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Tp ch Khoa hc 2012:23b 60-68

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NGHIN CU CH TO HT GEL CHITOSAN LIN KT NGANG KCH THC NH


L Thanh Phc1 v Bi V Thanh Phng1

ABSTRACT
Chitosan, derived from chitin in the shell of black tiger shrimp (Penaeus monodon), was chemically modified with glutaraldehyde to make cross-linked chitosan microspheres. In this study, the effects of preparation methods, stirring time and speed on the characteristics of chitosan microspheres have been studied. In the same experimental conditions, morphology of chitosan microspheres made by emulsification method was more uniform particle size than that of by mechanic method, and one-pot chemically modified method gave chitosan microspheres with the best desired charaterizations. The longer stirring time and quicker stirring rate, the more uniform particle and anticoagulation of the chitosan microspheres. The morphology of microspheres was studied using scanning electron microscopy (SEM) and it was shown that microspheres had a spherical shape and smooth surface. In the comparison of adsorption rate of Cu2+, larger (2.5-4 mm) cross-linked chitosan microspheres showed slower and smaller (2-10 m) cross-linked chitosan microspheres showed faster. Keywords: Chitosan, glutaraldehyde, cross-linked, microspheres Title: Study on the preparation of cross-linked chitosan microspheres

TM TT
Chitosan, sn xut t chitin ca v tm s (Penaeus monodon), c bin tnh ha hc vi glutaraldehyde to ht chitosan cha lin kt ngang c kch thc nh (ht vi cu). Trong nghin cu ny nh hng ca cc yu t khc nhau n qu trnh to ht nh: Phng php ch to, vn tc khuy, thi gian khuy c kho st. Trong cng iu kin th nghim, ht thu c t phng php nh tng c hnh dng cu tt hn phng php c hc v phng php bin tnh ha hc trc tip to ra ht c tnh cht mong mun tt nht. Vn tc khuy ln s lm gim kch thc ht, thi gian khuy di lm tng ng u v gim tnh kt khi ca ht. Hnh dng ca cc ht vi cu c xc nh bng nh hin vi in t qut (SEM) chng t rng chng c hnh cu u v b mt kh bng. S so snh tc hp ph Cu2+ gia ht chitosan lin kt ngang c kch thc ln (2.5-4 mm) v ht chitosan lin kt ngang c kch thc nh hn (2-10 m) chng t rng ht vi kch thc nh hn c tc hp ph nhanh hn. T kha: Chitosan, glutaraldehyde, ht vi cu, lin kt ngang

1 T VN Chitin l mt loi polymer sinh hc c ngun gc t nhin c tm thy trong mt s loi thc vt bc thp nh: nm, to (vi vai tr ging nh cellulose cc loi cy). Chitin cn xut hin trong v mai (b xng ngoi) ca loi ng vt khng xng sng nh: tm, cua, mc; lp bao ngoi ca cc loi cn trng cnh cng,...

Khoa Khoa hc T nhin, Trng i hc Cn Th

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Chitosan l dn xut ca chitin, c to thnh bi phn ng deacetyl ha chitin bi dung dch kim mnh nhit cao (120C). Chitosan c ng dng rt nhiu trong lnh vc dc phm v y hc do c nhng c tnh tt nh: Khng c, tnh tng hp sinh hc cao, c th t phn hy,... Do trong cu trc ca chitosan c s hin din ca nhm hydroxyl (-OH) v nhm amine (-NH2) c kh nng to lin kt vi cc nhm chc khc nn chitosan thng c dng lm cht mang trong ngnh dc phm. Bn cnh , chitosan cn c ng dng vo lnh vc mi trng, dng lm cht hp ph sinh hc tch cc ion kim loi nng ra khi nc thi. c mt s ti nghin cu cp n kh nng hp ph kim loi trong nc thi ca chitosan, ng thi kho st nhng yu t nh hng n hiu sut hp ph nh: pH, nhit mi trng, nng kim loi, thi gian tip xc ca cht hp ph vi ion kim loi, tc khuy,... nhng thng s dng cht hp ph l nhng ht chitosan lin kt ngang vi kch thc ln (t 2.5 mm n 4 mm) ( Th M Phng, V Cm T, 2005). pH di 5.5 chitosan to gel lm gim kh nng hp ph ion kim loi nng cng nh kh x l hn hp sn phm sau khi hp ph do vy mt s ha cht c s dng lm bn ht chitosan bng cch to lin kt ngang trong ht chitosan nh: Epichlorohydrin, glutaraldehyde, sodium tripoly-phosphate, Ht chitosan lin kt ngang kch thc nh thu c chng nhng khng tan trong dung dch acid m cn c tnh cht c l bn hn (Oliveiral B. F., et al., 2005; Rajesh R. Dubey et al., 2003). Trn l thuyt, khi ng knh ca ht cht hp ph cng nh th hiu sut hp ph cng ln do tng din tch b mt hp ph tng ln, do , kh nng hp ph ca ht chitosan lin kt ngang kch thc nh s ln hn ht c kch thc ln (s dng cng mt khi lng cht hp ph ban u). Ni dung bi vit ny s ch yu trnh by cc phng php to ht chitosan lin kt ngang kch thc nh vi glutaraldehyde, kho st cc yu t nh hng n hnh dng v kch thc ht, sau tin hnh th nghim hp ph kim loi trong dung dch so snh hiu sut hp ph gia ht thu c vi ht c kch thc 2.5 mm. 2 PHNG TIN V PHNG PHP 2.1 Nguyn liu V tm s c ly ti X nghip Ch bin Thu sn Cafatex, nm trn Quc l 1A, huyn Chu Thnh, tnh Hu Giang. Cc ha cht s dng: glutaraldehyde (Merck, c); cn li hydrochloric acid, sodium hydroxide, potassium permanganate, oxalic acid, ethanol c xut x t Trung Quc. 2.2 Phng php iu ch chitosan: Chitosan c iu ch (theo Nguyn Th Thy i, Nguyn Hong o, 2004) nh sau: V tm ti c ra sch, em phi kh trong nng ri nghin v ry to bt. Tin hnh loi khong vi dung dch HCl 5%, sau loi protein bng dung dch NaOH 1%, nhit khong 90-100C. Sn phm c ty mu bng dung dch KMnO4 0.1% v ty trng bng oxalic acid 0.2%. Sn phm ra v sy kh thu c chitin. Chitosan c to ra bng cch deacetyl ha
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chitin thu c vi dung dch NaOH 55% nhit 120-130C trong 2.5 gi. Lc v ra hn hp bng nc ct n pH = 7 sau ra li bng ethanol, sy kh thu c chitosan. T dung dch nht chitosan 2% wt/vol c to ra bng cch ha tan chitosan trong dung dch AcOH 5% theo t lng v c s dng cho ba phng php iu ch ht gel chitosan lin kt ngang c kch thc nh di y. iu ch ht gel chitosan lin kt ngang c kch thc nh To ht bng phng php c hc ri to lin kt ngang Ha tan dung dch nht chitosan 2% wt/vol vo nc ct theo t l 3 ml dung dch nht: 100 ml nc ct ri cho vo cc nha 250 ml v khuy vi tc 1600 vng/pht. Sau khi khuy c 60 pht, thm vo dung dch 1.6 ml NaOH 1N trung ha lng acid trong mui to ht chitosan khng tan. Sau 90 pht, gim vn tc khuy t 1600 vng/pht xung 600 vng/pht nhm duy tr trng thi l lng ca cc ht chitosan. Sau 30 pht, tt my khuy, gi n nh dung dch trong 2 gi cc ht chitosan lng xung y cc. Lc ly ht sau ra k vi nc ct n khi trung ha. Ngm ht chitosan thu c vo dung dch glutaraldehyde (Glu) 25 % (theo t l 1 kg chitosan: 0.15 kg Glu, c dng cho c ba phng php) trong 3 gi thc hin qu trnh to lin kt ngang. Lc v ra phn ht rn bng ether sau kh trong iu kin t nhin, thu c ht chitosan lin kt ngang di dng bt. To ht bng phng php nh tng ri to lin kt ngang To nh tng (theo Singla A. K., et al., 2001): S dng 50 ml dung dch nht chitosan 2% wt/vol lm pha phn tn. Pha lin tc gm 200 ml du paraffin v 1 g cht hot ng b mt Span 80. Hn hp trn c khuy trong 20 pht hnh thnh nh tng nc trong du. Sau , 15 ml dung dch NaOH 1 N c thm vo hn hp. Tc khuy c duy tr vn tc 1600 vng/pht trong 2.5 gi na. Kt thc th nghim, hn hp c cho vo bnh chit v n nh qua mt m. Di tc dng ca trng lc, cc ht chitosan s lng xung y bnh chit. Phn bng cn ly ra t bnh chit c ly tm trong 2 pht, vn tc 2500 vng/pht loi b phn du tha cn st li, sau tip tc ra vi nc ct n khi trung ha. To lin kt ngang cho ht chitosan thu c bng Glu 25% trong 3 gi ri ra li bng ether. kh trong mi trng t nhin s thu c bt chitosan. To ht bng phng php bin tnh ha hc trc tip Ha tan 2 g chitosan vo 100 ml dung dch AcOH 5% hnh thnh dung dch nht chitosan. Ly 100 ml du paraffin v 1 ml cht hot ng b mt Span 80 cho vo cc nha 250 ml. Khuy vi vn tc 500 vng/pht. Thm vo hn hp 3 ml dung dch nht trn, ng thi tng tc khuy ln 1600 vng/pht. Sau khi khuy 5 pht k t lc kt thc qu trnh thm dung dch nht chitosan vo hn hp, tip tc thm vo 0.012 ml dung dch Glu 25%, tc khuy khng i. Sau 5 pht, gim tc khuy v 500 vng/pht. Trong 2 gi tip theo, 0.024 ml dung dch Glu 25% c thm vo, mi ln 0.012 ml v cch nhau 60 pht. Tip tc duy tr vn tc khuy khng i trong 1 gi na ri ngng. Hn hp lc ny chuyn thnh dng huyn ph vi cc ht chitosan lin kt ngang phn tn trong du c yn trong 1 gi cho cc ht ny lng xung. Lc ly phn kt ta em
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ly tm loi b phn du tha. Ra vi ln vi ether, kh trong khng kh, thu c bt chitosan. Bt chitosan thu c trong cc th nghim trn c chp nh hin vi SEM kho st hnh dng, kch thc v cu trc b mt ca ht. 3 KT QU V THO LUN 3.1 nh hng ca phng php ch to ln kch thc v hnh dng ht chitosan 3.1.1 Phng php c hc Bt chitosan lin kt ngang thu c t phng php ny c mu nu nht, khng mi. Kt qu chp nh hin vi SEM ca th nghim ny th hin trong hnh 1a.

Hnh 1: nh SEM ht chitosan hnh thnh bng phng php c hc (a - tri) v bng phng php nh tng (b phi)

Chitosan to ra khng t c hnh dng cu nh mong mun. B mt gm nhiu lp vy nh xp chng ln nhau. Do nh hng ca lc khuy nn xut hin nhng vt ui ko di pha sau ht. Kch thc v hnh dng ht khng n nh. Nh vy, phng php ny khng cho kt qu tt khi khuy vi vn tc 1600 vng/pht. 3.1.2 Phng php nh tng Kt qu chitosan thu c c mu nu nht, l mt khi ln gm nhng ht nh kt dnh li vi nhau (Hnh 1b). Ht to ra theo phng php ny da trn c ch hnh thnh nhng ht nh cha dung dch chitosan bn trong nn c th thy cc ht thu c c hnh dng trn hn so vi phng php c hc. Tuy nhin, do b mt ht gel chitosan lc ny rt mm v dnh, b mt ht b bin dng v xy ra hin tng kt khi.

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3.1.3 Phng php bin tnh ha hc Kt qu bt chitosan thu c c mu vng m (Hnh 2; 3).

Hnh 2: Bt chitosan

Hnh 3: nh SEM ca ht chitosan ch to bng phng php bin tnh ha hc

Phng php ny to c ht chitosan rt trn, ng knh bin thin trong khong t 5 m n 13 m. Chnh nhng lin kt ngang Glu lm tng lc lin kt gia cc mch polyme ca chitosan, lm b mt ht tr nn cng hn so vi ht ch to bng phng php nh tng nn trnh c hin tng kt khi khi ly tm. So snh vi kt qu to ht chitosan lin kt ngang bng phng php sy phun (Spray-Drying) m Oliveira B. F. et al. (2005) s dng, ht thu c t th nghim c ng dng cao hn v khong bin thin kch thc nh hn. Tuy nhin, cc ht chitosan vn cha tch ri nhau hon ton do thi gian khuy thp, ph hp vi kt lun ca Rajesh R. Dubey et al. (2003) khi s dng vn tc khuy nh hn 2000 vng/pht. Nh vy, trong cng mt iu kin th nghim (nguyn liu ban u, nhit , tc khuy 1600 vng/pht), phng php bin tnh ha hc trc tip cho kt qu tt nht, ht to ra t c hnh dng nh mong mun. Phng php ny s c s dng trong cc th nghim tip theo kho st nh hng ca qu trnh khuy n s hnh thnh ht chitosan. 3.2 Kho st nh hng ca s khuy trn n kch thc v hnh dng ht 3.2.1 nh hng ca tc khuy Tc khuy mi th nghim c thay i theo cc mc: 1200, 1400, 1600 vng/pht. Khong thi gian tnh t lc kt thc thm dung dch nht chitosan n trc khi thm dung dch Glu ln th nht (khong thi gian to ht) c c nh l 5 pht trong c 3 th nghim. Tng thi gian khuy l 190 pht. em ht chitosan thu c i chp nh hin vi SEM, thu c cc kt qu nh hnh 4. Kt qu chp nh hin vi SEM ca ht hnh thnh bng phng php bin tnh ha hc, theo cc tc khuy khc nhau trong cng khong thi gian khuy to ht l 5 pht, ph hp vi gi thuyt ban u v nh hng ca tc khuy ln qu trnh to ht.

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Kch thc (gn ng) ca ht ln nht ghi nhn c tng ng vi cc vn tc khuy 1200, 1400, 1600 vng/pht l 55 m, 34 m, 25 m. Nh vy, vn tc khuy ln s lm gim kch thc trung bnh ca ht v cc ht t b kt dnh hn. Xc sut xut hin nhng ht c kch thc nh (nh hn 5 m) trong 3 hnh chp tng dn theo vn tc khuy. Vn tc khuy ln, lc khuy mnh lm ht b v ra thnh nhiu ht nh hn. Gia cc ht khng c s ng nht v kch thc do s phn b khng u ca lc khuy trong dung dch. Tng vn tc khuy s lm gim khong bin thin kch thc gia cc ht.

(a)

(b)

(c)

Hnh 4: nh SEM ca ht chitosan hnh thnh bng phng php bin tnh ha hc vi vn tc khuy 1200, 1400 v 1600 vng/pht; (a), (b), (c) tng ng

3.2.2 nh hng ca thi gian khuy Tuy vn tc khuy 1600 vng/pht cho kt qu tt nhng cng gy hao ht mt lng ln dung dch do dung dch b vng ra ngoi trong khi khuy. Do , vn tc khuy 1000 vng/pht c chn s dng trong c 3 th nghim. Khong thi gian tnh t lc kt thc thm dung dch nht chitosan n trc khi thm dung dch Glu ln th nht c thay i theo 3 mc: 60, 120, 240 pht. Th nghim tin hnh trong iu kin bnh thng ca phng th nghim. Kt qu chp nh hin vi SEM nh hnh 5:

(a)

(b)

(c)

Hnh 5: nh hin vi SEM ca ht chitosan hnh thnh bng phng php bin tnhha hc vi thi gian khuy 60, 120 v 240 pht; (a), (b), (c) tng ng

Th nghim ny cho thy r c tc ng ca thi gian khuy n qu trnh hnh thnh ht micro chitosan. T mt khi ln gm nhng ht nh dnh lin vo nhau ban u, khi thi gian khuy tng ln, ht dn tch khi nhau v n mt lc no th tch hn ra thnh nhng ht ring bit. S kt khi c nh hng rt ln n tnh cht ca ht (v d nh kh nng hp ph) do n lm thay i hnh dng, kch thc v lm gim din tch b mt ca ht. Kt qu cho thy rng, vi th nghim c thi gian khuy 60 pht v 120 pht, tc 1000 vng/pht khng sc to c ht cu c dnh b mt thp nh khi s dng tc 1200 vng/pht trong 5 pht. iu ny chng minh nhn nh: tc
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khuy cng nh th kh nng kt khi gia cc ht cng ln (Rajesh R. Dubey et al., 2003). Tuy nhin, iu m hai ng cha cp n l khi thi gian khuy ln, ht s tch ri khi nhau hon ton nh kt qu ca th nghim s dng thi gian khuy 240 pht th hin trong hnh 5a. Nh vy, mun gim tnh kt khi ca ht, thay v s dng vn tc khuy ln (n 3000 vng/pht trong th nghim ca Rajesh R. Dubey et al., 2003) chng ta c th ko di thi gian khuy ra (tc khuy ln nht m my khuy trong phng th nghim t c l 2000 vng/pht ). Tuy vy, vi tc khuy tng th thi gian to ht c kch thc nh s gim. S lng ht c kch thc khong 5 m n 6 m xut hin trong 240 pht cng nhiu hn so vi 60 v 120 pht. T y rt ra kt lun, khi thi gian khuy c ko di, s c nhiu ht ln ban u b v nh n kch thc ti thiu tng ng vi lc m n chu tc ng, do lm tng ng u gia cc ht vi nhau. 3.3 So snh kh nng hp ph Cu2+ ca ht chitosan lin kt ngang kch thc nh vi ht c kch thc ln hn Ht chitosan lin kt ngang c kch thc nh (2-10 m) thu c bng phng php bin tnh ha hc trn, s dng vn tc khuy 1600 vng/pht v thi gian khuy l 240 pht s c s dng kho st kh nng hp ph Cu2+ (o bng my cc ph CPA-HH3) ri so snh vi kh nng hp ph ca ht c kch thc 2.5 mm ( Th M Phng, V Cm T, 2005). iu kin tin hnh th nghim nh sau: - C nh yu t nhit : cho ht chitosan hp ph Cu2+ trong dung dch Cu2+ 50 mg/L nhit phng. - Cn 4 g ht chitosan cho vo cc cha 68 ml dung dch Cu2+ 50 mg/L. B tr lm 3 cc ( thu ly kt qu trung bnh), mi cc s l mt ln lp li. - C sau 1 gi, mu 3 cc c ly ra mt t o hm lng Cu2+ cn li trong dung dch. o lin tc trong 10 gi. Kt qu th nghim trnh by trong bng 1.
Bng 1: Kt qu hp ph Cu2+ bng ht micro chitosan

Thi gian hp ph (h) 1 2 3 4 5 10

Nng Cu2+ cn li Trung bnh trong dung dch (mg/L) (mg/L) Cc I Cc II Cc III 6.41990 5.81482 5.80632 6.01368 5.53700 5.33381 5.52167 5.46416 5.09620 5.16558 5.33012 5.19370 4.71649 5.04134 4.97997 4.91260 4.70510 5.03095 4.97040 4.90215 3.90128 4.04670 3.89146 3.94648

Hiu sut hp ph (%) 87.87 88.98 89.52 90.09 90.12 92.04

Hiu sut hp ph ca ht micro chitosan trong 1 gi u tin v sau 10 gi ln lt l 87.87% v 92.04% (so vi ht 2.5 mm l 73.19% v 86.84%). Sau 1 gi, hiu sut m ht micro chitosan t c ln hn ht 2.5 mm hp th trong 10 gi. th so snh hiu sut hp ph gia ht micro chitosan v ht chitosan kch thc 2.5 mm c trnh by trong hnh 6.
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100 80 60 40 20 0 1
Ht micro chitosan Ht chitosan 2.5 mm

Hi u su t h p ph (%)

10

Thi gian hp ph (h)

Hnh 6: th so snh hiu sut hp ph gia ht micro chitosan v ht chitosan kch thc 2.5 mm

T th ta thy rng, hiu sut hp ph ca ht chitosan lin kt ngang c kch thc nh tng rt nhanh trong 1 gi u tin, sau tng chm dn cho n gi th 10, cn hiu sut hp ph ca ht 2.5 mm tng dn sau 10 gi. Kt qu ny c gii thch da trn s khc bit v kch thc gia hai loi ht. Ht micro chitosan c kch thc rt nh nn t c tng din tch b mt hp ph ln hn nhiu ln so vi ht 2.5 mm s dng cng mt khi lng. Do vy, tc hp ph ca ht micro chitosan ln v nhanh chng t c hiu sut ti u ch trong mt thi gian ngn, sau n nh dn. 4 KT LUN Nhng yu t nh hng n qu trnh to ht chitosan lin kt ngang c kch thc nh nh: phng php ch to, vn tc khuy, thi gian khuy c kho st. Trong cng iu kin th nghim, vi 3 phng php iu ch nu, phng php bin tnh ha hc trc tip cho kt qu tt nht, ht thu c c kch thc nh (5-13 m), hnh cu trn u v t b kt khi. Cc kt qu th nghim cng chng t mi quan h gia qu trnh khuy v s hnh thnh ht. Vn tc khuy ln (1600 vng/pht) s lm gim kch thc ht, thi gian khuy di (240 pht) lm tng ng u v gim tnh kt khi ca ht. Hiu sut hp ph Cu2+ ca ht chitosan lin kt ngang kch thc nh tc hp ph s tng ln, do , nu dng chng tch kim loi nng trong nc thi s va tng c hiu sut hp ph va rt ngn thi gian v ht bn d x l.

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TI LIU THAM KHO


Th M Phng, V Cm T, Nghin cu nh hng ca kch thc ht gel chitosan lin kt ngang v nhit ln kh nng hp ph ng trong nc, Lun Vn Tt Nghip i hc, i hc Cn Th, Cn Th, 2005. Nguyn Th Thy i, Nguyn Hong o, Ti u ha quy trnh trch ly chitin theo m, Lun Vn Tt Nghip i hc, i hc Cn Th, Cn Th, 2004. Oliveira1 B. F., Santana M. H. A., R M. I., Spray-Dried Chitosan Microspheres CrossLinked With D, L-Glyceraldehyde As A Potential Drug Delivery System: Preparation and Characterization, 2005, 22, 353-360. Rajesh R. Dubey, Rajesh H. Parikh, Two-Stage Optimization Process For Formulation of Chitosan Microspheres, Vallabh Vidyanagar, Gujarat, 2003. Singla A. K., Sharma M. L., Dhawan S., Nifedipine loaded chitosan microspheres: Internal structure characterization, Biotech Histochem, 2001, 76, 165-171.

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