calik, M.; Ozdemir, M. Synthesis and controlled release of curcumin-β-

cyclodextrin inclusion complex from nanocomposite poly (N-isopropylacrylamide/sodium alginate) hydrogels Journal of Applied Polymer Science, 2019, 136, 47554.


22

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


Table of Contents (ToC) Graphic


23

Antibacterial porous microcarriers with pathological state responsive

Recommend Documents