Enzymatic preparation of modulated– biodegradable hydrogel nanocomposites based chitosan/gelatin and biphasic calcium phosphate nanoparticles - Nguyen Tien Thinh

Journal of Science and Technology 55 (1B) (2017) 185–192 ENZYMATIC PREPARATION OF MODULATED– BIODEGRADABLE HYDROGEL NANOCOMPOSITES BASED CHITOSAN/GELATIN AND BIPHASIC CALCIUM PHOSPHATE NANOPARTICLES Nguyen Tien Thinh1, Nguyen Thi Phuong2, Bui Thanh Thai2, Nguyen Trong Tri3, Nguyen Huynh Bach Son Long3, Tran Quoc Son3, Nguyen Tri Phu2, Nguyen Cuu Khoa2, Nguyen Dai Hai2, Tran Ngoc Quyen1, 2, * 1School of Medicine and Pharmacy, TraVinh University 126 National Road 53, Ward 5, Tra Vinh City, Tra Vinh Province, Vietnam 2Institute of Applied Materials Science, VAST 1 Mac Dinh Chi Street, Ben Nghe Ward, District 1, Ho Chi Minh City, Vietnam 3Department of Chemical Engineering, Lac Hong University 10 Huynh Van Nghe Street, Buu Long Ward, Bien Hoa City, Dong Nai Province, Vietnam *Email: tnquyen979@gmail.com Received: 30 December 2016; Accepted for publication: 6 March 2017 ABSTRACT In the study, injectable chitosan–4 hydroxyphenylacectamide acid (CHPA) and gelatin– tyramine (GTA)–based hydrogels were enzymatically prepared, in which could encapsulate biphasic calcium phosphate nanoparticles (BCP NPs) for enhancing bone regeneration. The in situ formation of hydrogel composite was varied from 35 to 80 seconds depending on concentration of H2O2. Collagenase–mediated biodegradation of the hydrogel composite could be modulated from 3 days to over one month depending on amount of the formulated CHPA. Live/dead cell viability assay indicated that the hydrogel composite enhanced bone marrow mesenchymal stem cells (MSCs). The obtained results show a great potential of the hydrogel composites for bone regeneration due to its adjustable biodegradation, biocompatibility and enhancement in new bone formation. Keywords: chitosan, gelatin, horseradish peroxidase (HRP), hydrogel, collagenase. 1. INTRODUCTION Recently, biological hydrogels have played an important role in the advanced biomaterials for tissue regeneration and drug delivery systems. Several kinds of the injectable hydrogels performed an effective encapsulation of drugs/cells and convenience for applying the minimally invasive implant surgery [1]. The hydrogels play a role as an artificial extracellular matrix (ECM) inside body for cell migration and proliferation allowing transportation of nutrients En 18 sub en pro Fo by clo inj enz Ch adh for of the rat co as 2.1 MW dim Co 2.2 2.2 sol of stir (m ch NM C3 zymatic prep 6 stances an capsulated liferation. T r enzyme–fa –products in sely integra ectable hydr yme and H itosan–base esion, anti– biomedical cell attachm hydrogels ios of chitos llagenase. Th reducing lim . Materials Chitosan 100kD), ethylamino llagenase an . Preparatio .1. Prepara In a flask ution (0.50 solution wa ring for 24 olecular we itosan soluti R (D2O)/pp +4+5+6, chitos aration of m d by–produ with peptid hese hydrog bricated hyd network ting artificia ogel compo 2O2. It is w d gels posse infective ac applications ent and prol are quickly an and gelat e hydrogel itation of ge (100 kD, 75 4–hydroxy propyl) car d HRP enzy nof polyme tion of 4–hyd , chitosan ( mL). 4–Hyd s adjusted t h. The solu ight cut–of on was lyop m: δ 2.05 ( an); δ 2.89 ( odulated–bi cts from c e, growth els have bee rogels, they formation. T l materials w sites from C ell–known ss several b tivity and en due to its h iferation [5, degraded by in, the hydro composite c latin–based 2. –85% deace lphenylacet bodiimide me (type VI rs roxyphenyl 1g) was diss roxyphenyla o 5 and the tion was dia f (MWCO) hilized to ob s, –COCH3, d, –CH2–, H Figure 1 odegradable ell metabo factor and n prepared v used some herefore, t ith biologi HPA and G that chitosa eneficial pro hancement igh biocom 6]. Gelatin– collagenase gel could ad ould be pote materials in EXPERIM tylation), ge ic acid (H (EDC), wh ) were purch acetic acid c olved in the cetic acid (0 n EDC (0.9 lyzed again 6000–8000 tain CHPA chitosan); δ PA); δ 6.89 . Synthetic sc hydrogel na lism [2]. T etc. for e ia physical, specific cro he approach cal entities [ TA in the n and gela perties for t of cell attach patibility, fa based hydro within 3–4 just its biod ntial in diff biomedical ENTAL latin from p PA) and ich were o ased from S onjugated c solution of .45g, 2.9 mm 0 g, 4.7 mm st deionized ) for 3 da as shown in 3.22 (m, –C và 7.22 (d, – heme of CHP nocomposit hese scaffo nhancing c chemical or ss–linked re performs 3].In this stu presence of tin are bioc issue regene ment [4]. G st biodegrad gels were b days [7]. U egradation r erent tissue applications orcine skin tyramine (T btained fro igma–Aldric hitosan (CHP 40 mL DI ol) was add ol) added t water usin ys. Subsequ Figure 1 (th 2(H), chitos CH=CH–, H A. es based lds could ell attachm enzymatic r actions whic characterist dy, we intr the BCP N ompatible m ration such elatin has b ability, enha io property; sing differen ate in the pr regeneration . (Bloom 300 A), 1–eth m Acros O h. A) water and H ed into the o the reacti g membrane ently, the e yield was an); δ 3.43– PA). also be ent and eactions. h avoids ics more oduce an Ps, HRP aterials. as tissue een used ncement through, t weight esence of s as well , type A, yl–3–(3– rganics. Cl 1.0M flask. pH on under dialysis modified 0.9g). 1H 3.92 (m, 2.2 of 24 (M GT CH 2.2 tric 7 t sol cal 2.3 Th int hy of wa 2.4 mg in wt sol CH BC sam 0.2 .2. Prepara Gelatin sk the mixture h. Then, t WCO6000– A as shown =CH– of TA .3. Prepara BCP were alcium pho o obtain a w ution. The p cination wa . Preparatio GTA (40 en, enzyme o each tube. drogel was p 0.05–0.2 wt s 2 wt/wt%. . Preparatio Precursor ) were disso DI water (16 /vol%) was utions conta PA hydroge P nanoparti ples were wt/vol%. tion of tyram in (2g) and was adjusted he solution 8000) for 3 in Figure ), δ 2.65 an tion of BCP synthesized sphate salts w hite suspens recipitate w s carried out n of gelatin mg) was di HRP (30 µL GTA hydro repared by /vol%) were The gelation n of chitosa polymer so lved comple 0 µL). 30 µ added into B ining HRP a ls at 8 wt/w cles (10 wt studied bas ine conjuga TA (1.00 g, to 6 follow was dialyz days. Subse 2. Theyield d 2.88 (m, – Figure 2 using an ul ith molar r ion. The pH as washed w at 750 °C. and chitos ssolved in D of 0.07 mg/ gel was form a same abov added into e time was d n/gelatin–b lutions were tely in DI w l HRP (0.07 , D. A was nd H2O2 we t% of the po /wt%) was p ed on variat ted gelatin ( 7.3 mmol) ing addition ed against quently, the was 1.80 g CH2CH2–, T . Synthetic sc trasonic assi atio of Ca/P solution wa ith DI wate an–based hy I water (30 mL) and H2 ed by mixin e process. H ach tube. Th etermined b ased hydro prepared in ater (150 µL mg/mL) wa mixed with re interfuse lymer conce repared by ion of the c GTA) were dissolv of EDC (0.5 deionized dialyzed so . 1H NMR ( A). heme of GTA sted process = 1.57 for 1 s maintaine r and dried drogels 0 µL) and O2 (30 µL o g the solutio RP (30µL o e final conc y using the v gels, hydrog four vials. ). In vial C s added into C, B was m d together to ntration. Th the same m oncentratio Ngu ed in DI wa 0 g, 2.5 mm water using lution was D2O)/ppm: . . The calciu 2 h at 50 °C d by adding in an oven a separated in f 0.03–0.07 w n of 10 wt/w f 0.05 mg/m entration of ial tilting m el composit In each via and D, CHP A, C and 3 ix with D. F create in si e hydrogel c anner. The n of H2O2 f yen Tien Th ter (30 mL) ol) under st membrane lyophilized δ 6.75 and m chloride r under contr of sodium h t 70 °C. Fin to two vials t/vol%) we t% polyme L) and H2O the polymer ethod. es l A and B, A (10 mg) d 0 µL H2O2 ( inally, two tu formation omposites co gelation tim rom 0.05, 0 inh, et al. 187 . The pH irring for dialysis to obtain 7.11 (d,– eacted to olled pH ydroxide ally, the equally. re added r. CHPA 2 (30 µL solution GTA (20 isslolved 0.05–0.2 polymer GTA or ntaining e of the .07, 0.1, Enzymatic preparation of modulated–biodegradable hydrogel nanocomposites based 188 2.5. In vitro biodegradation study The in vitro biodegradation of hydrogel, hydrogel composites were studied immersing hydrogels and hydrogel composites in PBS solution with the presence of collagenase (0.2 U/mL) at 37 °C and then monitored their weight–losses following different incubation times. The enzymes were prepared in a PBS 0.01 M, pH = 7.4 solution. The samples with different mass ratios were accurately weighted before immersing in 1 mL of enzymatic solution. At the predetermined intervals, the samples were removed from the incubation medium. Then the weight of degraded hydrogels, hydrogel composites (Wt) was measured to determine the weight of the remaining the samples. Degradation rate (rate of weight loss %) ൌ ௐ௜ିௐ௧ௐ௜ . 100 % Wi and Wt are initial weights of hydrogels or hydrogel composites and degraded hydrogels or hydrogel composites, respectively. 2.6. Characterization The structures of CHPA and GTA were determined by using NMR at Institute of Chemical– VAST (Varian, 400 MHz, U.S.A) at 37 °C and an UV–Vis spectrophotometer (JASCO V–570, Japan). Morphology of BCP was determined by using Field–emission scanning electron microscope (FESEM) JSM–635F, JEOL. The measurement was conducted at Institute of Chemical Technology–VAST. The phase analysis of the BCP NPs was identified using an X– ray diffractometer (XRD, D8/Advance, Bruker, UK) with CuKα, (λ = 1.5406 Å) at Institute of Applied Materials Science–VAST. 3. RESULTS AND DISCUSSION 3.1. Characterizations of polymers Recent years, the HPR enzymatically cross–linked reactions have played a crucial role in preparation of several polysacharide–based hydrogels [3]. Conjugation of HPA on chitosan formed a phenolic derivative enable to exploit for enzyme–mediated cross–linking reaction. The HPA–conjugated chitosan was confirmed from resonance signals of aromatic protons of HPA at 6.89 and 7.22 ppm (Figure 3, top). The signals at 2.89 ppm were assigned to methylene protons of HPA. Overlapped, broad resonance signals of D–glucosamine of chitosan were observed in the interval 3–4 ppm. GTA can be also synthesized by the coupling reaction using EDC. Tyramine grafted gelatin was determined by the resonance signals (2.65 ppm and 2.88 ppm) of the methylene protons of tyramine. Peaks of aromatic protons of tyramine appeared at 6.75 and 7.11ppm. Some signals of amino acids in 1H NMR spectrum were shown (Figure 3, bottom): δ 4.55 and 4.68 (–CH2–, proline); 4.27 (methine proton of hydroxyproline); 3.88 (–CH2–, alanine); 1.34 (–CH3, alanine); 3.57 (–CH2–, glycine); 2.23 (–CH2–, glutamic acid); 1.60(–CH2–, arginine); 3.14, 7.23 and 7.29 methine proton of phenylalanine). These 1H NMR results could confirm the successful preparation of two phenolic precursors for fabricating the hydrogels. F 3.2 igure 3.1H NM . Character R spectrum izations of B F of chitosan 4 (G CP igure 4. SEM –hydroxyphen TA, bottom) image of th ylacetic acid in D2O. e BCP nanopa Ngu (CHPA, top) rticles. yen Tien Th and gelatin–t inh, et al. 189 yramine En 19 ult ran ult ult go 3.3 cou sol H2 an of 0.0 nu GT ge 6c an hy OH res com cel gro de zymatic prep 0 Figure 4 s rasound irra ging from 6 rasonic cavi rasound–ass od mixing o . Character As menti pling react utions occur O2 into radic d formed a s Figure 6a H2O2 and H 5 wt% conc mbers of phe A gel, an i lation time b indicated tha d half minut drogel and h , COOH gr ulting in inc posites dec l–favorable ups should creased in th aration of m hows the S diation. Th 0 to 100 nm tation impro isted metho f the precurs izations of h oned, using ion is intere s by couplin als which in trong and hi Figure , bindicate t RP (0.05 m entration of nolic group ncrement of ecause mor t CHPA–G es dependin ydrogel com oups of gela reasing cros reased. In t range that be matched e process of odulated–bi EM images e synthesize . The ultra ves the ma d can synth ors. ydrogels, h phenolic m sting appro g of pheno itiate the fo ghly elastic g 5. Formation hat the lowe g/mL) in 12 H2O2 and 0. s coupled to H2O2 conc e phenolic r TA hydroge g on amount posite that tin and ami s–linking de he study, it doesn’t indu each other. the hydroge odegradable of BCP na d BCP pow sound prom terial transfe esize smalle ydrogel com oieties con ach to prep l moieties [3 rmation of g el (Figure 5 of gelatin and st gelation t s for CHPA 07 mg/mL c GTA were entration at adicals were ls and hydro of H2O2. It contributes ne groups ch nsity of hyd is important ce cell apo In the fact, l formation hydrogel na no powders ders had otes chemic r at particle r particle si posites and taining pol are hydroge ]. In case, H el. Gelation ). chitosan–ba ime was obt hydrogel a oncentration less than to C the fixed H produced i gel compos is a lightly from presen itosan link rogel compo to use an am ptosis. So m concentratio due to oxida nocomposit which wer a spherical al reactions surfaces. T ze and high gelation ti ymers and ls. The gela RP promote formed with sed hydrogels ained at 0.0 nd in 50 s of HRP. Th HPA. In ca RP could l n their polym ites could b difference in ce of BCP N with OH gro site so gelat ount of hy olar ratio n of H2O2 c tion of pheno es based e synthesiz shape and and physica herefore, u er uniformit me HRP/H2O2– tion of the s the degra in a few pe . 7 wt% conc for GTA hy is could res ses of CHPA ead to exten er solution e formed be gelation tim Ps. Functio ups of HAp ion time of drogen pero of H2O2 an ould be sign l moieties. ed using diameter l effects; se of the y due to mediated polymer dation of riod time entration drogel at ult in the gel and ding the s. Figure low one e of the nal NH2, in BCP hydrogel xide in a d phenol ificantly F g 3.4 tis ma rat at for of de exp cro cro igure 6. Effe el, b) 0.05 mg . In vitro bio The prote sue regener terials with e decreased a mass ratio the mass ra 1C:10G; 1C gradation rat lained that ss–linking r ss–linking d Figure 7b. ct of H2O2 con /mL HRP fo degradatio olytically de ation. Figur different ma following th of 0C:10G tio of 0.5C: :5G; 1C:2.5 e of all hyd presence of eaction with ensity. In vitro biode GTA– centration on r CHPA gel a n study gradable pr e7 shows t ss ratios of e decrease i were comple 10G. In con G were not u rogel compo calcium and amine and gradation rat CHPA (botto gelation tim nd c) 0.07 mg operty of th he collagen formulated c n amount o tely degrade trast, chitosa tterly degra site sample phosphate carboxylat e of hydrogel m) in presen e of hydrogel /mL HRP for e artificial m ase–mediate hitosan (C) f gelatin in h d after 42 h n/gelatin–b ded within 7 in comparis ions release e groups in s GTA–CHPA ce of collagen Ngu with a) 0.07 m CHPA–GTA atrix plays d degradati and gelatin ydrogel. Fo ours and deg ased hydrog 62 hours. Th on with hyd d from BCP polymers re (top) and hy ase enzyme. yen Tien Th g/mL HRP f hydrogel com a crucial ro on behavio (G). The deg r instance, h raded after els at the ma ere was a p rogels. This NPs partici sulting in in drogel compo inh, et al. 191 or GTA posite. le in the r of the radation ydrogels 90 hours ss ratios rolonged could be pating to creasing site Enzymatic preparation of modulated–biodegradable hydrogel nanocomposites based 192 This result may be explained by the fact that gelatin–based materials have a fast degradable profile. Incorporating with chitosan, the hydrogel could adjust its biodegradation rate in the presence of collagenase. The preliminarily obtained results are significant because the hydrogel composites could be selected to implant into human body to regenerate every specific tissue. 4. 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