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Ying Chuin Yee, [Takeshi Mori](https://orcid.org/0000-0002-1821-5427), [Shima Ito](https://orcid.org/0000-0002-3233-617X), [Tetsushi Taguchi](https://orcid.org/0000-0003-2541-2530), Yoshiki Katayama

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This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s44211-024-00643-2[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[Impact of hydrophobic modification on biocompatibility of Alaska pollock gelatin microparticles](https://mdr.nims.go.jp/datasets/9a272db2-ca6f-4211-a25e-a229b7b989af)

## Fulltext

1  Impact of hydrophobic modification on biocompatibility of Alaska pollock gelatin microparticles 1  2 Ying Chuin YEE a, Takeshi MORI a,b†, Shima ITO g,h, Tetsushi TAGUCHI g,h†, Yoshiki KATAYAMA a,b,c,d,e,f † 3  4 a Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-5 ku, Fukuoka 819-0395, Japan. 6 b Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. 7 c Center for Molecular Systems, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. 8 d Centre for Advanced Medicine Open Innovation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-9 8582, Japan. 10 e Department of Biomedical Engineering, Chung Yuan Christian University, 200 Chung Pei Rd., Chung Li, 32023 11 ROC, Taiwan. 12 f Department of Biomedical Engineering, Chung Yuan Christian University, 200 Chung Pei Rd., Chung Li, 32023 13 ROC, Taiwan. 14 g Graduate School of Science and Technology, Degree Programs in Pure and Applied Sciences, University of 15 Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan. 16 h Biomaterials Field, Research Center for Macromolecules and Biomaterials, National Institute for Materials 17 Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. 18  19 † To whom correspondence should be addressed. 20 Email: mori.takeshi.880@m.kyushu-u.ac.jp; taguchi.tetsushi@nims.go.jp; katayama.yoshiki.958@m.kyushu-21 u.ac.jp   22 Manuscript Click here toaccess/download;Manuscript;Manuscript_revised2.docxClick here to view linked References 1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 mailto:mori.takeshi.880@m.kyushu-u.ac.jpmailto:taguchi.tetsushi@nims.go.jpmailto:katayama.yoshiki.958@m.kyushu-u.ac.jpmailto:katayama.yoshiki.958@m.kyushu-u.ac.jphttps://www2.cloud.editorialmanager.com/ansc/download.aspx?id=38405&guid=3c1fdb13-3af6-410e-a901-a82b0668bc2f&scheme=1https://www2.cloud.editorialmanager.com/ansc/download.aspx?id=38405&guid=3c1fdb13-3af6-410e-a901-a82b0668bc2f&scheme=1https://www2.cloud.editorialmanager.com/ansc/viewRCResults.aspx?pdf=1&docID=2037&rev=2&fileID=38405&msid=b7ad497c-3342-473a-afcc-ac02714511a62  Abstract 23 This study investigates the impact of hydrophobic modification on the immunogenicity, cytotoxicity, and 24 inflammatory response of Alaska pollock gelatin (ApGltn) microparticles (MPs). Gelatin, known for its inherent 25 biocompatibility, was modified with decyl group (C10) to explore potential alterations in its interaction with the 26 immune system.  Immunogenicity was evaluated through the measurement of material-specific IgM and IgG 27 responses, indicating no significant increase post-modification. Cytotoxicity against Caco-2 cell lines and NF-kB-28 mediated LPS-induced inflammation were also assessed, revealing no exacerbation by the modified MPs. 29 Furthermore, C10-modification with different types of linkage such as secondary amine and amide structure did 30 not influence immune reactivity. These findings suggest that C10-modification maintains the non-immunogenicity 31 and biocompatibility of gelatin MPs, supporting their potential use in biomedical applications. 32  33 Keywords: Alaska pollock gelatin microparticle, decyl-group modification, immunogenicity, biocompatibility  34  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3  1. Introduction 35 Gelatin, a versatile biopolymer derived from collagen, has been widely used in various industries, including food, 36 pharmaceutical and cosmetics, owing to its remarkable properties that enhance viscosity, texture, and stability of 37 products [1]. Traditionally, gelatin has been sourced primarily from pig skins, bovine hides, and bones. However, 38 recent attention has shifted towards alternative sources, particularly fish and fish by-products, such as fish bones 39 and skins, as promising biomaterials for gelatin production [2]. The distinctive characteristics of fish gelatin, such 40 as its lower gelling and melting points compared to gelatin derived from porcine or bovine sources [3], enable its 41 use in applications requiring higher fluidity at room temperature. 42  43 Alaska pollock gelatin (ApGtln) has been explored as a basic material for biodegradable surgical sealants due to 44 its low transition temperature, low imino acids content and low viscosity [4]. Introduction of several hydrophobic 45 groups such as cholesterol, alkyl chains with various length have been demonstrated to enhance interfacial strength 46 under wet conditions [5–7]. Among these modifications, the incorporation of the decyl group (C10) onto ApGltn 47 has been highlighted for its notable enhancement of interfacial strength with blood vessels [6]. Interestingly, 48 microparticles formed by the self-assembly of C10-modified ApGltn swelled into a stable hydrogel layer in 49 physiological saline, which was demonstrated to protect the wound surface and facilitate the tissue regeneration 50 process in wound healing models [7]. Notably, in addition to internal gastrointestinal tissue, efficacy in preventing 51 postoperative adhesion on duodenum serosal tissue was also demonstrated [8]. 52  53 Given that most C10 modifications were targeted to the primary amine groups of ApGltn, the exposure of primary 54 amine groups may have implications for immunogenicity and inflammatory response. For example, primary 55 amines on polymers have been reported to form hydrogen bonds and electrostatic interactions with phosphate 56 groups in lipids, disrupting the organization of lipid bilayers and potentially increasing toxicity [9]. Furthermore, 57 in vivo toxicity associated with cationic nanoparticles such as chitosan and bPEI nanoparticles, which induce 58 complement activation and Toll-like receptor 4 (TLR-4) activation respectively, underscores the importance of 59 understanding the impact of primary amine exposure. Interestingly, modifying primary amines to secondary and 60 tertiary amines has shown promise in mitigating these adverse effects [10], suggesting a potential strategy for 61 enhancing biocompatibility and minimizing risk of potential inflammation in biomedical applications. 62  63 Thus, in this study, C10-modification was performed on amine groups in ApGltn with different types of linkage 64 formation: amide (C10-am-ApGltn) and secondary amine (C10-sa-ApGltn), prior to microparticle fabrication, 65 allowing a comprehensive evaluation of the immunogenicity, cytotoxicity and inflammatory response associated 66 with these microparticles. By investigating the interactions between these modified microparticles and biological 67 systems, insights can be gained into their biocompatibility post-modification and potential suitability for 68 biomedical applications.  69  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4  2. Materials and Methods 70 2.1 Materials 71 Alaska pollock gelatin (ApGtln, molecular weight (MW) = 34,352 Da, amine group content: 364 µmol/g and MW 72 = 34,323 Da, amine group content: 355 µmol/g) was purchased from Nitta Gelatin Inc. (Osaka, Japan). Decanal 73 and decanoic anhydrate were obtained from Tokyo Chemical Industry Co., Ltd. (Osaka, Japan). Ethanol and 2-74 picoline borane were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Dulbecco's Modified Eagle 75 Medium (DMEM), Antibiotic-Antimycotic Mixed Stock Solution, MEM Non-Essential Amino Acids Solution, 76 Dulbecco's Phosphate-buffered Saline (DPBS) and dimethyl sulfoxide (DMSO) were purchased from Nacalai 77 Tesque, Inc. (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from Nichirei Bioscience Inc. (Tokyo, 78 Japan). Trypsin-EDTA was purchased from Gibco (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was 79 purchased from Dojindo Laboratories (Kumamoto, Japan). Sodium carbonate, 4-Nitrophenyl phosphate disodium 80 salt hexahydrate, albumin from mouse serum (MSA), albumin from chicken egg white (OVA), lipopolysaccharide 81 (LPS) from Escherichia coli O111:B4 and TWEEN® 20 were purchased from Sigma-Aldrich (Saint Louis, MO, 82 USA). Sodium hydrogen carbonate, magnesium chloride hexahydrate, G418 sulfate and sulfuric acid were 83 purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Anti-mouse horseradish peroxidase 84 (HRP)-conjugated IgM and IgG were purchased from Bethyl Laboratories, Inc (Montgomery, TX, USA).  3, 3', 85 5, 5' tetramethyl benzidine (TMB) was purchased from BioLegend, Inc. (San Diego, CA, USA).  86  87 2.2 Synthesis of C10-sa-ApGltn 88 C10-sa-ApGltn was synthesized following a previously established method [11, 12]. In brief, ApGltn (50 g) was 89 dissolved into 175 mL of ultrapure water at 50 °C under continuous stirring at 400 rpm. Subsequently, 35 mL of 90 ethanol and 35.5 mmol of decanal (two equivalent molar ratio of amine groups in ApGltn) dissolved into ethanol 91 were added into the ApGltn solution, followed by 1 hour of stirring at 50 °C. Then, 53.3 mmol of 2-picoline borane 92 (1.5 equivalent molar ratio of decanal), used as a reductant, was added to the ethanol solution under continuous 93 stirring at 400 rpm. The resulting solution had a 20 w/v% ApGtln concentration (water: ethanol = 175:75 (mL)). 94 After stirring the reaction for 17 hours, the solution was dropped into 2,500 mL of chilled ethanol (maintained at 95 −7 to 4 °C) to induce precipitation. The precipitate was washed three times with 1,250 mL of ethanol to remove 96 residual decanal and 2-picoline borane, then vacuum dried overnight (< 3 mbar) to obtain C10-sa-ApGltn powder. 97 The degree of substitution (DS) was measured by TNBS method where residual amine groups were labeled by 98 2,4,6-trinitrobenzene sulfonic acid (TNBS) to measure the absorbance [13]. 99  100 2.3 Synthesis of C10-am-ApGltn 101 C10-am-ApGltn was synthesized through a reaction with primary amine and decanoic anhydrate. Briefly, ApGltn 102 (10 g) was dissolved in 35 mL of 0.1 M phosphoric acid buffer solution (pH 8.0) at 50 °C with continuous stirring 103 at 400 rpm. Subsequently, 7.06 mmol of decanoic anhydrate (2-equivalent molar ratio of amine groups in ApGltn) 104 was dissolved in 15 mL of ethanol and added into the ApGltn solution. The resulting solution had a 20 w/v% 105 ApGtln concentration (water: ethanol = 35:15 (mL)). After stirring for 1 hour, the obtained solution was dropped 106 into 500 mL of iced ethanol (maintained at −7 to 4 °C) to reprecipitate. The precipitate was washed three times 107  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5  with 250 mL of ethanol to remove residual decanoic acid. The washed precipitate was dissolved in a water/ethanol 108 mixture (35/15 mL) and then dialyzed against 2 L of a water/ethanol mixture at a ratio of 7/3 (v/v) for 2 days at 109 room temperature. The dialyzed solution was freeze dried to obtain C10-am-ApGltn powder. The DS was 110 measured by TNBS method. 111  112 2.4 Preparation and characterization of C10-sa-MPs and C10-am-MPs 113 C10-sa- and C10-am-MPs were prepared using the coacervation method, following a previous method [7, 8, 12, 114 14]. In this process, a poor solvent for C10-sa- and C10-am-ApGltn was used to induce coacervation. C10-sa- or 115 C10-am-ApGltn was dissolved in ultrapure water at a concentration of 5 w/v%. An equal volume of ethanol, 116 serving as a poor solvent, was added dropwise to the ApGltn solution, inducing the formation of a coacervate. The 117 resulting coacervate solutions were freeze dried to remove water and ethanol. Subsequently, thermal treatment at 118 150 °C for 3 hours under vacuum conditions (< 3 mbar) was applied to facilitate the formation of chemical bonds 119 between C10-sa- or C10-am-ApGltn molecules. Org-MPs derived from Org-ApGltn, non-modified Alaska 120 pollock gelatin, were prepared using the same procedure. The obtained MPs were examined using scanning 121 electron microscopy (SEM; JCM- 7000, JEOL, Japan) and the particle diameter was determined using Image J 122 software. 123  124 2.5 Mice 125 Female Balb/c mice (6 weeks old) were purchased from Kyudo Co. Ltd. (Saga, Japan) and acclimatized for one 126 week before the commencement of experiments. All animals were housed under standard laboratory conditions 127 on a 12-hour light-dark cycle. During the acclimatization and experimental periods, mice were provided with CE-128 2 (CLEA Japan, Inc.) and had ad libitum access to water. The diet and water were replaced and replenished as 129 needed to ensure the well-being of the animals. Experiment was conducted with approval from the Institutional 130 Animal Care and Use Committee of Kyushu University. 131  132 2.6 Immunogenicity of MPs 133 Female Balb/c mice were randomly divided into groups of five. Each group received four consecutive 134 subcutaneous injections per week with the following substances: Org-MPs (0.4 mg/mL), C10-am-MPs (0.4 135 mg/mL), C10-sa-MPs (0.4 mg/mL), MSA (0.4 mg/mL) and OVA (0.4 mg/mL), respectively. Following the final 136 injection, mice sera were collected one week later for analysis.  137  138 2.7 Enzyme-Linked Immunosorbent Assay (ELISA) 139 ELISA was conducted following the protocol published by Li et al. [15] with slight modifications. In brief, ELISA 140 plates were coated with 100 μL of sample solution (10 μg/mL), containing the substances to be tested (Org-MPs, 141 C10-am-MPs, C10-sa-MPs, MSA and OVA, respectively), prepared in 0.1 M sodium carbonate buffer (pH 10.5) 142 and incubated at 4°C overnight. Following the coating procedure, plates underwent five washes with PBS-T and 143 were subsequently blocked with 1% BSA for 1 hour at room temperature. After blocking, plates were washed 144 again five times with PBS-T before being incubated with diluted mice sera (in 1% BSA solution) for 1 hour at 145  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6  room temperature. Following this incubation, plates were washed five times with PBS-T and then incubated with 146 diluted HRP-conjugated IgM and IgG (1:50,000) for 1 hour at room temperature. Subsequently, plates were 147 washed five times with PBS-T and incubated with TMB substrate for 15 minutes at room temperature. The 148 reaction was stopped by adding 0.2 M H2SO4 solution. Absorbance was measured at 450 nm with a reference 149 wavelength of 570 nm using Infinite® 200 PRO M Plex (Tecan, Switzerland). 150  151 2.8 Cell cultures 152 Human colorectal adenocarcinoma (Caco-2) cells were cultured and maintained in complete DMEM, 153 supplemented with 10% heat-inactivated FBS, 1% antibiotic-antimycotic mixed stock solution and 1% MEM non-154 essential amino acid solution. RAW264.7 macrophages transfected with secreted alkaline phosphatase (SEAP) 155 gene under the transcriptional control of NF-κB responsive promoter, were cultured and maintained in complete 156 DMEM, supplemented with 10% heat-inactivated FBS, 1% antibiotic-antimycotic mixed stock solution and 500 157 μg/mL G418. All cell lines were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2.  158  159 2.9 Cytotoxicity of MPs 160 Cytotoxicity against Caco-2 cells was assessed using Cell Counting Kit-8 assay. Briefly, Caco-2 cells were seeded 161 at a density of 2 x 104 per well in a 96-well plate and allowed to pre-incubate for 24 hours at 37°C in a 5% CO2 162 atmosphere. Subsequently, the cells were treated with MPs at concentrations ranging from 6.25 to 200 μg/mL or 163 with 10% DMSO (v/v) for either 24 hours or 48 hours, at 37°C in a 5% CO2 atmosphere. MPs solutions were 164 prepared by first dispersing it in DPBS, followed by 2-fold serial dilutions using DPBS. After the treatment period, 165 CCK-8 was added to each well according to the manufacturer’s instruction and incubated for 2 hours at 37°C in 166 a 5% CO2 atmosphere. Absorbance was then measured at 450 nm using Infinite® 200 PRO M Plex (Tecan, 167 Switzerland). Cell viability was calculated using the following formula: - 168 % viability =  𝐴1 − 𝐴0𝐴𝑐 − 𝐴0 × 100 169 where A0 is the absorbance value of wells lacking the presence of CCK-8, A1 is the absorbance value of wells 170 containing both samples and CCK-8, and Ac is the absorbance value of wells containing CCK-8 alone, without 171 any samples. 172  173 2.10 NF-κB-mediated inflammatory response of MPs 174 The NF-κB-mediated inflammatory response of MPs was assessed by quantification of secreted alkaline 175 phosphatase (SEAP) activity based on the hydrolysis of para-nitrophenylphosphate (pNPP) into para-nitrophenol 176 (pNP), a yellow compound detectable at 405 nm. Briefly, RAW264.7 macrophages transfected with the SEAP 177 gene (SEAP-RAW264.7) were seeded at a density of 2 x 104 per well in a 96-well plate and pre-incubated for 24 178 hours at 37°C in a 5% CO2 atmosphere. Subsequently, the cells were treated with MPs at concentrations ranging 179 from 2 to 200 μg/mL for 6 hours at 37°C in a 5% CO2 atmosphere. MP solutions were prepared by first dispersing 180 it in DPBS, followed by 10-fold serial dilutions using DPBS. After the 6-hour treatment period, LPS was added 181 to each well at a final concentration 20 ng/mL, and the cells were further incubated for 18 hours at 37°C in a 5% 182 CO2 atmosphere. Following incubation, culture supernatants were collected and heated at 65°C for 5 minutes to 183  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7  inactivate other alkaline phosphatases in the cells and serum [16]. An equal volume of culture supernatant and 4-184 Nitrophenyl phosphate (1 mg/mL) in substrate buffer (composed of 13.2 mM Na2CO3, 35 mM NaHCO3, 1mM 185 MgCl2 at pH 9.6 [17]) was mixed and incubated at 37°C for 3 hours in the dark. Absorbance was then measured 186 at 405 nm using Infinite® 200 PRO M Plex (Tecan, Switzerland).  187  188 2.11 Statistical analysis 189 Statistical analyses were conducted using GraphPad Prism 9 (La Jolla, CA, USA). One-way ANOVA was 190 employed to assess the significance of means across all groups, followed by Dunnett's multiple comparisons test 191 to compare each mean to the control group. Significance levels were denoted as follows: *P < 0.05, **P < 0.01, 192 ***P < 0.001, ****P < 0.0001.  193  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8  3. Results and Discussion 194 3.1 Characterization of MPs 195 The DS of C10-sa-ApGln and C10-am-ApGltn were 47 mol% and 44 mol% against total amount of primary amine. 196 As reported previously, C10-sa-ApGltn and C10-am-ApGltn were successfully synthesized based FT-IR spectra 197 and 1H NMR spectra [13]. In the paper, the increased C-H bond stretching peak of -CH2- structure at 2,927 cm-1 198 in both C10-sa-ApGltn and C10-am-ApGltn FT-IR spectra compared to Org-ApGltn spectra and increased C-H 199 peak at 1.24 ppm in both C10-sa-ApGltn and C10-am-ApGltn 1H NMR spectra were confirmed. From these result 200 and literature, the modification of decyl group was considered successful.  201  202 Afterward, three types of microparticles (MPs) were successfully prepared by coacervation method as described 203 previously [7, 8, 12, 14]. Scanning electron microscopy (SEM) observations of all MPs revealed the consistent 204 formation of monodispersed, spherical, and smooth MP structures (Figure 1b-d). The average diameter of MPs 205 prepared by Org-ApGltn was approximately 2.26 μm (Figure 1e). However, MPs prepared by C10-sa-ApGltn and 206 C10-am-ApGltn exhibited a slight increase in average diameter, with a difference of approximately 0.8 μm (Figure 207 1f, g). Through C10 modification, the increase in hydrophobicity of ApGltn introduces additional sites for 208 intermolecular interaction within the gelatin matrix. This promotes stronger interactions between ApGltn chains 209 and particles, leading to the formation of larger clusters of ApGltn molecules and, consequently, resulting in an 210 increase in microparticle size. However, there was no difference in particle size between C10-sa-MPs and C10-211 am-MPs, indicating that the type of linkage between C10 group and ApGltn does not significantly influence the 212 self-assembly process of microparticle formation. This suggest that the overall hydrophobicity of the ApGltn, 213 rather than the type of C10 group modification method, is the primary factor that drive the observed change in 214 particle size.  215  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9   216 Figure 1: (a) Synthesis of C10-sa-ApGltn and C10-am-ApGltn to prepare C10-sa-MPs and C10-am-MPs. 217 Characterization of Org-, C10-sa-, C10-am-MPs. (b-d) Scanning electron microscopy (SEM) images of Org-, 218 C10-sa-, C10-am-MPs. The scale bars represent 5 µm. (e-g) Particle diameter distribution of Org-, C10-sa-, C10-219 am-MPs. Particle diameter analysis was performed by ImageJ software. 100 particles were analyzed for each 220 analysis. 221  222 3.2 Immunogenicity of MPs 223 While previous studies have highlighted the superior characteristics of microparticles (MPs) as a surgical sealant, 224 it is crucial to assess their potential risk in triggering unnecessary immune responses. This consideration is 225 especially significant due to the inherent risk associated with polymers, which often induce the production of 226 material-specific antibodies that may compromise their effectiveness in biomedical applications. To evaluate the 227 immunogenicity of MPs, subcutaneous injection in mice was employed. The subcutaneous route was selected 228 based on its relevance, as anti-drug antibodies are frequently associated with drugs administered via this route 229 [18].  230  231  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10  To evaluate the production of MPs-specific antibodies, mice received a total of four subcutaneous injections of 232 the respective MPs, administered once per week. Serum samples were collected, and a series of diluted serum 233 samples were used to quantify antibody titers using a sandwich ELISA with plates coated with the respective MPs 234 [15]. In this experiment, ovalbumin (OVA) is used as a positive control due to its well-known immunogenic 235 properties [19], providing a standard reference for evaluating the immunogenicity of all test MPs. Conversely, 236 mouse serum albumin (MSA) was used as negative control as it is a common protein native to mice itself, and its 237 immunogenicity is expected to be minimal [20]. This was confirmed in Figure 2, where OVA induced a high titer 238 of OVA-specific IgG while MSA activity was negligible.  239  240 By comparing to OVA treatment group, Org-MPs, which was prepared from unmodified ApGltn, did not induce 241 specific IgM or IgG responses, demonstrating the non-immunogenic nature of ApGltn. Additionally, specific IgG 242 response to C10-modified ApGltn, either at its linkage structures: amide (C10-am-MPs) or secondary amine (C10-243 sa-MPs), were not observed. While the length of alkyl side chain has been previously demonstrated to confer 244 immunogenicity to peptide vaccine [21], it itself did not induce any immunogenic responses in this study.  245  246 On the other hand, in the IgM production evaluation, specific IgM seemed to be detected in mice injected with 247 C10-modified ApGltn. However, similar levels of IgM were also detected in the C10-modified ApGltn-untreated 248 group (Figure S1), suggesting that the observed IgM may be due to nonspecific adsorption and not directly related 249 to the substance itself.  250  251 In other words, these results suggest that all tested MPs did not induce targeted antibody responses and are unlikely 252 to induce an immune reaction or sensitization upon exposure. 253  254 Figure 2: Immunogenicity of Org-, C10-sa-, C10-am-MPs. (a) MP-specific IgM levels (b) MP-specific IgG levels 255 measured by ELISA. 5 mice were used for each treatment group. Ovalbumin (OVA) and mouse serum albumin 256 (MSA) were used as positive and negative control, respectively. The error bars represent the standard deviation 257 of the mean for a sample size of n = 5.  258  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 11  3.3 In vitro cytotoxicity of MPs 259 The potential cytotoxic effects of MPs were evaluated on Caco-2 cell line to provide valuable insights into its 260 cytocompatibility. The viability assay utilized 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-261 disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), which is reduced by cellular dehydrogenases to form 262 a water-soluble, orange-colored product (formazan). The amount of formazan dye produced correlates with the 263 number of living cells, providing a measure of cell viability [22].  264  265 According to Figure 3, the cytotoxicity assay against Caco-2 cell lines revealed that all MPs, when compared to 266 the untreated group, did not exhibit cytotoxicity up to a concentration of 200 μg/mL. This observation suggests 267 that C10-modification with different linkage type: amide and secondary amine does not compromise the non-268 cytotoxic characteristic of hydrophilic ApGltn. Furthermore, cell viability was not significantly affected at both 269 the 24-hour and 48-hour time points, indicating the long-term safety of MPs. Interestingly, tendency of increase 270 in cell viability was observed upon treatment with Org-MPs and C10-sa-MPs at the 24-hour timepoint; however, 271 this effect did not extend to the 48-hour mark. This indicates that these MPs treatment show some beneficial effect 272 on cell viability in the early stages, potentially promoting cell proliferation or enhancing cellular functions [23], 273 while C10-am-MPs do not possess this effect. 274  275 Figure 3: Cytotoxicity against Caco-2 cell lines after 24 hours (a-c) and 48 hours (d-f) of exposure to (a, d) Org-276 MPs, (b, e) C10-sa-MPs, and (c, f) C10-am-MPs. 10% DMSO were used as positive control. The error bars 277 represent the standard deviation of two independent experiments, each conducted with a sample size of n = 3.  278  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12  3.4 Anti-inflammatory properties of MPs 279 Immune activation begins when receptors on phagocytes are engaged, initiating signaling cascade. For instance, 280 lipopolysaccharides (LPS), the major components of outer membrane of Gram-negative bacteria, bind to Toll-like 281 receptor-4 (TLR-4) on macrophages, initiating an inflammatory response. This binding triggers the activation of 282 nuclear factor kappa B (NF-κB) pathway, a crucial mechanism in inflammation [24]. RAW264.7 macrophages 283 transfected with the SEAP (alkaline phosphatase) reporter gene (SEAP-RAW264.7) were utilized to evaluate NF-284 κB activity. The inflammatory response induced by MPs was evaluated by quantifying secreted alkaline 285 phosphatase (SEAP) activity, measured based on the hydrolysis of para-nitrophenylphosphate (pNPP) to para-286 nitrophenol (pNP), a yellow-colored compound detectable by absorbance. 287  288 The effect of SEAP secretion upon exposure to varying concentration of each MPs was depicted in Figure 4. In 289 the absence of LPS, SEAP activity was negligible compared to the control group (PBS). As expected, the addition 290 of LPS increased SEAP activity, indicating the initiation of an inflammatory response. Treatment of RAW264.7 291 macrophages with C10-sa-MPs and C10-am-MPs tended to increase SEAP activity compared with Org-MP 292 treatment.  Considering that hydrophobic C10 chains are predominantly localized on the surface [7], these 293 hydrophobic counterparts may interact more readily with the cell membrane. Consequently, this interaction leads 294 to an observable trend of increased SEAP activity.  295  296 In addition, treatment with Org-MPs showed dose-dependent changes in inflammatory responses. At the highest 297 concentration (200 μg/mL) of Org-MPs, SEAP activity mirrored that of the control (PBS), but at the lowest 298 concentration (2 μg/mL), SEAP activity was significantly decreased. Although a decreasing trend was also 299 observed in C10-sa-MPs and C10-am-MPs, it was not statistically significant. The decreasing trend suggests that 300 Org-MPs have the potential to suppress inflammatory responses. However, at higher concentrations, Org-MPs 301 may induce cellular stress, which could counteract its anti-inflammatory activity. Overall, all three MPs did not 302 exhibit an additional effect on SEAP activity beyond that induced by LPS alone, suggesting their neutral impact 303 on LPS-induced inflammation.  304  305 Figure 4: Anti-inflammatory properties of MPs (a) alone and (b) LPS-induced NF-κB activation in SEAP-306 RAW264.7 macrophages. PBS was used as negative control. The error bars represent the standard deviation of 307 two independent experiments, each conducted with a sample size of n = 3.  308  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13  4. Conclusion 309 In conclusion the non-immunogenic, non-cytotoxic, and anti-inflammatory characteristics of C10-modified MPs 310 were demonstrated. While C10 modification improves the interfacial strength of MPs under wet conditions in 311 previous studies, these findings demonstrate that C10-modification with amide or secondary amine linkage 312 showed similar properties and do not compromise the inherent biocompatibility of ApGltn, underscoring their 313 potential safe use in biomedical applications. Further investigation is required to elucidate the underlying 314 mechanisms and to solidify their implications for biomedical applications.   315  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14  Statements and Declarations 316 The authors declare that they have no conflicts of interest relevant to this manuscript. 317  318 Data availability 319 The data presented in this study are available upon request from the corresponding authors. 320  321 Acknowledgements 322 This work was supported by Grant-in-Aid for Transformative Research Areas (A) (20H05876, 20H05872). Y. C. 323 Y. gratefully acknowledges the Ministry of Education, Culture, Sports, Science and Technology (MEXT) 324 Scholarship for financial support during the course of this research.  325  326 References 327 1. Stevens, P.: Gelatine. 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