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Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan

Biomaterials24(2003)5015–5022

Preparation and modi?cation of N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier Yongmei Xu,Yumin Du*,Ronghua Huang,Leping Gao

Department of Environmental Science,Wuhan University,Wuhan,Hubei430072,China

Received14October2002;accepted30May2003

Abstract

N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride(HTCC)is water-soluble derivative of chitosan(CS),synthesized by the reaction between glycidyl-trimethyl-ammonium chloride and CS.HTCC nanoparticles have been formed based on ionic gelation process of HTCC and sodium tripolyphosphate(TPP).Bovine serum albumin(BSA),as a model protein drug,was incorporated into the HTCC nanoparticles.HTCC nanoparticles were110–180nm in size,and their encapsulation ef?ciency was up to90%.In vitro release studies showed a burst effect and a slow and continuous release followed.Encapsulation ef?ciency was obviously increased with increase of initial BSA concentration.Increasing TPP concentration from0.5to0.7mg/ml promoted encapsulation ef?ciency from46.7%to90%,and delayed release.As for modi?ed HTCC nanoparticles,adding polyethylene glycol (PEG)or sodium alginate obviously decreased the burst effect of BSA from42%to18%.Encapsulation ef?ciency was signi?cantly reduced from47.6%to2%with increase of PEG from1.0to20.0mg/ml.Encapsulation ef?ciency was increased from14.5%to 25.4%with increase of alginate from0.3to1.0mg/ml.

Keywords:Chitosan;Quaternized chitosan;Bovine serum albumin;Nanoparticles;In vitro

1.Introduction

Chitosan(CS)is a deacetylation derivative of chitin, the second abundant polysaccharide present in nature. Thanks to its biocompatibility and mucoadhesivity,CS has been formulated as?lms,beads,intragastric?oating tablets,microspheres,and nanoparticles in the pharma-ceutical?eld[1,2].N-(2-hydroxyl)propyl-3-trimethyl ammonium chitosan chloride(HTCC)can be prepared by a relatively easy chemical reaction of CS and glycidyl-trimethyl-ammonium chloride(GTMAC). Quaternized CS is potential to be used as an absorption enhancer across intestinal epithelial due to its mucoad-hesive and permeability enhancing property[3].CS microspheres were chemically modi?ed by quaterniza-tion in several https://www.wendangku.net/doc/0110573164.html,pared with the CS micro-spheres modi?ed by reagents to introduce aliphatic and aromatic acyl groups,those quateraminated by GTMAC display the highest adsorption of antigen and indomethacin due to quaternary aminonium group of HTCC strongly electro-static attraction with the nega-tively charged antigen and indomethacin[4,5].But the literature about quaternary CS nanoparticles is less. Solubility of CS is poor above pH6,it will lose the charge when precipitating from solution,rendering it unsuitable for ionic adsorption in neutral and basic environments,and HTCC has excellent water solubility over wide pH range[6].Besides,compared with CS salts, the idea of making particles consisting solely of soluble polymers becomes appealing in order to avoid negative effects of the organic solvents and high-energy sources required for the formation of nanoparticles on delicate macromolecular[7].

Alginate and polyethylene glycol(PEG)have been widely used in biomaterial application.Alginate,a naturally occurring copolymer of guluronic and man-uronic acid,has been modi?ed with polycationic polymer to increase the stability of capsules or to minimize the loss of encapsulated material[8–11].PEG is hydrophilic?exible,non-ionic and biodegradable, PEG-coated nanoparticles have been found to be of

*Corresponding author.Fax:+86-27-8768-6402.

E-mail address:duyumin@https://www.wendangku.net/doc/0110573164.html,(Y.Du).

important potential in therapeutic applications as injectable colloidal systems for the controlled release of drugs and site-speci?c drug delivery[12–15]. Therefore,the major goal of the work presented here is to create new nanoparticles appropriately modi?ed and to evaluate their potential as protein carriers.The physicochemical structure of HTCC nanoparticles was analyzed by FTIR and transmission electron micro-scopy(TEM).We investigated the in?uence of alginate and PEG modi?cation on improving the stability of bovine serum albumin(BSA)encapsulation and modu-lating their encapsulation and release property.

2.Material and method

2.1.Materials

CS from a shrimp shell was purchased from Yuhan Ocean Biochemical Co.(Zhejiang Yuhuan,China), deacetylation degree was92%,and molecular weight (Mw)was210,000.BSA with Mw68,000and PEG with Mw20,000were purchased from Sigma Chemical Co. (USA).HTCC was prepared according to Ref.[16].All other chemicals were of reagent grade.

2.2.Preparation of HTCC nanoparticles and

modi?cation

2.2.1.HTCC nanoparticles preparation

HTCC was dissolved in distilled water at various concentrations(0.8,1.2,1.6,2.0mg/ml),and then under stirring at room temperature2ml sodium tripolypho-sphate TPP aqueous solution with various concentra-tions(0.3,0.5,0.7,0.8,1.0,1.2mg/ml)was,respectively, added to5ml of HTCC solution.Three kinds of formations were observed:solution,aggregates and opalescent suspension.The zone of opalescent suspen-sion was further examined as nanoparticles.

2.2.2.Modi?cation of nanoparticles

PEG modi?ed nanoparticles were formed sponta-neously upon incorporation of2ml of the TPP solution (0.7mg/ml)and6ml of the HTCC solution containing various concentrations of PEG(1.0, 2.0, 5.0,10.0, 20.0mg/ml).Alginate modi?ed nanoparticles were formed upon incorporation of2ml of the TPP solution (0.3,0.5,0.7mg/ml)containing sodium alginate(0.3,

0.5,0.7mg/ml)and5ml of the HTCC solution

(1.6mg/ml).

The residual polysaccharides in clear supernatant of gelated suspension were determined according to Ref.

[17].About4ml undiluted sulfuric acid was added to the 2ml ultra-centrifuged supernatant of gelated suspen-sion;absorbance at490nm of the degraded solution was examined by UV–visible absorption spectrophotometry,which indicated indirectly the amount of residual polysaccharide.

2.2.

3.Preparation of BSA loading nanoparticles

BSA loading nanoparticles were formed upon incor-poration of TPP(0.5,0.8mg/ml)and HTCC solutions

(1.6mg/ml)containing BSA(0.2,0.5,0.8, 1.0, 1.5,

2.0mg/ml).BSA concentration was1.5mg/ml for the preparation of PEG and alginate modi?ed nanoparti-cles-loaded BSA.

Detailed preparation conditions were shown in the corresponding?gures and table.

2.3.Physicochemical characterization of HTCC nanoparticles

The particles size and morphological measurements of the nanoparticles were performed by TEM-100CXII. Zeta potential measurements of nanoparticles were performed by microelectrophoresis apparatus(Model BDL-B,Shanghai,China).IR spectra of CS,HTCC, HTCC nanoparticles,BSA-loaded nanoparticles and BSA were taken with KBr pellets on Perkin-Elmer spectrum on FTIR.

2.4.Determination of BSA encapsulation ef?ciency of nanoparticles

Encapsulation ef?ciency of the different formations was determined by ultra-centrifugation of samples at 20,000?g and15 C for30min,the amount of free BSA was determined in clear supernatant by UV spectro-photometry at280nm using supernatant of their corresponding non-loaded BSA nanoparticles as basic correction.The BSA encapsulation ef?ciency(AE)of the process was calculated from

AE?eAàBT=A?100;

where A is the total amount of BSA;and B the free amount of BSA.

2.5.BSA release from the nanoparticles in vitro

The in vitro BSA release pro?les of HTCC nanopar-ticles were determined as follows,the BSA-loaded HTCC nanoparticles separated from18ml suspension were placed into test tubes with6ml of0.9%(w/v) sodium chloride saline,and incubated at37 C under stirring.At appropriate intervals,samples were ultra-centrifuged,and4ml of the supernatant was replaced by fresh medium.The amount of BSA released from the nanoparticles was evaluated by modi?ed Coomassie Blue protein assay(Pierce Inc.,New York,NY,USA), which was different from the determinations of en-capsulation ef?ciency(UV spectrophotometry),so we investigated the error of both assay procedures,the

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results were in good agreement.The calibration curve was made using non-loaded BSA nanoparticles as correction.

All release tests were run in triplicate and the mean value was reported.

3.Results and discussion

3.1.Physicochemical characterization of HTCC nanoparticles

3.1.1.FTIR

Fig.1shows FTIR spectra of CS,HTCC,HTCC nanoparticles,BSA loading HTCC nanoparticles and BSA.There are three characteristic peaks of CS at 3434cmà1of n(OH),1094cmà1of d(C–O–C)and 1603cmà1of n(NH2)[18].Compared with CS,HTCC shows the disappearance of the NH2-associated band at 1600cmà1of the N–H bending in the primary amine; and appearance of a new band at1482cmà1,which is attributed to the methyl groups of the ammonium.The IR spectrum is consistent with the reported spectra [16,19,20].Characteristic peaks of alcohol and second alcohol between1160and1030cmà1are not changed in HTCC con?rming the lack of the introduction of an alkyl group at C-3and C-6of the CS[6,20].

The spectrum of HTCC nanoparticles is different from that of HTCC matrix.The intensity of1482cmà1peak becomes weaker,1637cmà1shifts to1655cmà1, and a new peak of1545cmà1appears.We observed a similar result in CS nanoparticles gelated TPP,and this is consistent with the result of CS?lm modi?ed by phosphate[21].So we suppose that TPP was linked with ammonium groups of HTCC in nanoparticles.Hydroxyl group absorption of CS at1243cmà1almost disappears in HTCC nanoparticles,which indicates that free hydroxyl groups form hydrogen bonding[22].Char-acteristic peaks of BSA have acetylamino I1657cmà1,II 1539cmà1and III1242cmà1,and3307cmà1of n(NH2) [18].Acetylamino I1657cmà1and II1539cmà1in BSA overlap with1655cmà1of d(NH)and1545cmà1in non-loaded HTCC nanoparticles,so more intensive peaks of both are shown in BSA loading HTCC nanoparticles,and the characteristic of1091cmà1of HTCC becomes weak.

3.1.2.TEM

Fig.2shows the morphological characteristic of nanoparticles.HTCC nanoparticles and HTCC nano-particles modi?ed by alginate have spherical shape and monodisperse.HTCC nanoparticles are about122nm in size.HTCC nanoparticles modi?ed by alginate are 113nm in size and more spherical and smoother.HTCC nanoparticles modi?ed by PEG are186nm in size and in irregular shape.Alginate and PEG play different roles in formation of nanoparticles.Alginate is anionic polymer, its COOàgroups can interact strongly with–N+(CH3)3 groups of HTCC,and the similarity of the polysacchar-ide structure of two biopolymer offers a great interac-tion,which results in a strong interchain reaction and more compact formation.Here alginate concentration was not high(0.3mg/ml),high alginate concentration easily results in aggregation.The size of alginate modi?ed nanoparticles is slightly smaller than that of pure HTCC,which may be due to the compact structure.Cenk Aral et al.also observed the same phenomenon when they prepared modi?ed CS bead based on TPP gelation process[23],smaller beads were obtained by addition of sodium alginate to TPP solution.PEG a non-ionic polymer cannot form particles with HTCC without TPP incorporation, PEG,interaction between the oxygen atom of PEG and HTCC ammonium groups is much weaker than that of alginate and HTCC,but it can affect nanoparticles formation.The structure of PEG modi?ed nanoparticles is looser,and the size is larger than that of pure HTCC.

3.2.In?uence of preparation and modi?cation on BSA encapsulation

3.2.1.Effect of initial concentration of BSA

Taking into account that the isoelectric point of BSA is4.8,we prepared nanoparticles in distilled water at pH 6.5,which favors the interaction of BSA and HTCC.

Fig. 1.FTIR of(1)CS,(2)HTCC,(3)HTCC nanoparticles,

(4)loaded BSA nanoparticles,and(5)BSA.

Y.Xu et al./Biomaterials24(2003)5015–50225017

The formation of nanoparticles is only possible for some speci?c concentration of HTCC and TPP,too high HTCC concentration (2.0mg/ml)led to clear solution,no nanoparticles formation,too high TPP (1.2mg/ml)led to aggregates with large size.As shown in Fig.3,the higher the initial concentration of BSA,the higher the encapsulation ef?ciency.As for preparation with TPP 0.8mg/ml,increasing BSA concentration from 0.2to

0.8mg/ml increased encapsulation ef?ciency of BSA from 71%to 92%.As for preparation with TPP 0.5mg/ml,increasing BSA concentration from 1.0to 2.0mg/ml increased BSA encapsulation ef?ciency from 46%to 74%.When concentration of HTCC and TPP was kept constant,too high BSA concentration (BSA 1.0mg/ml when TPP 0.8mg/ml,BSA 2.0mg/ml when TPP 0.5mg/ml)resulted in aggregates.TPP with higher concentra-tion can gelate more amount of HTCC,and so relatively greater amount of BSA can be associated in nanopar-ticles.

3.2.2.Effect of alginate modi?cation

Table 1shows that increasing alginate concentration promoted BSA encapsulation ef?ciency.Vandenberg has observed the similar phenomenon.Encapsulation ef?ciency of BSA was promoted by increasing one of the concentration of CS and alginate in the system of BSA-loaded alginate/CS coacervate microcapsules [11].The amount of residual polysaccharide in clear supernatant of encapsulated suspension is not increased with increase of alginate concentration in Fig.4,which indicates adequate alginate incorporated with HTCC during the process of entrapping BSA.

However,theoretically the negatively charged COO àof alginate may compete with BSA to form polyelec-trolyte complex with HTCC,which may lead to the decreased encapsulation ef?ciency of BSA.There are

(a)(b)

(c)(d)

Fig.2.TEM of nanoparticles (a)and (b)pure HTCC,(c)alginate modi?ed HTCC,and (d)PEG modi?ed HTCC.

BSA concentration (mg/ml)

B S A e n c a p s u l a t i o n e f f i c i e n c y

Fig.3.In?uence of BSA initial concentration on its encapsulation ef?ciency,date shown are the mean 7standard deviation (n ?4).

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three types of ionic interactions that contribute to the crosslinked networks of alginate modi?ed HTCC nanoparticles:the interaction between opposite charges of alginate and HTCC,junction formed by tripolypho-sphoric and àN +(CH 3)3of HTCC and interaction between COO àof BSA and –N +(CH 3)3of HTCC [24].Competition between alginate and BSA and physical encapsulation between alginate and HTCC exists during gelation process at the same time.Adding alginate results in increased BSA encapsulation ef?ciency,illustrating that physical encapsulation plays more important role in entrapping BSA than competition does.

3.2.3.Effect of PEG modi?cation

Fig.5shows that increasing PEG concentration from 1.0to 20.0mg/ml decreased encapsulation ef?ciency of BSA from 47%to 2%.The incorporation of PEG in CS gel system is through intermolecular hydrogen between the electro-positive amino hydrogen of CS and the electro-negative oxygen atom of PEG,thus forming CS and PEG semi-interpenetration network [25],so we suppose that PEG is attached to HTCC nanoparticles.PEG was added to HTCC solution prior to gelation,

without TPP incorporation,PEG cannot gelate with HTCC,but –N +(CH 3)3groups of HTCC can be occupied by the oxygen atom of PEG,which may compete in their interaction with HTCC ammonium groups,so the possibilities of ion interaction between the BSA and HTCC are reduced,the entanglement of PEG chain with HTCC molecule hinders BSA from encapsu-lating into the nanoparticles.

3.3.In vitro release of BSA from the nanoparticles The preliminary protein release test from HTCC or modi?ed HTCC nanoparticles in vitro proves that they had a sustained release form as shown from Figs.6–8.Ionic strength in release medium may affect signi?cantly the release properties of HTCC nanoparticles based on ionic gelation,so we choose 0.9%(w/v)sodium chloride saline as release medium which simulates body ?uid according to the literatures [26,27].The in vitro protein release pro?les obtained for each formulation showed four phases compositions [28]:

1.A ?rst initial burst release of 17–45%,due to the drug desorption from the particles surface.

2.A reversed release in following 12h,due to BSA readsorption onto nanoparticles surface again.As reported in relative literature,during this phase,erosion of polymer could occur through the reversal of the gelation reaction,ionic exchanges between polymer and release medium,which results in the solubilization of HTCC molecules,or through the degradation of the backbone into smaller Mw components [29].But we did not observe solution and degradation of nanoparticles in ?rst 12h by

r e s i d u a l p o l y s a c c h a r i d e s a b s o r b a n c e

alginate concentration(mg/ml)

Fig.4.In?uence of alginate on residual polysaccharides in gelated suspension (HTCC 1.6mg/ml,initial BSA 1.0mg/ml).

B S A e n c a p s u l a t i o n e f f i c i e n c y (%)

PEG concentration (mg/ml)

Fig. 5.In?uence of PEG modi?cation on BSA encapsulation ef?ciency,date shown are the mean 7standard deviation (TPP 0.7mg/ml,BSA 1.0mg/ml,n ?4).

Table 1

In?uence of TPP and alginate concentration on BSA encapsulation,date shown are the mean values (HTCC 1.6mg/ml,initial BSA 1.0mg/ml,n ?4)

Alginate concentration (mg/ml)TPP concentration (mg/ml)0.3

0.50.700a

47.6%90%

0.314.5%100%b

b 0.517.5%b b 0.7

25.4%

a Clear solution,no nanoparticles.b

Aggregates.

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GPC.HTCC nanoparticles swelled immediately in sodium chloride saline,swelling degree was up to 200%in Fig.9,the increased speci?city of particles may lead to a high amount of BSA readsorption and a low amount of free BSA in the supernatant [29].This phenomenon has also been observed on BSA-loaded PLGA microspheres by Crotts and Park [30],BSA-loaded alginate microspheres by Lemoine [9]and protein C-loaded MPEO-PLA nanoparticles by Zambaux [31].

3.A plateau for the following 3days,resulting from the only diffusion of the drug dispersed in the polymer matrix.

4.A constant sustained release of the drug,resulting from the diffusion of the protein through the polymer wall as well as its erosion.3.3.1.Effect of TPP concentration

As shown in Fig.6,the higher TPP concentration resulted in the lower release rate.The initial burst release from nanoparticles with TPP concentration of 0.5and 0.8mg/ml is 43%and 28%,respectively.The burst release of BSA occurred instantaneously,which indi-cates the burst released BSA residues on the surface of nanoparticles.The lower burst release from nanoparti-cles with highly crosslinked nanoparticles suggests that main BSA molecules are encapsulated inside the matrix,highly crosslinked networks enhance encapsulation.3.3.2.Effect of PEG and alginate modi?cation

In vitro protein release pro?les obtained for the modi?ed HTCC are illustrated by Figs.7and 8.The burst effect is less pronounced (18%of entrapped amount)for modi?ed HTCC nanoparticles than for pure HTCC nanoparticles (42%of entrapped amount).The much lower burst release from alginate modi?ed nanoparticles suggests that the main BSA are encapsu-lated inside the matrix.In the alginate nanoparticles system reported previously,addition of opposite poly-electrolyte to the gelation medium resulted in reduced drug loss and microcapsule swelling [32,33],improved stability [22,34]and reduced microcapsule permeability [35],positively charged amino groups of CS formed membranes through ionic interactions with carboxylic residues of the alginate [36].Adding alginate 1.0mg/ml in gelation medium,zeta potential of nanoparticles was decreased from 5.1842to 4.4572mV (HTCC 1.6mg/ml,TPP 0.7mg/ml,BSA 1.0mg/ml),which indicates that alginate incorporated with HTCC and modi?ed surface of particles,and is consistent with the result of residual polysaccharides analysis in clear supernatant of encap-sulated suspension.BSA was dissolved in HTCC solution before gelation,some –N +(CH 3)3of HTCC have been occupied by COO àof BSA,BSA Mw (68,000)is much smaller than alginate Mw (850,000),adding alginate and TPP in gelation medium,unoccu-

0C u m u l a t i v e B S A r e l e a s e (%)

Time (days)

Fig.6.In?uence of TPP concentration on BSA release,date shown are the mean 7standard deviation (initial BSA 1.0mg/ml,

n ?3).

0 C u m u l a t i v e B S A r e l e a s e (%)

Time (days)

Fig.7.In?uence of alginate modi?cation on BSA release,date shown are the mean 7standard deviation (TPP 0.3mg/ml,alginate 1.0mg/ml,initial BSA 1.0mg/ml,n ?

3).

0102030405060

C u m u l a t i v e B S A r e l e a s e (%)

Time (days)

Fig.8.In?uence of PEG modi?cation on BSA release,date shown are the mean 7standard deviation (TPP 0.7mg/ml,PEG 2.0mg/ml,initial BSA 1.0mg/ml,n ?3).

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pied groups of–N+(CH3)3may interact with alginate immediately,which results in entrapping ef?ciently BSA inside the nanoparticles.Though the ionic interaction of BSA with HTCC may be interfered by the presence of alginate in the gelation medium,which could lead to the decreased stability of BSA,the observed results illustrate physical encapsulation may play a leading role in entrapping BSA.The similarity of the polysaccharide structure of HTCC and alginate offers a strong interchain reaction,which results in the promoted stability of particles[37].Thus incorporation of alginate promotes the stability of nanoparticles surface,which reduces the burst release of BSA.

PEG also modi?es the release pro?le of HTCC nanoparticles,reduces the burst effect.

4.Conclusion

This study demonstrates that spherical and mono-dispersed HTCC nanoparticles with a mean diameter of 110–180nm can be prepared by an ionic gelation method.High encapsulation ef?ciency of BSA(90%) is achieved,and the release pro?le of BSA from nanoparticles has an obvious burst effect and a slowly continuous release phase followed.Higher initial BSA concentration promotes encapsulation ef?ciency of BSA.Increasing TPP concentration enhances BSA encapsulation and reduces the burst release.Adding alginate promotes the encapsulation and obviously decreases the burst release of BSA.Adding PEG decreases the encapsulation and the burst effect.All these investigated features reveal that HTCC nanopar-ticles are a potential vehicle for the administration of proteins.

Acknowledgements

We gratefully acknowledge?nancial support from the National Natural Science Foundation of China(Foun-dation No:29977014).References

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