Sustainable Dual Release of Antibiotic and Growth Factor from pH

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Sustainable dual release of antibiotic and growth factor from pH-responsive uniform alginate composite microparticles to enhance wound healing Ming Shi, Hao Zhang, Ting Song, Xiaofang Liu, Yunfen Gao, Jianhua Zhou, and Yan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04750 • Publication Date (Web): 29 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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ACS Applied Materials & Interfaces

Sustainable dual release of antibiotic and growth factor from pH-responsive uniform alginate composite microparticles to enhance wound healing Ming Shi,a,b Hao Zhang,a,b Ting Song,a,b Xiaofang Liu,a,b Yunfen Gao,a,b Jianhua Zhoua,b,c and Yan Lia,b* aGuangdong

Provincial Key Laboratory of Sensor Technology and Biomedical

Instrument, School of Biomedical Engineering, Sun Yat-sen University, Guangzhou 510006, Guangdong, P.R. China bGuangdong

Provincial Engineering and Technology Center of Advanced and Portable

Medical Devices, Sun Yat-sen University, Guangzhou 510006, Guangdong, P.R. China cDivision

of Engineering in Medicine, Department of Medicine, Brigham and Women's

Hospital, Harvard Medical School, Cambridge, MA 02138, USA *Corresponding author: Yan Li, Telephone: +86-20-39332146, Fax: +86-20-39332146, Email: [email protected]

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Abstract Hydrogel-based wound dressings provided a moist microenvironment and local release of bioactive molecules. Single drug loading along with fast release rates and usually in hydrogel sheets limited their performance. Hence, uniform Alginate/CaCO3 composite microparticles (~430 μm) with tunable compositions for sustainable release of drug and pH-sensitivity were successfully fabricated using microfluidic technology. Due to the presence of CaCO3 and the strong interactions with alginate molecules, lyophilized composite microparticles reverted to hydrogel state after rehydration. Regardless of microparticle states (hydrogel or lyophilized) and pH values (6.4 or 7.4), in vitro release rates of model drug were inversely related with CaCO3 concentrations and much lower than that for pure alginate microparticles. The release rate at pH 6.4 (simulating wound microenvironment) was always slower than that at pH 7.4 for the same type of microparticles. Rifamycin and basic fibroblast growth factor (bFGF) were independently encapsulated into AD-5-R and AD-40-F to achieve a fast release of rifamycin and a slower, more sustained release of bFGF, respectively; CD-F-R was a mixture of AD-5-R and AD-40-F at weight ratio 1/1. For AD-5-R and CD-F-R, inhibition zones of S. aureus were observed until day 5, showing a sustained antibacterial property. Based on in vitro wound healing model of NIH-3T3 cell micropattern on glass cover slips with hole array, it was found that AD-40-F and CDF-R significantly promoted cell proliferation and migration rates. In a full-thickness skin wound model of rat, CD-F-R microparticles significantly accelerated wound healing with higher granulation tissue thickness and better bioactivity to stimulate angiogenesis than Control group. Furthermore, CD-F-R microparticles demonstrated a good biocompatibility and biodegradability in vivo. Taken together, CD-F-R composite microparticles may ideally meet the requirements for different stages during wound healing and demonstrated a good potential to be used as dressing materials.

Key words: Alginate hydrogel microparticles, pH sensitivity, microfluidic, antibacterial, cell migration

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1. Introduction Wound treatment is a major health issue and has attracted lots of attentions. Millions people suffered from acute and chronic wounds in the world every year 1. For extensive full-thickness wounds, a long period is usually needed to heal 2. Thus, dressings as barriers should promote the healing process, which ideally meet the expectations of rapid wound closure and reduction of scar formation. Till date, various types of wound dressings which were made of rubber, cotton, electrospun nanofibers, membranes and hydrogels have been developed. Hydrogel dressings have been widely applied which usually provided a moist microenvironment, absorbed tissue exudates and promoted repair of damaged tissues 3. Hydrogel dressings from chitosan, gelatin, polyvinyl alcohol (PVA), poly (ethylene glycol) (PEG), and alginate have also encapsulated bioactive drugs

4-8.

Because of the porosity structure, the encapsulated therapeutic

substances were usually delivered to wound areas in a sustained manner 3. In addition to efficiency (i.e., targeted at wounds), such topical delivery route also minimized toxicity or side effects of the drug 9. For acute wounds, an orderly and timely separated process was usually followed to establish skin barrier functions, including coagulation and hemostasis, infection or inflammation, migration and proliferation of mainly fibroblast cells, and then remodeling phase. After injury, hemostasis should take place immediately to prevent exsanguination. Alginate has been reported to be often used as a hemostatic agent. After this stage, ideal wound dressings should have good antibacterial properties, which promoted wound healing via reducing pathogens numbers and inflammatory responses. For this purpose, antibacterial wound dressings have been developed either by incorporating antibacterial agents into hydrogels or directly using materials with inherent antibacterial activity

10.

When hemostasis was achieved and an immune

response successfully set in place, the acute wound healing shifted toward tissue repair. In this period growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) were applied as therapeutic agents to promote proliferation of fibroblasts and formation of new blood vessels 11. Besides sheets, hydrogels as microparticles and dispersions have been widely used as wound dressing. Because of the small size (1 - 1000 μm) and other properties such as bioadhesion, swelling, and ability to respond to environment stimuli, microparticles are 3



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adaptable to a wide range of dosage forms and product applications which were beneficial to wound healing. Furthermore, microparticles can be applied at irregular parts of the wound surface to achieve well matched local release of loaded drugs

12.

Such adaptability coupled with the ability to control drug release rates have been found 13.

to be highly advantageous in treatment of chronic wounds

It was reported that

doxycycline-loaded functionalized gelatin microspheres showed an effective antimicrobial activity against Staphylococcus aureus (S. aureus), exhibiting a complete wound healing within 15 days as compared to 24 days for the untreated control group 14.

Epidermal growth factor was first entrapped in alginate hydrogel and then was

further loaded in poly (lactide-co-glycolide) microspheres, which demonstrated a promising potential in promoting faster and more effective wound healing. It was reported that hydrogels containing

125I-labeled

bFGF were implanted subcutaneously

onto the backs of mice and residual radioactivity was measured. The decrement pattern of bFGF radioactivity in the hydrogel was in agreement with radioactivity for 14 days. It was also pointed out that in the subcutaneous injection of

125I-labeled

bFGF in

aqueous solution, about 80% of bFGF disappeared within 1 day15. These results lead us to believe the hydrogel sheet loaded bFGF as a dressing material can maintain biological activity and release bFGF effectively at Local wound area. Impaired and delayed healing occurred in chronic wounds due to complications such as ulcers 16. This requires a long-term controlled release of drugs. Hydrogel dressings such as alginate have a large pore size network, which was easy to be swollen and resulted in a rapid release of loaded drugs. To obtain more sustained release profiles, efforts on improving gelation properties via introducing other materials into hydrogel matrix have been made. It was worth noting that such incorporation usually did not affect hydrogel structure (i.e., gelation mechanism) and did not lead to cytotoxicity either 17. In recent studies, inorganic materials such as hydroxyapatite, brushite and calcium carbonate attracted broad attentions and were added into hydrogel matrix to limit the movement of polymer chains so as to prolong the drug release time

17-20.

Dabiri et al. fabricated

alginate-brushite hydrogel composites to avoid burst release associated with pure hydrogels and also to optimize release profile of ibuprofen strontium-substituted

hydroxyapatite

microspheres

21.

into

Li et al. incorporated alginate

composite

microspheres which showed a sustained release of vancomycin for ~7 days with a accumulative release of 40% 22. When CaCO3 microspheres were entrapped in alginate 4


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membranes, the drug release was reduced from 99% to 81%, indicating that the organicinorganic hybrid structure hindered permeability of the encapsulated drug and reduced drug release rates. However, very few findings have been reported regarding multifunctional wound dressings which did not only provide physical protection and maintain moisture microenvironment, but also improved healing process through influencing the timely and orderly separated stages during wound repairing. For such purpose, multiple drugs are often required to be delivered simultaneously to control infections and also cell proliferations. Furthermore, pH sensitivity which was important for localized release in wound area, was rarely taken into account. Therefore, rifamycin and bFGF independently loaded Alginate/CaCO3 microparticles of different compositions were fabricated using microfluidic technique to study the synergistic effects on promoting wound healing. The preparation conditions were intensively explored, including types of organic solvents as continuous phase, with or without surfactant, flowing rate and concentrations of CaCO3. Vaterite microspheres were composed of nano-aggregates

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and its rough surface may lead to a strong

interaction with alginate molecules. Vaterite also has antacid activity. Trypan Blue was first used as a model drug because it was easy to quantify the concentration and thus in vitro release of Trypan Blue from Alginate/CaCO3 microparticles of different compositions was investigated. Based on the release data, rifamycin and bFGF were separately encapsulated into composite microparticles of different compositions to achieve different release rates. The antibacterial properties were evaluated using a disk diffusion method against S. aureus; for cell proliferation studies, an in vitro wound healing model based on mouse fibroblasts (NIH-3T3) growing on patterned glass cover slips with hole array in medium at pH 6.4 were developed. Cell proliferation and migration behaviors were monitored. The in vivo performance of composite microparticles on wound healing was evaluated in a full-thickness skin wound model.

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2. Materials and Methods 2.1 Materials Sodium alginate and Calcein-AM were from Aladdin (Shanghai, China). Trypan Blue solution (0.4%) and casein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Calcium chloride (CaCl2), sodium carbonate (Na2CO3), Span 80, ethyl acetate (EA) and dichloromethane (DCM, 99.5%) were obtained from Guangzhou Chemical Reagent Co. (Guangzhou, Guangdong, China). Recombinant murine FGF-basic (bFGF) was from Peprotech (Rocky Hill, NJ, USA). High glucose Dulbecco's Modified Eagle Medium (DMEM), PS (penicillin/streptomycin antibiotics), fetal bovine serum (FBS) and trypsin–EDTA were supplied by Gibco (Carlsbad, CA, USA). Actin cytoskeleton and focal adhesion staining kit (FAK-100) was from Merck (Billerica, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Shanghai, China). All other chemicals and reagents were of analytical grade. All reagents were used as received. 2.2 Preparation of alginate hydrogel microparticles with different shapes and sizes Alginate microparticles were fabricated using a microfluidic device. The device was assembled by inserting a 31G needle vertically into the middle of a poly tetrafluoroethylene (PTFE) tube (internal diameter of 0.6 mm and outside diameter of 1.0 mm) and sealed with acrylic adhesive glue. The exit of PTFE tube was placed in CaCl2 solution. The inner phase was 1% sodium alginate solution at a flow rate of 10 μl/min and these parameters did not change in the following tests. The continuous phase was organic solvent: EA or DCM, with or without surfactant Span 80 (3%, v/v) and the flow rate was set as 50, 200, 400, or 800 μl/min. Flow rates of inner and continuous phases were independently controlled by injection pumps. Due to the shear force from the continuous phase, alginate solution was separated into uniform droplets, which along with the continuous phase flowed into the collecting bath (CaCl2 solution of 102 mM). In the bath, droplets were solidified for 30 min and alginate hydrogel microparticles were collected by centrifugation. 2.3 Preparation and characterizations of Alginate/CaCO3 composite microparticles CaCO3 vaterite microparticles were first prepared with a co-precipitation method 23. In brief, 50 mM CaCl2 solution was quickly added into the same volume Na2CO3 solution (50 mM with casein of 8 mg/ml) under stirring at 600 rpm. After stirring for 20 min, certain amounts of suspension were centrifuged and corresponding contents of CaCO3 6


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vaterite were 0, 5, 20 and 40 mg. Then 400 μl of deionized (DI) water (pH 10) and 100 μl Trypan Blue (TB) solution (model drug) was transferred into these tubes and CaCO3 were resuspended. Afterwards, 500 μl of 2% SA solution was added and suspension was stirred at a 600 rpm for 2 hr until CaCO3 microspheres were well dispersed in alginate solution. The microfluidic device for composite microparticles was the same as described above. The internal phase was 1% alginate solution containing different concentrations of CaCO3 at a flow rate of 10 μl/min; the continuous phase was DCM without surfactant at a flow rate of 200 μl/min. The formed microparticles were named as A-0-TB, A-5-TB, A-20-TB, and A-40-TB, respectively.

Fig. 1. Schemes for preparation of Alginate/CaCO3 composite microparticles and their applications in wound healing. (a) Fabrication of composite hydrogel microparticles using a microfluidic method; (b) S. aureus growth inhibition by the mixed lyophilized microparticles CDF-R; (c) establishment of an in vitro wound healing model and application of the mixed lyophilized microspheres CD-F-R on promoting cell migration and proliferation; (d) application of CD-F-R on a full-thickness skin wound of rat to fasten the healing process. CD-F-R contained microparticles loaded with rifamycin and microparticles loaded with bFGF. For both in vitro and in vivo tests, freeze-dried microparticles were applied; after rehydration, these microparticles returned to hydrogel state.

The morphology of Alginate/CaCO3 hydrogel composite microparticles was observed under a light microscope and particle size was calculated using a software (Image J). After lyophilization, surface morphology was observed under a scanning electron microscope (SEM) after sputter coated with gold. The internal structure was also observed after microparticles embedded in resin and cross-sectioned using an ultramicrotome.

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2.4 In vitro drug release test of Alginate/CaCO3 composite microparticles Antacid activity of Alginate/CaCO3 hydrogel composite microparticles was first investigated. For the test, 500 mg microparticles were collected, rinsed and 5 ml of hydrochloric acid solution (pH = 1) was added. Four groups of microparticles (A-0-TB, A-5-TB, A-20-TB, and A-40-TB) were placed in a 37°C thermostatic shaker at a speed of 60 rpm. The pH value of supernatant was measured 3 hr later. For each sample, four replicates were conducted. The loading efficiency of model drug (Trypan Blue) for composite hydrogel microparticles was measured. After solidification, 10 ml trisodium citrate solution at 10 mg/ml was added into 500 mg (collection time 50 min) of microparticles. After microparticles completely dissolved in 37°C water bath, 1 ml supernatant was taken out to measure the absorbance at 583 nm. The total drug content in these microparticles was represented by the absorbance value of 5 μl of Trypan Blue solution in 995 μl of trisodium citrate. The loading efficiency was calculated using equation (1). For each sample, four replicates were measured. Loading efficiency (%) = (absorbance for microparticles) / (absorbance for Trypan Blue) × 100%

(1)

Table 1. Drug loading efficiency and pH values after antacid test for Trypan Blue (TB) loaded alginate composite hydrogel microparticles Sample

Concentration

Encapsulation

name

of CaCO3

efficiency

pH value

(mg/ml) A-0-TB

0

60.1 ± 4.2%

1.13 ± 0.03

A-5-TB

5

63.6 ± 1.8%

1.33 ± 0.01

A-20-TB

20

72.5 ± 1.0 %

3.18 ± 0.02

A-40-TB

40

75.9 ± 0.5 %

4.16 ± 0.04

Composite hydrogel microparticles were then collected for drug release test in PBS following similar parameters reported elsewhere 22. 50 mg of microparticles (collection

8


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time 5 min) were resuspended in 2 ml of PBS at pH 6.4 or 7.4 and placed in a 37°C water bath. At predetermined time points, 1 ml of the supernatant was transferred into a 48-well plate and 1 ml fresh PBS was refilled. Small amount of EDTA was added into the well to completely dissolve any precipitate and minimize interference. The absorbance value at 583 nm was measured with a microplate reader (Synergy4, BioTek, USA). Similarly, the release behaviors of composite microparticles after lyophilization (AD-0-TB, AD-5-TB, AD-20-TB and AD-40-TB) were investigated. 2.5 Preparation of Alginate/CaCO3 composite microparticles loaded with antibiotic or growth factor For Alginate/CaCO3 composite microparticles loaded with antibiotic or growth factor, the same fabrication parameters were followed as described in Section 2.3. All chemicals were sterilized with filtration through a 0.22 μm filter and fabrication processes were carried out in a laminar flow hood. Rifamycin was first dissolved in PBS at 10 mg/ml and 500 μl was taken out to resuspended 5 mg CaCO3. After vortexing, 500 μl of 2% SA solution was then added and the suspension was further magnetically stirred for 2 hr. The well dispersed suspension was the internal phase to prepare rifamycin loaded composite microparticles (AD-5-R). Each group was collected for 5 min, 50 mg of hydrogel microparticle were obtained and further freeze-dried. Similarly, bFGF loaded composite microparticles were prepared. 40 mg CaCO3 microparticles were firstly resuspended in 300 μl PBS (containing 5% trehalose); 500 μl of 2% SA was added and stirred to form homogenous suspension. 200 μl of bFGF solution (10 μg/ml) was then added and the mixture was stirred for another 30 min for fabrication of bFGF-loaded composite microparticles (AD-40-F). AD-5 and AD-40 without rifamycin or bFGF were set as control groups. AD-5-R and AD-40-F were mixed at weight ratio 1/1 to form the dual-drug carrier sample CD-F-R. 2.6 Antibacterial test for Alginate/CaCO3 composite microparticles loaded with antibiotic The inhibition zone assay was adopted to quantify the antibacterial activity of microparticles against the growth of S. aureus (ATCC 25923), which was carried out using a disk diffusion method as described in United States Pharmacopeia. S. aureus were cultured in Luria-Bertani (LB) broth (yeast extract 5 g/l, NaCl 10 g/l and triptone 10 g/l) at 37°C, 200 rpm for 24 hr. 100 μl of bacterial suspension (8×108 CFU/ml) was 9



inoculated onto LB agar medium in a 90 mm Petri dish. AD-5-R or CD-F-R microparticles were first placed on a filter paper of diameter 9 mm, and then transferred onto the dish center. Filter paper, AD-5 and AD-40 were control samples. After incubation at 37°C for 24 hr, digital images were taken to quantify the zone of inhibition (ZOI). In order to evaluate the long-term antibacterial effect, samples on filter paper were transferred to a new dish every 24 hr until no ZOI was observed. The experiments were repeated three times. 2.7 In vitro cell experiments 2.7.1 Cytotoxicity test for Alginate/CaCO3 composite hydrogel microparticles In order to investigate cytotoxicity of composite hydrogel microparticles, A-40 group (without Trypan Blue) with the highest CaCO3 concentration was chosen. Mouse fibroblasts (NIH-3T3) were seeded in 24-well plates (DMEM with 10% FBS and 1% PS) at a density of 5 × 104 cells/well, and incubated at 37°C in a 5% CO2 incubator. After 24 hr, the old medium (1 ml/well) was replaced by the corresponding 1 ml fresh medium containing 2.5, 5, 20 and 50 mg of A-40 which were prepared from sterilized chemicals in a laminar flow hood. The one cultured in normal medium was the negative control (TCP), and the one cultured in 1 ml medium containing 0.2% Triton X-100 was the positive control group (X-100). Three replicates were prepared for each group. After 3 days, CCK-8 assay was used to characterize cell viability 24. 2.7.2 Cell proliferation studies for Alginate/CaCO3 composite microparticles loaded with antibiotic or growth factor In vitro wound healing model was first developed. Arrays of 6×6 circle holes were obtained on glass cover slips (14 mm in diameter) using a Nanosecond Laser (UV-3SSP, No:KYD6917082099, Han’S Laser, China) 25. Diameter of these circles was 600 μm and the center-to-center space was 1.5 mm. After laser ablation, glass cover slips were placed in 24-well plates, soaked in 75% ethanol for 4 hr for sterilization and washed with PBS three times. NIH-3T3 cells were seeded at a density of 105 cell/well. After cultured for 48 hr, the glass cover slips with adhered cells were gently transferred into another 24-well plate and the in vitro wound healing model was obtained. The initial cell viability was quantified using CCK-8 assay and this day was noted as D 0. After the test, the old medium was replaced by medium at pH 6.4 to simulate wound microenvironment. On day 3 and 7, cytoskeleton staining for actin filaments and 10


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nucleus were carried out to observe the migration of cells from glass cover slips onto the circle empty space on TCP. Manufactures’ descriptions were followed for staining process. After medium at pH 6.4 was added, three groups of microparticles AD-5-R, AD-40-F and CD-F-R were placed into transwell and the one without any microparticles was as the control group. AD-5-R and AD-40-F were lyophilized from 50 mg hydrogel microparticles, while CD-F-R was the freeze-dried combination of 50 mg A-5-R and 50 mg A-40-F hydrogel microparticles. For each group, three replicates were set up. On day 1, 3, 5, and 7, cell migration and proliferation in the pattern area was observed under a light microscope, and relative cell viabilities were measured with CCK-8 assay. 2.8 Application of Alginate/CaCO3 composite microparticles loaded with antibiotic and growth factor in full-thickness skin wound of SD rats Based on in vitro investigations, sample CD-F-R was selected for animal study which was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. A full-thickness skin wound model was selected mainly to investigate the performance of microparticles on hemostatic, wound healing and also the in vivo biodegradability. Sprague-Dawley rats (SD rats, 250-300 g, 6-8 weeks) were randomly divided into three groups and each group contained four rats (n = 4). After anaesthetized with 3% sodium pentobarbital solution, two circular full-thickness excisions with a diameter of 15 mm were created on back of each rat. The left wound was usually used as Control (no treatment), and CD-F-R microparticles (from 200 mg A-5-R and 200 mg A-40-F hydrogel microparticles) were applied onto the right wound; both wounds were then covered with a transparent dressing and further fastened with a self-adhesive bandage for protection. Sample CD-F-R was applied only once. On day 3, 7, 14 and 21, wounds were photographed using a digital camera and Image J was used to measure the wound area. The wound contraction was calculated using equation (2): wound contraction (%) = (A0 - At) / A0 × 100%

(2)

where A0 is the wound area on day 0 and At is the wound area at the indicated time point. On predetermined time points (7, 14 and 21 days), four rats in each group were sacrificed by injection of excessive 3% sodium pentobarbital solution. The full11



thickness skin around the wound (2 × 2 cm) was removed and fixed in paraformaldehyde solution for 24 hr. These harvest samples were paraffin embedded, sectioned and then stained with hematoxylin/eosin (H&E), Masson’s trichrome and anti-CD31 antibody to evaluate regeneration of skin tissue, collagen formation and neovascularization following procedures reported elsewhere 26. 2.9 Statistical analysis All data were shown as mean ± standard deviation and difference between groups was analyzed with student un-paired T-test. p < 0.05 indicated difference was statistically significant.

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3. Results and discussions 3.1 Effects of solvent, surfactant and flow rate of continuous phase on alginate microparticle morphology Microfluidic method was applied to fabricate alginate microparticles. Alginate microdroplets and external organic phase have different densities. When microdroplets in organic phase flowed into the collecting beaker, phase separation occurred. Organic phase accumulated in the beaker and alginate microdroplets started to cross-link with Ca2+; these droplets transited into gel state and fall onto the bottom of CaCl2 solution. The density and viscosity of continuous phase had a significant effect on the crosslinking process and it was found that deformation of alginate microdroplets happened when these microdroplets contacted with the cross-linking solution (CaCl2 solution) 27. Therefore, the effects of solvent types and with/without surfactant Span 80 on the morphology of alginate hydrogel microparticles were first investigated. EA of which the density was lower and DCM of which the density was higher than CaCl2 solution was selected as the continuous organic phase. As shown in Fig. 2 (a1-a2), when surfactant Span 80 was added into organic phase, microparticles of teardrop-like were obtained regardless of organic solvent type. When no surfactant was included, these microparticles from EA group were tadpole-like (Fig. 2a3); as for DCM group (Fig. 2a4) microparticles were still teardrop-like with slightly larger size. When EA was used as continuous phase, the addition of Span 80 changed the shape of the microparticles. For all microparticles the size was ~ 400 μm which was slightly affected by solvent type and surfactant. The sphericity of alginate hydrogel microparticles appeared to be positively related with the rate of microdroplets escaping from organic phase. The viscosity of EA was higher than DCM, which may result in slower separation rate of alginate droplets from EA and thus a tail structure. When surfactant Span 80 was added, viscosity of EA was reduced along with the interfacial tension, microdroplets were easier transferred from organic phase into gelling solution and teardrop-like microparticles were formed. Davarc et al. manipulated shape of alginate microparticles by adding different concentrations of surfactants (Tween 20) in a range of 0.01–1 g/L 28. Hu et al. changed the contacting velocity of droplets with gelling solution by varying the distance between tube tip and surface of crosslinking solution, leading to different shapes of alginate microgel such as tear drop, lamp-like, mushroom-like27. As compared with these reported methods, it 13



was easier to obtain nearly spherical alginate hydrogel microparticles when appropriate organic solvent such as DCM was used here.

Fig. 2. Fabrication of Alginate/CaCO3 hydrogel composite microparticles using microfluidic method. The effects of different organic solvents w/o surfactant Span 80 on microparticle morphology were first investigated when the flow rate for continuous phase was 200 μl/min. Optical microscope images of alginate hydrogel microparticles when (a1) EA + Span 80, (a2) DCM + Span 80, (a3) EA, and (a4) DCM was used as the continuous phase. The inset of (a4) was the corresponding particle size distribution. The effect of continuous phase flow rate on alginate hydrogel microparticle size was then studied when DCM was the continuous phase. Optical microscope images and particle size distribution of alginate hydrogel microparticle when different flow rates for continuous phase were applied, (b1) 50, (b2) 400, and (b3) 800 μl/min. After optimization, Alginate/CaCO3 hydrogel composite microparticles were fabricated with DCM as continuous phase at a flow rate of 200 μl/min. Optical microscope images and particle size distributions of these composite microparticles when different concentrations of CaCO3 were encapsulated, (c1) 5, (c2) 20 and (c3) 40 mg/ml. The corresponding alginate composite hydrogel microparticles were named as A-5-TB, A-20-TB, and A-40-TB. Scale bars are 300 μm.

In order to obtain teardrop-like hydrogel microparticles, DCM without surfactant was selected as the continuous phase and also to eliminate the influence of surfactant. The effect of flow rate of the continuous phase on microparticle size and applicability of the process was then studied, which was set as 50, 200, 400, and 800 μl/min, with other conditions unchanged. As shown in Fig. 2 (a4, b1-b3), at each rate, size distribution was narrow and all variances were smaller than 10 μm. With the increase of flow rate

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for continuous phase, the average diameter decreased from 474 to 344 μm. This trend was similar to other reported data when microfluidic device was used to fabricate microparticles. Hu et al. fabricated alginate microspheres in a range of 300 - 240 μm by changing the flow rate from 2 to 10 ml/h 27. Choi et al. obtained polycaprolactone microparticles of size 80-235 μm via changing the flow rate of continuous phase in a range of 0.1~2 ml/min

29.

This was due to the increase in shear stress imposed on

microdroplets when flow rate for the continuous phase increased. Here, when the flow rate was 800 μl/min, tail structure of microparticles appeared to be longer than others. When microdroplets became smaller, it was more difficult to separate from organic phase because the effect of surface tension was more profound; thus a longer tail structure was formed during the gelling process. 3.2 Surface morphology and internal structure of Alginate/CaCO3 composite microparticles After optimizing fabrication parameters, Alginate/CaCO3 composite microparticles were prepared using DCM as the continuous phase without surfactant at a flow rate of 200 μl/min. As shown in Fig. 2(c1-c3), all hydrogel composite microparticles were in teardrop morphology with a narrow size distribution; with the increase of CaCO3 concentration from 5 to 40 mg/ml for sample A-5-TB, A-20-TB and A-40-TB, tail structure appeared to be elongated and microparticle size slightly increased from 423 to 441 μm. This was probably related with the slight increment in suspension viscosity along with CaCO3 concentration. As for A-0-TB, the light microscope image was shown in Fig. 2a4 and microparticle size was 437 ± 8 μm, which was very close to that for A-5-TB and A-20-TB. The influence of CaCO3 on surface morphology and internal structure of alginate composite microparticles were further studied using SEM after freeze-dried. For pure alginate microparticles, shrunken and collapsed microparticles in irregular shape of a much smaller size than 400 μm was observed (Fig. 3A), similar to other reported results 30.

The enlarged image shows that the surface of AD-0-TB microparticles was crimped

and smooth (Fig. 3a), which was very different from rough surface for microparticles prepared using DCM with surfactant Span 80 (Fig. S1). For AD-5-TB, AD-20-TB and AD-40-TB (Fig. 3B-D), due to the addition of CaCO3, freeze-dried microparticles appeared to be much more porous than that for AD-0-TB as particle sizes were much 15



larger. When compared with hydrogel microparticles, their morphologies were no longer teardrop-like and aspect ratios were higher, while tail structure was still maintained. The surfaces of these composite microparticles were rough (Fig. 3b-d). In Fig. 3b, CaCO3 adhered tightly onto microparticle surface and well dispersed. With the increase of CaCO3 concentration, more CaCO3 microparticles were present on microparticle surfaces, and aggregates were also observed. When comparing with asprepared CaCO3 microparticles, which were well dispersed with an average diameter of ~1 μm (Fig. S2) and similar to our previous results 31, these on alginate composite microparticles were still round shaped and bonded tightly with alginate matrix.

Fig. 3. Surface morphologies and internal structures of lyophilized Alginate/CaCO3 composite microparticles. SEM images of (A)(a) AD-0-TB, (B)(b) AD-5-TB, (C)(c) AD-20-TB, and (D)(d) AD-40-TB microparticles. After embedded in epoxy and cryosectioned, representative SEM images of internal structure of (E)(e) AD-0-TB and (F)(f) AD-40-TB microparticles.

The cross-section views of representative samples are shown in Fig. 3 (E-F). Pure alginate microparticles AD-0-TB were flattened after freeze-drying and there was no porous structure was observed (Fig. 3e). For AD-40-TB, the cross-section was circle shaped, further showing that with the encapsulation of CaCO3, alginate composite microparticles were still in porous structure even after lyophilization. When looking at a higher magnification (Fig. 3f), it was found that vaterite were well dispersed inside composite microparticle and homogenously embedded in alginate matrix.

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Due to the addition of CaCO3, three-dimensional structure of lyophilized composite microparticles remained very well, close to the shape of hydrogel microparticles. There might be two reasons contributed to the fact, where strong interactions were present between CaCO3 and alginate molecules, and CaCO3 microparticles may form a robust framework inside alginate microparticles. Here, CaCO3 was in vaterite phase composed of nano-aggregates 23, with a rough surface and a high specific surface area, which may result in a strong and tight interaction between CaCO3 and alginate matrix. Furthermore, lyophilized Alginate/CaCO3 composite microparticles returned to hydrogel state after rehydration, providing a possibility to absorb extrude from wounds and be beneficial for sustained release of encapsulated drugs. Hence, not only were the release rates of composite hydrogel microparticles with different CaCO3 contents compared, but also were the release behaviors of freeze-dried microparticles studied. 3.3 In vitro release behaviors of Alginate/CaCO3 composite microparticles The antacid activity of Alginate/CaCO3 hydrogel composite microparticles was evaluated 32 since CaCO3 has been extensively used as an antacid. After 3 hr incubation in hydrochloric acid solution, pH values for composite microparticles were much higher than that of pure alginate microparticles A-0-TB (Table 1). The addition of CaCO3 significantly improved antacid ability of composite microparticles. The higher the CaCO3 content was, the better the effect was. CaCO3 was composed of nano-aggregates and thus demonstrated good antacid activity, which was similar to the literature 33. In addition, after the test, microparticle sizes were all slightly decreased probably due to more hydrogen bonds formed

34

and morphologies were all well maintained; for

composite microparticles, small bubbles appeared inside microparticles because of the fast reaction between CaCO3 and HCl. Even though the conditions for the test were harsh, the results demonstrated that such property of composite microparticles may be beneficial for controlling drug release at different pH values. The loading efficiency of model drug Trypan Blue in alginate composite microparticles was determined (Table 1). For pure alginate microparticles (A-0-TB), the loading efficiency was 60.1± 4.2%, where drug loss was mainly due to the porous structure of alginate network and thus fast diffusion of drug molecules during the gelling process. With the increase of CaCO3 content in composite microparticles, the loading efficiency increased from 63.6 ± 1.8% to 75.9 ± 0.5 %. This was similar to previous studies where 17



calcium phosphate was encapsulated into alginate hydrogels in order to increase drug loading efficiency

21.

This increment may be due to two reasons. The presence of

CaCO3 microparticles in alginate matrix hindered the diffusion of model drug. Furthermore, the addition of CaCO3 may limit the movement of molecular chains in hydrogel network and also increase the degree of cross-linking of alginate, further hampered the diffusion of model drug. Hence, the loss of model drug was reduced during the preparation process and the loading efficiency was significantly improved eventually. Such effect may also lower drug release rate.

Fig. 4. Cumulative release of model drug Trypan Blue from Alginate/CaCO3 composite microparticles at hydrogel state (a1, a2) or after lyophilization (b1, b2) at pH 6.4 (a1, b1) or 7.4 (a2, b2). For each sample, three replicates were conducted. Data = mean ± SD. (c) Representative optical microscope images of AD-40-TB microparticles after incubation in PBS (pH 6.4) at 37°C for various time. The image at 0 hr was freeze-dried microparticles. Scale bars are 200 μm.

The drug release behaviors of Alginate/CaCO3 composite microparticles at different pH values and states were studied and Trypan Blue was used as a model drug due to

18


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the simplicity. Since acute wounds, wounds with pus or necrotic tissue and chronic wounds during healing process show an acidic pH in the range of 5.5 - 6.5, while pH for normal tissue is 7.4 35-36, PBS at pH 6.4 and 7.4 were used to simulate wound area and normal tissue microenvironment, respectively. As shown in Fig. 4, regardless of pH values and microparticles states, pure alginate microparticles always showed much faster release rates than others, where in less than 12 hr all entrapped drug was completely released. At hydrogel state, for composite microparticles in PBS at pH 6.4 (Fig. 4a1), after the initial burst release, a sustained release was followed which lasted longer than 96 hr. The drug release rate of A-40-TB group was slower than that for A5-TB. When pH changed to 7.4 (Fig. 4a2), for composite microparticle nearly all drug was released within 10 hr at a slower rate than that for the group A-0-TB. When compared with reported hydrogels with similar composition, where for IBU-CaCO3alginate hybrid hydrogel drug release lasted for 4 hr (pH 7.4, PBS) 37, the release time here was longer probably because of the presence of CaCO3 along with strong interactions between CaCO3 microspheres and alginate network. These factors may also contribute to a much more sustained release from composite microparticles than from pure alginate microparticles at pH 6.4. After lyophilization, microparticles exhibited similar release behaviors but slower release rates than that in hydrogel state. When at pH 6.4 (Fig. 4b1), drug release lasted for 6 days. Furthermore, these composite microparticles always showed a slower release rate at pH 6.4 than at pH 7.4. A significant pH-dependent response was also observed in alginate/CaCO3 hybrid membranes containing poly (urethane-amine). The cumulative release of indomethacin was 81% after 12 hr in PBS at pH 7.4, whereas the value was less than 5% in PBS at pH 2.1 38. Although the structure and compositions of hydrogel systems and pH values were different, the trend was similar, where at lower pH the release rate was lower. As shown in Fig. 4c, after rehydration, the volume of AD-40-TB microparticles gradually increased with incubation time. After 24 hr, diameters were ~400 μm, which was close to the size of as-prepared hydrogel microparticles (Fig. 2c3). The lyophilized microparticles nearly returned to hydrogel state because of water absorption. As for AD-0-TB microparticles, water uptaking was fast; within 30 min, the loaded model drug Trypan Blue was completely released and alginate matrix was dissolved (Fig. S3). When at pH 7.4 (Fig. 4b2), AD-0-TB still had a very fast release rate, while for AD-5TB and AD-40-TB, after the initial burst, sustained drug release lasted more than 2 days. 19



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When cumulative release reached 100%, pure alginate microparticles were broken into segments or dissolved no matter in hydrogel or lyophilized state. For composite microparticles, the morphology was well maintained. As shown in Fig. S4, A-40-TB microparticles were swollen with a diameter of ~800 μm which was nearly twice as that of as-prepared microparticles. After lyophilization, the surface was found to be porous probably due to microparticles swollen and degradation of alginate; the presence of CaCO3 vaterite on surface was still observed. The much higher stability of Alginate/CaCO3 composite microparticles than pure alginate microparticles were probably because of the strong interactions between rough surfaces of CaCO3 microparticles and alginate matrix. In summary, Alginate/CaCO3 composite microparticles demonstrated sustained and pH-responsive drug release profiles. With the increase of CaCO3 concentration, the initial burst release and following release rate was significantly reduced. The presence of CaCO3 microparticles not only hindered the diffusion of drug molecules, but also improved stability of microparticles. Compared with hydrogel microparticles, lyophilized composite microparticles recovered hydrogel state after rehydration which made they were possible to effectively absorb exudate from wounds, and also continuously release internal drug, showing a better application potential for wound healing. 3.4

Antibacterial

property

of

rifamycin-loaded

Alginate/CaCO3

composite

microparticles To fasten wound healing process, besides suppressing bacterial infection at the first place, improving proliferation and migration of fibroblast cells should be followed. According to the in vitro drug release results (Fig. 4), pure alginate microparticles demonstrated a fast drug release rate which were not suitable for encapsulating antibiotic or growth factor, and among composite microparticles, release rate of AD-5TB was the highest and for AD-40-TB it was the lowest. Since release rate was believed to be negligible influenced by drug type for the composite hydrogel system, these two compositions were selected to entrap rifamycin and bFGF independently to obtain a fast release of rifamycin and a sustained release of bFGF. Dual-drug carrier composite

20


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sample CD-F-R was prepared and the efficacy of these microparticles on bacterial inhibition and cell proliferation was investigated.

Fig. 5. Evaluation of sustained antibacterial activity of AD-5-R and CD-F-R microparticles using disk diffusion method. The diameter of inhibition zone is marked on the image and the scale bar is 20 mm.

S. aureus is the main bacterial species of wound infection 39. To evaluate antibacterial properties of composite microparticles, inhibition zone was quantified using a disk diffusion method. For AD-5-R, the added amount of rifamycin was 160 μg for each group and the loading efficiency was believed to be close to 63.6 ± 1.8% (Table 1). Thus, rifamycin in each group was calculated to be 101.8 μg. After applying AD-5-R and CD-F-R microparticles onto the agar plate for 24 hr, diameters of inhibition zones were nearly twice as that for filter paper with microparticles (diameter of 9 mm), showing that the antibacterial effects were strong. Every 24 hr, the filter paper with antibiotic-loaded microparticles were transferred to a new agar plate, and inhibition zones with gradually decreasing diameters were observed, demonstrating that the bacteriostatic effect lasted for 5 days as shown in Fig. 5. Only filter paper and blank microparticles did not possess any antibacterial properties (Fig. S5). The group AD-5R and CD-F-R showed similar antibacterial properties. These observations were consistent with the in vitro drug release results (Fig. 4). In the first two days, the inhibition zones were relatively large and the encapsulated drug was released rapidly from composite microparticles containing CaCO3 at 5 mg/ml. After the second day, the antibacterial effect gradually decreased, which was consistent with the slower release rate (Fig. 4b2).

21



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3.5 In vitro cell proliferation behaviors after applied Alginate/CaCO3 composite microparticles Wound dressings should not be cytotoxic or negatively affect cellular growth. In vitro cell test is usually first conducted for preliminary studies

40.

The presence of a high

concentration of CaCO3 in alginate composite microparticles may have a negative effect on cells and sample A-40, the one with the highest concentration of CaCO3 was selected as the representative. After 72 hr co-culturing with different qualities of A-40, relative cell viabilities for A-40 group even at a high concentration of 50 mg/ml were close to the value for TCP group, while the relative cell viability of the positive group triton X-100 was 16.4% (Fig. 6a1). Fig. 6a2 showed that cells grew very well around the microparticle in the co-culture system, showing no contact cytotoxicity. The results reflected

that

alginate

hydrogel

composite

microparticles

have

a

good

cytocompatibility, which may be related to the good biocompatibility of sodium alginate and vaterite 41-42. To investigate the effects of bFGF-loaded Alginate/CaCO3 composite microparticles on cell proliferation and migration behaviors, in vitro wound healing model was first developed based on cell micropatterns on glass cover slips with hole array. The glass cover slip with laser ablated hole array was shown in Fig. S6. The rounded edges of holes were rough and uneven, which well mimicked not only irregular shapes of wound tissues but also cell migration paths during healing process. After NIH-3T3 cells seeded glass cover slips transferred to 24-well plates and stained with Calcein-AM, it was found that living cells fully occupied the glass slip, cells were also adhered at the edges of holes and no cells were present on TCP (Fig. 6b1-b2). To mimic wound microenvironment, medium at pH 6.4 was added after co-culturing with microparticles. As shown in Fig. 6c, for all groups cells migrated onto the empty circular area on TCP, and different ingrowth rates were exhibited due to the influence of rifamycin and bFGF released from microparticles. At each time point, the proliferation and migration rates for AD-5-R were the lowest while for AD-40-F the rates were the fastest. On day 3, empty circle areas with small amounts of cells were observed for all groups while on day 7, except AD-5-R group, empty areas on TCP were nearly full of cells. The CD-F-R group exhibited a similar proliferative tendency to the AD-40-F group. 22


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Fig. 6. In vitro performance of Alginate/CaCO3 composite microparticles on cell proliferation and migration. (a1) Relative cell viabilities of NIH-3T3 after co-culturing with different concentrations of A-40 microparticles for 72 hr in medium at pH 7.4. Values were normalized against the TCP group. (a2) Light microscope image of cells growing under composite microparticles for 72 hr. (b1) Fluorescence microscope image of cells seeded cover slip just after transferred into a new 24-well plate which was developed to mimic wound healing process; and (b2) the enlarged image showed the adhesion of cells on the edge of hole on cover slip. (c) Monitoring cell proliferation and migration using the glass cover slip based wound healing model to assess the impact of different samples AD-5-R, AD-40-F and CD-F-R. After adding microparticles, medium at pH 7.4 was replaced by medium at 6.4 to mimic wound microenvironment. At predetermined time points, light microscope images of cells just beneath microparticles were taken. The control group was to culture cells in medium without any microparticles. (d) Monitoring proliferation of NIH-3T3 cells in the model after co-culturing with different microparticles using CCK-8 assay. The relative absorbance values on D 0 represented the initial cell viabilities on patterned glass cover slips which were just transferred into 24-well plates before replacing medium with medium at pH 6.4 and including microparticles. The measured absorbance values at each time point (D 1-7) were normalized with the

23



corresponding value on day 0. CLSM images (e1-e4) of cells after migrating from glass cover slip onto TCP and cytoskeleton staining for actin filaments (red) and nucleus (blue). During proliferation and migration, cells aligned along the circumference of circle area on TCP (e1) and eventually occupied the entire circle area on TCP (e2) with a growth depth of ~120 μm (e3e4). Data = mean ± SD; n = 3. * indicates p < 0.05 when compared with the Control group on the same day.

Relative cell viabilities were further monitored using CCK-8 assay. Before replacing medium at pH 7.4 with medium at pH 6.4, the initial cell viabilities were first measured (marked as D 0). After one day culture in medium at pH 6.4, the relative cell viability was significantly reduced, which may be due to the fact that appropriate pH values for fibroblasts were between 7.1 to 7.6 while medium at pH 6.4 that mimicked the wound area resulted in a certain reduction in fibroblast viabilities 43. From day 1 to 7, relative absorbance values gradually increased with culturing time, showing that cells gradually proliferated for all groups. At each time point, the relative absorbance value for AD40-F group was the highest due to the release of bFGF and the value for AD-5-R group was comparable with that for the control group even though slower proliferation and migration rates were observed as shown in Fig. 6c. The proliferation trend was well supported by the cumulative release profile as shown in Fig. 4b1 that model drug was sustainable released from AD-40-TB for 6 days. As for CD-F-R group, the relative cell viabilities were always slightly smaller than that for AD-40-F group within 7 days. The observation may be due to possible negative effects resulted from rifamycin and a high concentration of CaCO3 on fibroblasts which partially counteracted the enhancing effect of released bFGF on cell proliferation. Even so, CD-40-F group still had higher relative cell viabilities than that for TCP and AD-5-R groups in the first 5 days. In in vitro would healing model, cells proliferated and migrated from glass cover slips onto TCP. As shown in Fig. 6e1, cells were oriented along the circumference of empty circle on TCP during the process and cells eventually fully filled up the empty surface on TCP (Fig. 6e2) with a migration depth of ~120 μm (Fig. 6e3-e4). This in vitro model effectively mimicked cell proliferation and migration behaviors during wound healing processes, which was very different from other frequently used models. A linear thin scratch “wound” (creating a gap) was usually created (i.e., using a 200 μL pipette tip 40)

in a confluent cell monolayer and subsequently images of cell filling the gap were

captured at regular time intervals for analysis of cell migration. When compared with the model here, even though those scratch models were simpler and easier to be created,

24


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all cells were on the same surface and may not well mimic in vivo cell migration during healing process. This model based on cell micropatterns on glass cover slips with hole array was very suitable to evaluate the efficacy of composite microparticles on wound healing. In summary, the release of bFGF from AD-40-F group promoted cell growth and migration; while rifamycin from composite microparticles showed a negligible inhibitory effect on cell proliferation since the release duration was short. Hence, for CD-F-R microparticles, both bioactive molecules were released independently and both antibacterial and promoting cell proliferation effects were obtained. 3.6 In vivo effects of CD-F-R microparticles on wound healing

Fig. 7 In vivo performance of CD-F-R composite microparticles on wound healing. (a) Representative digital images of wound at predetermined time points for Control and CD-F-R group from the same SD rat. (b) Traces of wound-bed closure during 21 days. (c) Wound contraction for each group, n = 3, data = mean ± SD.

A full-thickness skin wound model was used to evaluate the effects of CD-F-R 25



microparticles on healing process. As shown in Fig. 7a-b, for both groups wound area gradually decreased with healing time. Hemostasis is the first phase of wound healing which occurs upon injury

44-45

. After applied CD-F-R microparticles onto wound

surface, effective hemostasis was observed when compared with Control group and these microparticles also absorbed wound exudate (Fig. 7a); after 3 days, such difference was more notable. This was similar to the reported results that a wound dressing adhered onto the surrounding tissue and served as a bleeding-arrest agent 44-45. On day 7, with the release of bFGF, CD-F-R group had a smaller wound area than Control group. On day 14, the difference was more pronounced and after 21 days, CDF-R group nearly healed completely, while for Control group the wound area was still ~10% of the initial size. When comparing wound contraction between these two groups (Fig. 7c), CD-F-R groups always had higher values than Control group. This was consistent with the in vitro result that proliferation and migration of fibroblast cells were fastened by CD-F-R microparticles (Fig. 6). All these observations demonstrated that CD-F-R microparticles accelerated wound healing, which was probably attributed to the combined effects of hemostatic performance, moist wound microenvironment 46 and release of encapsulated antibiotic and growth factor. Wound healing involves several stages: hemostasis, inflammatory response, cell migration and proliferation, and remodeling

47.

After gross analysis, wound cross

sections were investigated using H&E staining, Masson's trichrome staining, and immunohistochemical staining of CD31. As shown in Fig. 8, newly formed tissues of weaker color intensity than native tissue were observed in panoramic images of H&E and Masson's trichrome staining. For CD-F-R group, on day 7, granulation tissue filled around microparticles (spherical cavity in the image) including fibroblast cells and a few inflammatory cells. On the 14th day, neo-epidermis layer was formed and thick dermis layer was also observed which grew over microparticles; higher collagen expression was observed in area close to microparticles. The encapsulated bFGF was slowly released, which was a potent mitogen for fibroblasts and collagen in dermis was mainly secreted by fibroblast 48. Hence, more collagen was deposited. After 21 days, the wound was nearly completely healed. The newly formed skin was smooth; the epidermis and dermis structures were evident with plenty of collagen expression. Microparticles were more deeply embedded under dermis layer than that on day 14. As for Control group, until day 21, there was still wound area without formation of neo26


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epidermis layer.

Fig. 8 H&E and Masson's trichrome staining of wound section for Control and CD-F-R groups on day 7, day 14 and day 21.

The thickness of granulation tissue for CD-F-R and Control groups were further compared (Fig. 9a). On day 7, the value of CD-F-R group was nearly as twice as that for Control group. After 14 days, neo-epidermis layer already formed in CD-F-R group and the thickness correspondingly decreased; for Control group, granulation tissue continued to form and the thickness also increased. These results confirmed the observation as shown in Fig. 7 that CD-F-R microparticles fastened wound healing process. Based on the immunohistochemical staining of CD31 (Fig. 9c), vascular density was compared between these two groups (Fig. 9b). On day 7, the density for CD-F-R group was higher than that for Control group. The sustained release of bFGF which was a potent mitogen and chemoattractant for endothelial cells was beneficial for the formation of blood vessels. This was similar to the reported result that bFGF promoted the growth of new blood vessels during wound healing 49. On the 14th day,

27



the density decreased slightly for CD-F-R group while for Control group, the value still increased. The trend was probably due to the fact that vascularization mainly occurred in the early stage of wound healing along with formation of granulation tissue. On day 14, for CD-F-R group, neo-epidermis layer already formed and many blood vessels were perpendicular to skin surface. As for Control group, few of blood vessels were in such alignment on both day 7 and 14, consistent with the observation that granulation tissue was still present on day 14 (Fig. 8).

Fig. 9 Effect of CD-F-R microparticles on angiogenesis during the early stage of wound healing process. (a) Granulation tissue thickness and (b) vessel density on day 7 and 14 for CD-F-R and Control groups. (c) Immunohistochemical staining of CD31 for both groups on day 7 and 14. Scale bars are 100 μm.

The enhancing effect of CD-F-R microparticles on wound healing may be attributed to several reasons. Lyophilized Alginate/CaCO3 composite microparticles reverted to hydrogel state after rehydration. When applied onto wound surface, CD-F-R microparticles may offer a moist microenvironment after hemostasis. These microparticles then also functioned as scaffold materials to facilitate cell adhesion and migration. Because of the sustained release of bFGF, angiogenesis and collagen expression was enhanced. Thus, CD-F-R microparticles promoted granulation, vascularization and epithelialization, which are important steps during wound healing

28


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49.

Fig. 10 Investigation of biocompatibility and biodegradability of CD-F-R microparticles during wound healing process.

Biomaterial scaffolds usually acted as extracellular matrix, providing growth space and mechanical support for new tissues 50. The controlled degradability of a biomaterial was key during tissue regeneration since degraded material made room for the growth of new tissues 51-52. After applied onto wound-bed, CD-F-R microparticles were probably swollen based on the spherical cavity size as shown in Fig. 8. On day 7, slight acute inflammatory response was noted mainly around microparticles as evidenced by neutrophils according to H&E and CD31 staining 29

53

(Fig. 10). From day 14 to 21,



Page 30 of 35

neutrophils were still observed on microparticle surface; the thickness of new tissue around microparticles gradually increased, indicating that microparticles slowly degraded along with the growth of new tissue. On day 21, the newly formed tissue had a prominent thickness with cells embedding in tissue layer. In addition, cells were oriented around these microparticles. From these observation, it can be concluded that CD-F-R microparticles demonstrated a good biocompatibility similar to other reported results. Thrombin-loaded alginate-calcium microspheres were fabricated for transcatheter arterial embolization, which were of 350 μm and from 3% alginate solution. After implantation, even though these microspheres demonstrated a slight and temporary inflammation, they were non-irritating and non-genotoxic

54.

After

subcutaneous implantation, thrombin-loaded alginate-calcium microspheres degraded with time, where after 4 weeks microsphere size was ~60 μm smaller than that for asprepared and no residual was identified on week 10 55. Sodium alginate solution (1.5%) containing CaCO3 microspheres (ranging from 3 to 10 μm) was injected into groin subcutaneous to investigate the degradability and biocompatibility. After 12 weeks the diameter of samples decreased from 6 to 2 mm. Newly formed tissues invaded into remaining samples and very few inflammatory cells were observed

53.

Our research

showed similar degradation results. On day 21, residues of CD-F-R microparticles were still present, probably because of the much shorter observation time than other reported results; it was believed these microparticles were eventually absorbed and replaced by newly formed tissue. All these results showed that CD-F-R microparticles may ideally meet requirements of different stages during wound healing, absorbing wound exudates first, rapidly releasing antibiotic, then following a slow and steady release of bFGF to achieve hemostasis, antibacterial, and promoting granulation, vascularization and reepithelialization to fasten the healing process. These microparticles degraded with time, showing a good biocompatibility and biodegradability. 4. Conclusions Alginate/CaCO3 composite microparticles were successfully developed using a microfluidic method. With the encapsulation of CaCO3 microspheres, the 3D structure of lyophilized composite microparticles was better maintained, and the initial burst release was effectively reduced as well as a longer release time associate with pH 30


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sensitivity. The application of composite microparticles in wound healing was then explored. In vitro experiments demonstrated the mixed microparticles CD-F-R did not only facilitated a fast release of rifamycin to inhibit S. aureus growth but also obtained a sustained release of bFGF to promote cell proliferation and migration. In vivo animal tests proved that in addition to drug carriers, these microparticles also functioned as scaffold materials to enhance the formation of granulation tissue along with angiogenesis, epithelium regeneration and collagen deposition, promoting wound healing eventually. These microparticles demonstrated a good biocompatibility and biodegradability. In future, sodium alginate may be modified so as to accelerate the degradation rate in vivo. Supporting information Supporting Information is available online or from the author. Summary of main fabrication parameters, characteristics and tests conducted for all samples; SEM images of the surface of alginate microparticles prepared by adding surfactant Span 80 into the continuous phase DCM; SEM image of CaCO3 vaterite microparticles; Light microscope images of AD-0-TB microparticles after incubation in PBS (pH 6.4, 37°C) for various time; Light microscope image and SEM image of A-40-TB microparticles after cumulative release reaching 100%; Digital image of agar plate after incubating with control groups for 24 hr. Acknowledgements This work was supported in part by Guangdong Natural Science Foundation (2018A030313885), National Key R&D Program of China (2017YFE0102400), and the Australia-China Joint Institute for Health Technology and Innovation. References [1] Brem, H.; Stojadinovic, O.; Diegelmann, R. F.; Entero, H.; Lee, B.; Pastar, I.; Golinko, M.; Rosenberg, H.; Tomic-Canic, M. Molecular markers in patients with chronic wounds to guide surgical debridement. Molecular Medicine 2007, 13 (1-2), 30-39. [2] Xu, R.; Luo, G. X.; Xia, H. S.; He, W. F.; Zhao, J.; Liu, B.; Tan, J. L.; Zhou, J. Y.; Liu, D. S.; Wang, Y. Z.; Yao, Z. H.; Zhan, R. X.; Yang, S. S.; Wu, J. Novel bilayer wound dressing composed of silicone rubber with particular micropores enhanced wound re-epithelialization and contraction. Biomaterials 2015, 40, 111. [3] Anumolu, S. S.; Menjoge, A. R.; Deshmukh, M.; Gerecke, D.; Stein, S.; Laskin, J.; Sinko, P. J. Doxycycline hydrogels with reversible disulfide crosslinks for dermal wound healing of mustard injuries. Biomaterials 2011, 32 (4), 1204-1217. [4] Tavares de Figueiredo, I. S.; Ramos, M. V.; Pontes Silva Ricardo, N. M.; da Costa Gonzaga, M. L.; Paiva

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Sustainable Dual Release of Antibiotic and Growth Factor from pH

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