Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent

Journal Pre-proof Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent

Guobao Chen, Runan Wei, Xiang Huang, Fuping Wang, Zhongmin Chen PII:

S0141-8130(19)36999-5

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.122

Reference:

BIOMAC 13903

To appear in:

International Journal of Biological Macromolecules

Received date:

30 August 2019

Revised date:

4 November 2019

Accepted date:

13 November 2019

Please cite this article as: G. Chen, R. Wei, X. Huang, et al., Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.122

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© 2019 Published by Elsevier.

Journal Pre-proof

Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent

School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing

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Guobao Chen a, Runan Wei a, Xiang Huang a, Fuping Wang a, Zhongmin Chen a,*

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400054, P. R. China

Mr. Guobao Chen, E-mail: [email protected]

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Ms. Runan Wei, E-mail: [email protected]

Ms. Xiang Huang, E-mail: [email protected]

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Mr. Fuping Wang, E-mail: [email protected]

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Ms. Zhongmin Chen, E-mail: [email protected]

*Corresponding author:

Dr. Zhongmin Chen, Professor

School of Pharmacy and Biological Engineering, Chongqing University of Technology No. 69 Hongguang Avenue, Banan District, Chongqing 400054, China E-mail: [email protected]

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Journal Pre-proof ABSTRACT Transcatheter arterial chemoembolization (TACE) is well known as an effective treatment for hepatocellular carcinoma (HCC). In the present study, a novel embolic agent of sodium alginate (SA)-modified silk fibroin (SF) microspheres was successfully prepared by emulsifying cross-linking method. The SA-modified SF microspheres were evaluated for the ability of embolization by investigating the morphology, particle size, swelling ratio, degradation, cytotoxicity, blood compatibility, and in vivo embolization. The results found

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that SA-modified SF microspheres had smooth surfaces and good sphericity. Swelling ratio

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of the microspheres can meet the requirements of arterial embolic agent and have pH and

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temperature sensitivity. Furthermore, hemolytic and anticoagulant studies have proved that

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the microspheres have good blood compatibility. Cytotoxicity tests indicated that the microspheres could promote the proliferation of fibroblasts and HUVEC. In vivo

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embolization evaluation of microspheres revealed that the arteries could be embolized by SA-

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modified SF microspheres in 3 weeks. The ability of drug loading and releasing of microspheres was proved by the controlled release profile of Adriamycin hydrochloride. The

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findings indicated that the SA-modified SF microspheres can be used as a potentially biodegradable arterial embolic agent for liver cancer therapy. Keywords: Silk fibroin, Sodium alginate, Microspheres, Transcatheter arterial embolization, Adriamycin

1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant cancer with high morbidity and mortality rates [1]. Although surgery is the most effective method for the treatment of HCC at present, most HCC is diagnosed in the intermediate and advanced stages and is not suitable for surgical treatment [2, 3]. Transcatheter arterial embolization (TAE) or

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Journal Pre-proof transcatheter arterial chemoembolization (TACE) is used for some patients with HCC that cannot be treated by surgical resection. TAE is a typical noninvasive vascular interventional therapy for HCC by introducing embolic agents into the blood vessel through a catheter to cut off the nutrition and oxygen supply of tumor by blocking the blood flow [4]. The success of TAE depends heavily on the design of embolic agent, which is the key step in the development of TAE. The ideal embolic agent should have the following characteristics: (1) non-toxic or low-toxic, non-antigenic, excellent biocompatibility, and

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biodegradation; (2) size of embolic agent can adapt to the blood vessel diameter of the target

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site and quickly block the blood supply; (3) raw material source is wide, easy to handle, and

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can pass through the catheter smoothly; (4) has X-ray visibility; (5) potential impair the

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angiogenesis [5]. At present, a number of types of embolic agents have been developed, which can be divided into liquid embolic materials, solid embolic agents and mechanical

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embolic agents according to their physical properties; biodegradable and non-degradable

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mechanical embolic agents by their degradability [6]. Among them, calibrated microspheres are often preferred due to their controllable size distribution and spherical shape, which may

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improve embolization outcomes [7, 8]. In addition to the direction embolic effect, the volume of biodegradable microsphere can be reduced in size to enter a thinner blood vessel for further embolization after injection. At the same time, after the microsphere is degraded, the blood vessels can be recanalized, which is beneficial to the treatment of secondary embolization. Furthermore, the microsphere degrades under the action of enzymes in the body to release the chemotherapeutic drugs, and functions as a drug pump. At present, biodegradable microspheres for embolization include starch microspheres [9], gelatin microspheres [10], chitosan [11] and their derivative microspheres [12], etc. The non-biodegradable microspheres include poly (vinyl alcohol) (PVA) microspheres [13, 14], superabsorbent polymer (SAP) microsphere [15], poly (acrylic acid) (PAA) microspheres

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Journal Pre-proof [16], etc. Some of these microspheres have been commercialized and widely used in clinical practice, but all kinds of microspheres have strengths and weaknesses. For example, PVA microspheres used as non-biodegradable materials are regarded as permanent embolic agents, but PVA particles with irregular shapes cannot be calibrated and tend to aggregate, which cause catheter obstruction and large vessel occlusion [17, 18]. Furthermore, nonbiodegradable embolic microspheres should always be considered for removal from the body after use. Of course, attention should also be paid to the deficiencies and limitations in the use

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of biodegradable embolic microspheres. For instance, a starch based commercial

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biodegradable microspheres (Embocept®) were only suitable for vessel embolization for a

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short time (less than 1 h) due to size limitation (less than 100 μm) [19]. In addition, some

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degradation products of degraded microspheres may also cause an inflammatory reaction [20]. Therefore, the development of the ideal new embolic microspheres is still the focus and

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hot spot of TAE research, such as with the appropriate size, controllable degradation and drug

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release profile, good biocompatibility, blood compatibility, non-aggregate and so on. Silk fibroin (SF) is a natural polymer degummed from silk. Due to its good

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biocompatibility and biodegradability, good flexibility and tensile strength, as well as its strong plasticity, it has received much attention in the field of biomedical materials research [21, 22]. In addition, due to its versatile processability, SF solutions can be prepared into various morphologies of biomaterials, such as films, fibers, hydrogels, mats, microparticles [23]. These various biomaterials based on SF are also widely used in the repair of damage of tissues and organs such as skin [24], bone [25], cartilage and meniscus [26], blood vessels [27]. Ratanavaraporn and colleagues [28] prepared gelatin-SF microspheres encapsulating curcumin by emulsification cross-linking method. The results showed that the microspheres hardly degraded on the 14th day. Regenerated SF materials have shown poor mechanical properties, slow degradation, and poor hydrophilicity [29]. Although SF microsphere has

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Journal Pre-proof good globularity, the particle size is small and the swelling ratio is low, which is not suitable for artery embolic agent alone. Sodium alginate (SA) is a polysaccharide extracted from algae or kelp, with good biocompatibility, biodegradability, certain pH sensitivity, good hydration, as well as good particle size and swelling ratio [30, 31]. However, pure SA is poorly spherical, which is not conducive to the application of embolism. Therefore, the SF can be modified by SA to prepare a modified SF artery embolic microsphere with suitable particle size, spherical shape, and swelling ratio.

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In this study, the emulsified cross-linking method was used to modify SF with SA to

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prepare modified SF artery embolic microspheres. The structure and composition of the

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modified SF microspheres were characterized by inverted optical microscope, scanning

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electron microscope (SEM), infrared spectroscopy and laser particle size analyzer. The swelling ratio, in vitro degradability, pH sensitivity, and temperature sensitivity were also

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evaluated. In addition, L929 mouse fibroblasts and human umbilical vein endothelial cells

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(HUVEC) were used to evaluate the cytocompatibility of the modified SF microspheres. The blood compatibility was evaluated by hemolytic test and dynamic coagulation test. New

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Zealand white rabbit was selected as an animal model to carry out embolic experiment on the arteries in its ears. Finally, Adriamycin hydrochloride-loaded SF microspheres were prepared, and its drug loading, encapsulation efficiency, and in vitro release profile were determined.

2. Materials and methods 2.1. Materials Silk cocoon was bought from Chongqing Chemical Fiber Institute (Chongqing, China). Calcium chloride, SA, isopropanol, glutaraldehyde, span 80, and liquid paraffin were purchased from Chengdu Cologne Chemicals Co., Ltd. (Chengdu, China). Adriamycin

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Journal Pre-proof hydrochloride was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Dialysis bags (molecular weight cut offs: 8 kD-14 kD) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

2.2. Preparation of SF solution Wash the silkworm cocoons with tap water and then boil for 3 hours with 0.4% sodium carbonate solution. After that, rinse it with boiling water three times and dry the sample at 37

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°C. Prepare the CaCl2/C2H5OH/H2O solution (in the molar ratio of 1:2:8, that is 55.5 g CaCl2,

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60 mL C2H5OH, 72 mL deionized water of per 10 g cocoon), put the dried cocoon into the

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solution ,and then heat it in 80 °C water bath for 30 min until the SF is completely dissolved.

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The sample was filtered with a slow filter paper to obtain a clear solution free of impurities. The filtered solution was placed in a dialysis bag having a molecular weight cutoff of 8 kD to

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14 kD, dialyzed with deionized water for three days, and water was changed every morning

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and evening. Finally, the dialysis solution is concentrated to obtain a 2% SF solution.

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2.3. Preparation of pure SF microspheres and pure SA microspheres Pure SF microspheres were prepared by following procedure. 20 mL of liquid paraffin and 1 mL of span 80 were mixed and stirred (400 r/min) for 40 min in a 26 °C water bath to form a homogeneous emulsion. 2 mL of 16% SF solution was added to continue stirring (300 r/min) for 1 h, and 0.6 mL of glutaraldehyde was added to continue the stirring. After stirring (300 r/min) for 3 h, the supernatant was decanted, centrifuged, washed with isopropyl alcohol 3 times, after vacuum drying dried to obtain a light-yellow powder. The method of making pure SA microspheres is as follows. Mix 30 mL of liquid paraffin and 1 mL of span 80, then stirred (500 r/min) in a 40 °C water bath for 20 min to form a homogeneous emulsion, add 30 mL of 2% SA solution and continue stirring (500 r/min) for

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Journal Pre-proof 30 min to form a uniform water in oil emulsion. The emulsion was added to 5 mL of 3% calcium chloride (CaCl2) solution and stirred (300 r/min) for 30 min. The supernatant was centrifuged and dried under vacuum to obtain a white powder.

2.4. Fabrication of SA-modified SF microspheres SA-modified SF microspheres were prepared using water-in-oil emulsion technique (Fig. 1). Briefly, SA was dissolved in deionized water to obtain 2% (w/v) SA solution. Similarly,

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the 2% (w/v) SF solution was well prepared in a beaker, and then 0.75% (w/v) CaCl2 was

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added to formulate the SF-CaCl2 solution (pH 3.5). Meanwhile, 50 mL liquid paraffin

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solution containing 4% (v/v) span 80 was stirred for 30 min with electric mixer at 400 rpm in

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a 35 ℃ water bath to obtain a homogeneous emulsion that served as the oil phase followed by the addition of 30 mL SA solution and 20 mL SF-CaCl2 solution. Finally, adding 1 mL

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glutaraldehyde which was used as emulsifying crosslinker. The mixture was stirred for 3 h at

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400 rpm in a 35 ℃ water bath. Then leaved it at room temperature overnight. The next day, the supernatant was discarded through centrifugation at 4000 rpm for 15 min, washed the

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sediment with isopropyl alcohol, after three rounds, dried the sediment under vacuum resulting in yellow microspheres.

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Fig. 1. Schematic illustration of the preparation process of SA-modified SF microspheres for arterial

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embolization.

2.5.1. SEM

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2.5. Characterization of microspheres

The surface morphology of microspheres was observed via SEM (LEO-supra35, ZEISS, Germany). The sample should be evenly spread on the platform and sputter-coated with gold before observation.

2.5.2. Fourier transform infrared spectrometer (FTIR) Pure SA, SF microspheres and SA-SF microspheres were regarded as experimental groups which were analyzed using FTIR (spectrum 100, Perkin Elmer) with the wave number ranged of 4000-600 cm-1.

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2.5.3. Particle size distribution Mixing defined number of microspheres with PBS (pH 7.4) in a shaker at 100 rpm in the 37 ℃ till the sample reaching swelling equilibrium. Taking out 50 mL suspension into the sample pool. The average particle size of microspheres was measured accurately by laser diffraction particle size anaylzer (Bettersize 2000, China).

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2.5.4. Swelling ratio

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Swelling ratio of SA-modified SF microspheres was measured by calculating the

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diameter changes before and after the swelling experiment. In order to evaluate the effect of

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pH and temperature on swelling ratio of microspheres, the samples were immersed in PBS with different pH (1.0, 5.8, and 7.4) at 37 ℃ or immersed in PBS with different temperatures

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(4 °C, 37 °C, and 60 °C) at pH 7.4 with rotational speed of 100 rpm. The swelling

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microspheres were taken out at certain time points to measure their diameter. The swelling ratio of the microspheres was calculated according to the equation:

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Swelling ratio% = (Dt-D0)/D0 × 100%

where D0 is the initial diameter of microspheres and Dt is the diameter of microspheres after swelling.

2.5.5. In vitro hemolysis assay and coagulation test The hemolytic activity of the SA-modified SF microspheres was determined as follows. Prior to testing, 50 mg microspheres and 100 mg microspheres were soaked separately into 10 mL of PBS and fully dissolved. Positive and negative controls were also prepared by using deionized water and saline, respectively. Every group was added with 250 µL of diluted anticoagulant solution (3.8% sodium citrate: blood: 2% potassium oxalate: 0.9% sodium

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Journal Pre-proof chloride is equal to 0.1: 0.9: 0.05: 1.5) and kept at 37 °C for 1 h. The samples were centrifuged at 1000 rpm for 5 min. 200 µL of the supernatant was coated in a 96-well plate, furthermore measured its absorbance at 540 nm using an enzymatic marker (Multiskan GO Thermo Scientific, USA) and the hemolysis rate was calculated according to the following equation: Hemolysis rate% = (Ni-Na)/(Nb-Na) where Ni, Na, and Nb respectively represented the absorbance values of the experimental

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group, the negative control group and the positive control group.

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For clotting time measurement, the microspheres were swelling completely in saline and

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the surface moisture was absorbed by a filter paper. 0.5 g of swelling microspheres were

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weighed into a beaker as an experimental group, and no microsphere was added as a blank control group, and placed in a constant temperature water bath shaker at 37 ° C for 5 min.

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Then dropped the diluted anticoagulant solution (250 μL) onto the surface of these

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microspheres, followed by adding 20 μL CaCl2 solution (0.02 mol/L). After 10 min, 20 min, 30 min, 40 min, and 50 min, 100 mL of distilled water was added to stop the coagulation

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reaction, and then kept at 37 °C for 10 min in a constant temperature water bath shaker. 200 μL of the supernatant was taken from each group and added to a 96-well plate. The OD value was measured at a wavelength of 540 nm using a microplate reader, and the blood clotting index (BCI) was calculated according to the following equation: BCI = (Nt/N0) × 100% where Nt and N0 represent absorbance values of experimental group and control group.

2.5.6. Degradation in vitro The degradation of microspheres in vitro was tested by following procedure. 750 mg of dried microspheres were equally divided into 15 centrifuge tubes and the weight of each tube

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Journal Pre-proof was recorded as M0, followed by the addition of 10 mL PBS (pH 7.4). The microspheres are separated from solution after fully swelled, then 4 mL of 50 μg/mL the trypsin solution was added into the tubes. Shaking all tubes in a constant temperature oscillator at 37 °C and 50% of the solution was replaced every two days to maintain the activity of trypsin. The microspheres were taken out at specific time (2 days, 5 days, 7 days, 14 days, 21 days), washed with deionized water, dehydrated with ethanol and finally dried in vacuum. Recording the whole weight combining the biodegraded microspheres and centrifuge tubes as

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Mt. Degradation ratio was calculated according to the following equation:

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Degradation ratio%= (M0-Mt)/M0 × 100%

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where M0 is the initial weight of microspheres and Mt is the weight of microspheres after

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2.6. In vitro cytotoxicity assay

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degradation.

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The cytotoxicity of SA-modified SF microspheres with different concentrations on L929 mouse fibroblasts and HUVEC was evaluated by using the CCK-8 assays kit (Biosharp,

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Hefei, China) according to the manufacturer's protocols. Mixing sterilized microspheres with complete medium resulting in gradient concentration (1000 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL) of microspheres extract solution, then filtered that with a 0.22 μm filter membrane and stored at 4 °C. Adding 100 μL of 3ⅹ104/mL HUWEC and L929 fibroblasts suspensions to the 96-well plate followed by incubated at 37 °C till all the cells became adherent, then 100 μL microsphere leaching solution above was added. Added 10 μL CCK-8 solution per well every day and then cultured for 2 h before measured its 450 nm absorbance.

2.7. In vitro release from microspheres

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Journal Pre-proof Dynamic dialysis method was used to simulate the drug release process. In brief, 20 mg Adriamycin hydrochloride-loaded microspheres and 1 mL PBS (pH 7.4) solution were added to the dialysis bag (3000 kD), then transferred into a brown wide-mouth flask containing 50 mL PBS before oscillating at 37 °C. At different time points, 3 mL of dialysis bag external buffer was taken from the bottle in a quartz dish, and its absorbance value at wavelength 481 nm was measured by a UV spectrophotometer. The amount of drug released from the

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microspheres was calculated from the standard curve.

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2.8. In vivo vessel embolization

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New Zealand white rabbits with an average weight of 2.0 ± 0.3 kg were provided by the

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Animal Experimental Center of Chongqing Medical University. The Institutional Animal Care and Use Committee approved all of the animal experimental protocols. The experiment

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started with ear vein anesthesia of rabbit by 2% sodium pentobarbital (30 mg/kg). Then

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clamping the marginal vein and artery aimed at filling the rabbit ear blood vessels to facilitate the insertion of a puncturing needle directly into the artery. Subsequently 0.3 mL glycerol and

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microsphere suspension (20 mg/mL) mixture were slowly injected into the central artery of rabbit ear with syringe. Removing the arterial clip, the edema and thrombosis were observed at different days after surgery.

2.9. Statistical analysis Each experiment was performed at least three times. All data were presented as the mean ± standard deviation (SD). Statistically significant differences between two groups were determined by a Student’s t-test. p < 0.05 and p < 0.01 indicate significant difference and extremely significant difference, respectively.

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Journal Pre-proof 3. Results and discussion 3.1. Morphology of pure SF microspheres and pure SA microspheres As shown in Fig. S1, the pure SF microspheres have good sphericity (Fig. S1A) and pure SA microspheres have poor sphericity (Fig. S1B). The SF microspheres have a small particle size and a small swelling ratio, which is not suitable for hepatic artery embolization alone, and the particle size and swelling ratio of the SA microspheres are suitable for hepatic artery embolization (Tab. S1). Therefore, neither of these microspheres can be used as an embolic

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microsphere for hepatic artery embolization alone. The SF can be modified by SA to improve

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its performance, and the particle size and swelling ratio are suitable to form a spherical shape,

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non-adhesive phenomenon of modified SF arterial embolization microspheres.

3.2 Characterization of SA-modified SF microspheres

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3.2.1. Morphology of SA-modified SF microspheres

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Fig. 2A shows the macroscopic morphology of dry SA-modified SF microspheres under light microscopy. It can be seen that the microspheres have good sphericity. Fig. 2B is the

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macroscopic morphology of the microspheres after swelling, which shows that the surface of the microsphere is smooth, the liquidity is good, and there is no sticking phenomenon. The micrographs of the external surface of the SA-modified SF microspheres are presented in Fig. 2C-2F. In the present work, the SF and calcium chloride solutions were adjusted to pH 3.5 by dilute hydrochloric acid. Previous study has found that lower pH can catalyze the formation of gel microspheres and enhance physical cross-linking [32]. A chemical crosslinker, glutaraldehyde, is then added which reacts with the carboxyl and hydroxyl groups of the SA. The stable three-dimensional structure is formed by acetal reaction [33]. SF binds to the electrons of the outer layer in the form of hydrogen bonds and ionic bonds, and adheres to the surface of the microspheres, making the entire microsphere structure more stable. At low

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Journal Pre-proof magnification of the microspheres presented spherical shape and almost no sticking phenomenon (Fig. 2C-2D). At high magnification of the microspheres had rough and porous surfaces (Fig. 2E-2F). These porous structures appearing on the surface of the microspheres may be caused by a freeze-drying process and facilitate the drug loading or release of the microspheres. In previous studies, SF solutions with various nanostructures were prepared to

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regulate scaffold formation in freeze-drying treatments [34].

Fig. 2. Morphology of SA-modified SF microspheres. Dry (A) and wet (B) SA-modified microspheres under optical microscope. Surface morphology of SA-modified SF microspheres at different magnifications under SEM. (A) Scale bar indicates 100 μm. (B) Scale bar indicates 200 μm. (C) Scale bar indicates 100 μm. (D) Scale bar indicates 50 μm. (E) Scale bar indicates 20 μm. (F) Scale bar indicates 10 μm.

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3.2.2. Particle size distribution of SA-modified SF microspheres Particle size distribution of SA-modified SF microspheres is shown in Fig. 3A. The curve displayed normal distribution, and the average diameter of microsphere was 141.839 μm. As can be seen from Fig. 3A, microspheres account for 16.56% of the total size of less than 100 μm, 68.47% of the total of 100-200 μm, 9.82% of the total of 200-300 microns, and more than 300 μm, accounting for 5.14% of the total. The size of embolic microspheres depends on

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the size of the blood vessel to be embolized. Previous studies have shown that the size of

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microspheres used for embolization of liver cancer ranges from 50 μm to 500 μm in diameter

suitable for liver embolization.

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3.2.3. FTIR spectra of microspheres

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[35, 36]. The results demonstrated that the particle size of SA-modified SF microspheres is

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FTIR spectra of SF, SA, and SA-modified SF microspheres were shown in Fig. 3B. In the infrared spectrum of SF, 1657 cm-1 is the absorption peak of amide I, 1544 cm-1 is the

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absorption peak of amide II, 1240 cm-1 is the absorption peak of amide III, and 618 cm-1 is the absorption peak of amide V [37]. In the infrared spectrum of SA, 3419 cm-1 is the absorption peak of O—H, 2924 cm-1 is the stretching vibration absorption peak of C—H, 1645 cm-1 and 1437 cm-1 is the opposition and symmetrical telescopic vibration absorption peak of the —COO-1 group, and 1033 cm-1 is stretching vibration absorption peak of O=C— O. In the infrared spectra of SA-modified SF microspheres, the O—H absorption peak of SA at 3419 cm-1 moved to 3411 cm-1 at low wavenumber. This is due to the interaction between N—H and O—H in SF and O—H in SA. The absorption peak at 1630 cm-1 is the absorption peak of amide I in SF and the antisymmetric stretching vibration absorption peak of —COO-1 group in SA. As a result, the peak broadens at 3411 cm-1, mainly because the strong

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Journal Pre-proof absorption peak of O—H in SA occurs at high wavenumber. After mixing the two peaks, the hydrogen bonding increases, which leads to the broadening of the peak shape in this band. Compared with the infrared spectrum of SF, the peaks of amide I and amide II in the infrared spectroscopy of SF-SA microspheres shift to a lower wavenumber, and the peak of amide V moves to the direction of long waves. This result indicates that some irregular curls in SF change to β-folding conformation after SA is added. There is no absorption peak of aldehyde group in the Fig. 3B, which indicates that glutaraldehyde has been cleaned completely during

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the washing process, and there is no residue in the microspheres.

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Fig. 3. Characterization of SA-modified SF microspheres. (A) Particle size distribution and percentage of microspheres, (B) FTIR spectra of SF, SA, and SA-modified SF microspheres.

3.2.4 Swelling ratio of SA-modified SF microspheres Swelling ratio of SA-modified SF microspheres at different pH values and different temperatures was shown in Fig. 4. As shown in Fig. 4A, the swelling tendency of microspheres in the environment of pH of 1.0, 5.8, and 7.4 was rapid swelling in the first 10 min, and reached the maximum swelling ratio at 20 min, which was 474.29%, 389.77%, and 96.29%. Meanwhile, the swelling ratio of SA-modified SF microspheres increased significantly as pH increased (from 1.0 to 7.4) (Fig. 4A). The pKa values of the components

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Journal Pre-proof of the SA molecular chain, mannuronic acid and guluronic acid, are 3.38 and 3.65, respectively. When the pH value of the solution changes, it affects the charge density of the carboxyl group on the molecular chain of sodium alginate, which affects the degree of microsphere solubility. When pH < 3.0, the pH value will be affected by the change of the strength of carboxyl protonation. The higher the pH value, the weaker the protonation strength. On the one hand, the hydrogen-bonding interaction will cause the polymer chain to shrink, thus reducing the swelling of the microspheres. On the other hand, the repulsion

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between the anions and cations becomes small, resulting in a decrease in the degree of

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swelling. When pH > 3.0, the higher the pH value, the more easily the carboxyl group is

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ionized, and the electrostatic repulsion between -COO- increases the degree of swelling.

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The swelling ratio of the SA-modified SF microspheres at different temperatures is shown in Fig. 4B. The swelling behavior of the microspheres at 4 oC, 21 oC, and 37 oC was rapid

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swelling in the first 10 min, and reached the maximum swelling degree at 20 min, which was

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362.86%, 348.84%, and 474.29%. The ambient temperature has a great influence on the swelling rate of the microspheres. As the ambient temperature increases, the swelling rate of

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the microspheres increases. When the temperature rises, the heat in the microspheres rises, which will break the intermolecular hydrogen bonds and destroy the hydrophobic action. At this time, the water molecules will quickly penetrate into the microspheres and the swelling rate of the microspheres will be higher when the temperature is higher. When the temperature is lowered, the water molecules move slowly, and the hydrophilic action of the microspheres is also affected. Therefore, the lower the temperature, the lower the swelling degree of the microspheres. In short, according to the influence trend of pH and temperature on microsphere solubility, it can be considered that before microsphere embolism surgery, the microsphere should be pre-treated with pH or temperature according to the different diameter of blood vessels, in order to promote the effect of surgery.

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Fig. 4. Swelling ratio of SA-modified SF microspheres. (A) Swelling curve of microspheres at different

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pH values. (B) Swelling curve of microspheres at different temperature.

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3.2.5 Degradation characteristics of SA-modified SF microspheres

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The weight loss rate of SA-modified SF microspheres in trypsin degradation is shown in Fig. 5. The mass loss of microspheres increased with incubation time. In the first 7 days, the

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microspheres degraded slowly, the degradation rate of degradation was 4.1%, and the degradation rate accelerated in the last two weeks. On the 21st day, the weight loss rate

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reached 20.8%. According to this degradation trend, the microspheres can be predicted to have good degradability, and the loaded drug can be slowly released while the blood vessels can be recanalized, and the secondary TACE can be performed.

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Fig. 5. Weight loss rate of SA-modified SF microspheres.

3.2.6 In vitro blood compatibility of SA-modified SF microspheres

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One of the critical features of the embolic agent for arterial embolization is that the agent

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should not cause hemolysis between agent and blood. Hemolysis rate and BCI of SAmodified SF microspheres is shown in Fig. 6. The hemolysis rate of the 5 mg/mL microspheres to red blood cells in the blood was 2.2237 ± 0.2762%, and the hemolysis rate of the 10 mg/mL microspheres to red blood cells in the blood was 3.5737 ± 0.1969% (Fig. 6A). It is shown that the hemolysis rate of microspheres increases with the increase of microsphere concentration from 5 mg/mL to 10 mg/mL (p = 0.0227), but not more than 5%, indicating that the hemolysis degree of the prepared microsphere is within the standard range and conforms to the national safety standard for biological materials [38]. Under the same experimental conditions, the greater the BCI value, the better the anticoagulant properties of the biomaterial [39]. As shown in Fig. 6B, the BCI values of SAmodified SF microspheres decreased with the increase in time, which indicated that the

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Journal Pre-proof ability of microspheres to resist blood clotting gradually decreases over time. At the 50th min of the anticoagulation experiment, the BCI value was 72.9878 ± 1.1325. It is shown that although the prepared microspheres have a certain coagulation effect, it is not easy to form a thrombus, which facilitates the smooth injection of the embolic agent during the embolization

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process.

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Fig. 6. Hemolysis rate (A) and BCI (B) of SA-modified SF microspheres. *p < 0.05.

3.3 Cytotoxicity of SA-modified SF microspheres

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Embolic microspheres may have an effect on the proliferation and apoptosis of vascular endothelial cells after entering the blood vessels, so it is necessary to evaluate the in vitro cytotoxicity of microspheres. To evaluate the cytotoxicity of SA-modified SF microspheres, L929 mouse fibroblasts and HUVEC were chosen to incubate with microspheres at various concentrations for 5 days by using CCK-8 assay. As shown in Fig. 7A, the growth trends of L929 mouse fibroblasts on microspheres with different concentrations were basically the same as those of the blank control group at 1, 2, 3 and 5 days, and no significant difference was observed. Only on the 4th day, a significant difference between the microsphere group and the blank group, indicating a slight inhibition of cell growth (Fig. 7A). Within 5 days, the microspheres of each concentration showed slight inhibition of HUVEC growth, and there

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Journal Pre-proof were significant differences at the 4th and 5th day (Fig. 7B). The number of cells decreased compared with the first 3 days, probably due to the cell density is large and the nutrient of the medium is consumed. According to the standard of toxicity grading, the cytotoxicity of SAmodified SF microspheres was in grade 0 (relative growth rates ≥100%) or grade I (relative growth rates within 75-99%) during the culturing process (Tab. S2 and Tab. S3). Grade 0 is considered to be no cytotoxicity and grade I is considered to be low cytotoxicity [40]. The

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results proved that SA-modified SF microspheres almost had no cytotoxicity.

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Fig. 7. Viability of L929 (A) and HUVECs (B) after treated by different concentrations of SA-modified SF microspheres. *p < 0.05 indicates significant difference statistically with respect to the blank control group. *p < 0.01 indicates extremely significant difference statistically with respect to the blank control group.

3.4 Animal study of vessel embolization In order to investigate the embolization efficiency of SA-modified SF microspheres in vivo, a rabbit ear model in the present study was used for assessing the new embolic agent due to its easy establishment and observation by macrography as well as its low cost and efficacy [18, 36]. As shown in Fig. 8A, it was found that there was congestion at the injection site, but no edema occurred in the rabbit ear, and microsphere embolization was observed in

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Journal Pre-proof the distal aorta on the first day of embolization. On the third day, the middle ear artery of the rabbit ear was embolized and no blood flowed, but the rabbit ear still had edema and accompanied by an inflammatory reaction (Fig. 8B). In the second week, acute inflammation occurs around the small arteries at the far end of the heart, with partial necrosis and blackening of the tissue (Fig. 8C). In the third week, the tissue around the distal end of the heart was necrotic, the epidermis atrophied and thickened, and the aortic atrophy disappeared (Fig. 8D). The results showed that microspheres could effectively prevent blood flow in

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blood vessels, causing necrosis of distant end tissue, which had a significant embolism effect

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on the middle ear artery of rabbits.

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Fig. 8. Gross observation of SA-modified SF microspheres embolized into rabbit ear artery at different time points. (A) 1 days. (B) 3 days. (C) 2 weeks. (D) 3 weeks.

Histopathological examinations for rabbit ears embolized by SA-modified SF microspheres at 3 weeks are shown in Fig. 9. Fig. 9A shows that the endothelial cells of the blood vessels have a shedding phenomenon. Inflammatory cells infiltrated around the blood vessels (Fig. 9B). Fig. 9C clearly displays a mixture of microspheres and thrombus in the rabbit ear artery. It indicates that the microspheres degrade slowly in the blood vessels of rabbit ears and can achieve the effect of continuous embolization in a long time. Fig. 9D shows that the growth of small arterial endothelial cells is obvious, the surrounding tissue has

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Journal Pre-proof a new vascular phenomenon, the epidermis on both sides thickened and the granulation tissue appears. In short, macroscopic observation showed that the modified SF arterial embolization microspheres had obvious embolization effect on the rabbit ear artery, and the

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histopathological results further confirmed the embolization effect.

Fig. 9. Histopathological examinations for rabbit ears embolized by SA-modified SF microspheres at 3 weeks. (A) Vascular endothelial cells have a shedding phenomenon in the vessel. (B) Inflammatory cells infiltrated around the blood vessels. (C) Microspheres are visible intact in the arteries. (D) Proliferation of endothelial cells in arterioles was obvious.

3.5 Kinetics of Adriamycin hydrochloride release The cumulative release profile of Adriamycin hydrochloride from SA-modified SF microspheres in different time points was presented in Fig. 10. It can be seen from the release curve that the microspheres are rapidly released within 30 h and then steady released at low rate. The release profile of the drug from the microspheres fits the diffusion law and the drug

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Journal Pre-proof can be totally released before the microspheres are degraded completely. At the initial stage of drug delivery, the water molecules will quickly enter the micropore of the microspheres to dissolve Adriamycin hydrochloride, which will release it rapidly, so there is an obvious burst effect in the initial stage. Subsequently, the microspheres swell after absorbing water and form a hydrophilic gel layer on the surface. The fast-release channel of Adriamycin hydrochloride is closed, and the drug can only be released from microspheres or through the degradation of microspheres. Furthermore, as the time increases, the hydrophilic gel layer

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gradually thickens, resulting in a decrease in the release rate of the microspheres [41]. These

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findings indicated that the Adriamycin hydrochloride loaded SA-modified SF microspheres

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had controllable releasing effect and could be used for drug carrier delivery.

Fig. 10. Adriamycin hydrochloride release from microspheres in vitro.

4. Conclusions The aim of work was to investigate SF microspheres as a biodegradable embolic agent for arterial embolization applications. In the present study, biodegradable SF microsphere modified with SA were successfully fabricated using emulsified cross-linking method. The microspheres were spherical with porous structures and had desirable particle size for the

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Journal Pre-proof injection through a catheter for TAE treatment. Swelling ratio of SA-modified SF microspheres can meet the requirements of arterial embolic agent and has pH and temperature sensitivity. The results of hemolysis assay in vitro showed the microspheres exhibited good blood compatibility. Cytotoxicity analysis showed that microspheres could stimulate the growth of fibroblasts and HUVEC, and microspheres almost had no cytotoxicity on the cells. In vivo embolization evaluation of microspheres in a rabbit ear model revealed that the arteries could be embolized by SA-modified SF microspheres in 3 weeks and

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ischemic necrosis on the ear was visible due to the vascular occlusion. Adriamycin

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hydrochloride, as a model drug, was successfully loaded into the microspheres for drug

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release study in vitro and exhibited excellent controllable release properties. All the findings

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indicated that the SA-modified SF microspheres can be used as novel potential biocompatible

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and biodegradable embolic agents for TAE.

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Supporting Information.

Additional experimental results are included in the support information, including the

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morphology of pure SF microspheres (A) and pure SA microspheres (B) under optical microscope, particle diameter, swelling ratio and sphericity of pure SF microspheres and pure SA microspheres, and relative growth rate and cell toxicity grading of L929 and HUVEC on SA-modified SF microspheres.

Author Contributions The manuscript was written through contributions of all the authors. All the authors have given approval to the final version of the manuscript. Conflict of interest The authors have no competing financial interests.

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Journal Pre-proof Acknowledgments This work was supported by grants from the National Natural Science Foundation of China

(11702044),

Chongqing

Science

&

Technology

Commission

Project

(cstc2016shmszx0635), the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201901114), and Scientific Research

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Foundation of Chongqing University of Technology (2019ZD49).

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