《chitosan.docx》由会员分享，可在线阅读，更多相关《chitosan.docx（38页珍藏版）》请在三一文库上搜索。
1、chitosanAvailable online at http:/ International Journal of Pharmaceutics349(2008)226233Pharmaceutical NanotechnologyIn vitro and in vivo study of N-trimethyl chitosannanoparticles for oral protein deliveryFu Chen,Zhi-Rong Zhang,Fang Yuan,Xuan Qin,Minting Wang,Yuan Huang?Key Laboratory of Drug Targe
2、ting of Ministry of Education,West China School of Pharmacy,Sichuan University,No.17,Block3,Southern Renmin Road,Chengdu610041,PR ChinaReceived8February2007;received in revised form18July2007;accepted30July2007Available online2August2007AbstractIn this study,the effects of alginate modi?cation on ab
3、sorption properties of FITC-BSA loaded TMC nanoparticles were investigated on an in vitro model of GI epithelium(Caco-2cells).The feasibility of applying TMC nanoparticles loaded with a model vaccine urease in oral vaccination was also studied.Alginate modi?ed TMC nanoparticles showed higher FITC-BS
4、A permeate ef?ciency than non-modi?ed TMC nanoparticles. However,alginate modi?cation barely had any effect on TMC nanoparticlesproperty of decreasing TEER or enhancing drug paracellular transport. Mice s.c.immunized with urease loaded TMC nanoparticles showed highest systematic immune response(IgG
5、levels)but the lowest mucosal response(secretory IgA levels).In the contrast,mice i.g.immunized with urease loaded TMC nanoparticles showed much higher antibody titers of both IgG and secretory IgA than those with urease solution or urease co-administrated with TMC solution.These results indicated t
6、hat TMC nanoparticles are potential carriers for oral protein and vaccine delivery.?2007Elsevier B.V.All rights reserved.Keywords:N-Trimethyl chitosan;Nanoparticles;Protein carriers;Oral vaccination1.IntroductionOral drug delivery is considered as the preferred route of administration because of its
7、 non-invasive nature.It reduces the risk of infection,and do not require trained personnel.Moreover, the micro-fold(M)-cells,mainly located within the epithelium of Peyers patches,called Follicle Associated Epithelium(FAE), are specialized for antigen sampling,which represent a potential portal for
8、oral delivery of peptides and for mucosal vaccina-tion,since they possess a high transcytotic capacity and are able to transport a broad range of materials,including nanopar-ticles(Frey and Neutra,1997;Clark et al.,2000).However, the bioavailability of orally administered biotechnology drugs, such a
9、s proteins and vaccines,are usually poor because of the hostile gastric and intestinal environments and also the poor gastrointestinal(GI)mucosal permeability.For the last10years,many strategies have been developed to enhance oral protein and vaccine delivery(Wang,1996;?Corresponding author.Tel.:+86
10、2885501617;fax:+862885501617.E-mail address:huangyuan0http:/ and Lee,2001;Steffansen et al.,2004;des Rieux et al., 2006).Among these approaches,nanoparticulate systems have attracted especial interests for the following reasons.First,they can protect drugs from degradation(Lowe and Temple,1994). Sec
11、ond,they improve drug transmucosal transport(Janes et al., 2001)and transcytosis by M cells(Gullberg et al.,2000;Clark et al.,2001).Third,they can provide controlled release properties for encapsulated drugs(Galindo-Rodriguez et al.,2005).Among these systems,chitosan nanoparticles have received part
12、icular interest because it is a very ef?cient and non-toxic absorption enhancer for both orally and nasally administered peptide drugs(Fernandez-Urrursuno et al.,1999;van der Lubben et al.,2001).But when put in physiological pH,chi-tosan will lose this capacity which can only be achieved in its prot
13、onated form in acidic environments(Kotze et al.,1999a). Recently,people turn their attentions to N-trimethyl chitosan chloride(TMC),a quaternized chitosan derivative,since it has perfect solubility in water over a wide pH range.It also shows absorption enhancing effects even in neutral and basic-pH
14、envi-ronments(van der Merwe et al.,2004).Moreover,recent studies showed that the use of TMC solution and nanoparticles in nasal vaccination(Baudner et al.,2004;Amidi et al.,2007),and TMC0378-5173/$see front matter?2007Elsevier B.V.All rights reserved. doi:10.1016/j.ijpharm.2007.07.035F.Chen et al./I
15、nternational Journal of Pharmaceutics349(2008)226233227microspheres in oral vaccination(van der Lubben et al.,2002) could enhance the immunogenicity of the vaccine,and improve the systemic and local immune response compared to free anti-gen.The local and systemic immune responses are mainly initi-at
16、ed by delivering antigens to M cells located in peyers patches. It is generally believed that particles above1?m are taken up by M cells and trapped in peyers patches(Jani et al.,1990),while nanoparticles(particles smaller than1?m)are taken up by M cells and delivered in the basal medium(des Rieux e
17、t al.,2005), thus the optimal size for particles to be transcytosed by M cells would be below1?m(Gullberg et al.,2000;Florence,2004; Shakweh et al.,2005).Although TMC microspheres had been studied for oral vaccination(van der Lubben et al.,2002),these microspheres had a diameter of around2?m.Therefo
18、re,it was necessary to study the feasibility of applying TMC nanoparticles in oral vaccination.Although TMC nanoparticles have many interesting features suitable for protein and vaccine delivery,such as high protein loading capacity,low cytotoxicity,and enhanced nanoparticle internalization(Amidi et
19、 al.,2006;Sandri et al.,2007),the release feature showed a high initial burst caused by the adsorp-tion of protein drugs on the particle surface(Amidi et al.,2006; Chen et al.,2007).This may compromise the drug bioavail-ability,because the attack of the drug by enzymes or acidic substances from the
20、body?uids may occur during the travelling of nanoparticles in GI tract(Borges et al.,2005).In a previ-ous study,we successfully reduced the initial burst by alginate modi?cation,but the incorporation of alginate had also changed particle size and zeta potential(Chen et al.,2007)while these two facto
21、rs are very important for particle uptake by intestinal cells (des Rieux et al.,2006),and the presence of negatively charged alginate at the surface of TMC nanoparticles may in?uence their ability to enhance drug permeation through paracellular path-way.Thus it is very important to investigate the e
22、ffect of alginate modi?cation on the absorption properties of TMC nanoparticles.Based on the above-mentioned reasons,the aims of the present work were to further investigate the intestinal epithe-lial absorption properties of TMC nanoparticles,especially the effect of alginate modi?cation on their a
23、bsorption properties using an in vitro model of GI epithelium(Caco-2cells),and the feasibility of applying TMC nanoparticles in oral vaccina-tion.Fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA)and Urease,a vaccine protein against Helicobacter pylori infection,were used as model pr
24、otein and vaccine.2.Materials and methods2.1.MaterialsChitosan(with95%degree of deacetylation(DD)and a molecular weight(M w)of200kDa)used for synthesiz-ing TMC was purchased from BoAo Biochemical Company (Shanghai,China).TMC with a degree of quaternization (DQ)of37%was synthesized by methylation of
25、chitosan using CH3I in a strong base(NaOH)and analyzed by1H nuclear magnetic resonance(NMR)spectroscopy as previously described(Sieval et al.,1998).The degree of quaterniza-tion(DQ)was calculated using the following equation,DQ (%)=(?TM/?H)1/9100(Thanou et al.,2000),where ?TM is the integral of the
26、trimethyl amino group(quaternary amino group)peak at3.3ppm and?H is the integral of the1H peaks from4.7to5.7ppm.FITC-BSA was obtained by labeling BSA(BoAo Biochemical Company,China)with FITC (Amresco,USA)according to a standard method(Schauenstein et al.,1978).Urease and Lucifer Yellow(LY)were pur-c
27、hased from Sigma(USA).Goat anti-mouse IgG-horseradish peroxidase conjugate(IgG-HRP)and avidin-horseradish perox-idase conjugate(Avidin-HRP)were purchased from Chengwen Biochemical Company(Beijing,China).Biotinylated goat anti-mouse IgA(IgA-biotin)was purchased from Kirkegaard& Perry Laboratories(USA
28、).Aprotinin and tetramethyl benzi-dine(TMB)were purchased from Amresco(USA).All other materials used were of analytical or pharmaceutical grade.2.2.Preparation of TMC nanoparticlesThe TMC nanoparticles were prepared by the ionic gelation of TMC with TPP anions.Ten milligrams of TMC and2mg of FITC-BS
29、A were dissolved in5ml of water.Subsequently, 2ml of TPP solution(0.6mg/ml)was added drop-by-drop to the above solution under magnetic stirring at room tempera-ture.Urease loaded TMC nanoparticles were prepared by the same method described above,except that only0.5mg of urease and7.5mg of TMC was ad
30、ded.Alginate-modi?ed FITC-BSA loaded nanoparticles were prepared by dissolving sodium algi-nate(0.6mg)in2ml of TPP(0.6mg/ml)and then adding to the mixture solution of TMC and FITC-BSA.TMC nanoparticle suspensions were centrifuged for10min at10,000g and10?C on a10?l glycerol bed,and then resuspended
31、in5mM HEPES (pH7.4)or Hanks balanced salt solution(HBSS)for characteri-zation,permeability investigation or immunization respectively.2.3.Characterization of TMC nanoparticlesThe size and zeta potential of the TMC nanoparticles were measured with a Malvern Zatasize NanoZS90(Malvern Instru-ments Ltd.
32、,Malvern,UK).The particle-size distribution is reported as a polydispersity index(PDI).The amounts of protein (FITC-BSA or urease)loaded in the nanoparticles were deter-mined as described before(Chen et al.,2007),only the amount of FITC-BSA remaining in the supernatant was assayed by measur-ing?uore
33、scence using a spectro?uorophotometer(RF-S301PC, SHIMADZU,Japan)atex=498nm andem=527nm.The amount of urease was correlated to the amount of ammonia released from urea in the phenol-hypochlorite urease assay. Therefore,the determination of urease was carried out accord-ing to previous literature with
34、 slight modi?cation(Mcgee et al., 1999).Supernatants were added to urease buffer(0.05M PBS pH7.5plus25mM urea)in a25ml?nal volume and were incubated at37?C for20min.The reaction was stopped by removal of an aliquot which was added to a cuvette containing 1.5ml of solution A(38g/l of phenol and400mg/
35、l of sodium nitroprusside).An equal volume(1.5ml)of solution B(NaOH228F.Chen et al./International Journal of Pharmaceutics349(2008)226233 0.5MNaClO0.044%,vol/vol)was added at the same time.Following incubation at37?C for30min,the absorbance wasmeasured at625nm using Cintra10e UVvis Spectrometer(GBC
36、Scienti?c Equipment,Australia).Loading ef?ciency(LE)and loading capacity(LC)were calculated as follows:LE=Total amount of protein?free proteinTotal amount of protein100%LC=Total amount of protein?free proteinnanoparticles dry weight100%2.4.Permeability studies of FITC-BSA loaded TMC nanoparticles us
37、ing Caco-2cell monolayerCaco-2cells(passages23)were seeded on tissue culture treated polycarbonate?lters(area4.7cm2)in Costar Transwell 6-well plates,respectively(Costar,USA)at a seeding den-sity of4105cells/cm2.Dulbeccos Modi?ed Eagles Medium (DMEM,pH7.40;Sigma,USA),supplemented with1%non-essential
38、 amino acids,10%foetal bovine serum,benzylpenicillin G(160U/ml)and streptomycin sulphate(100mg/ml)(all obtained from Sigma),was used as culture medium,and added to both apical and basolateral side of the cell monolayers.The medium was changed every second day and cell cultures were kept at a tempera
39、ture of37?C in an atmosphere of95%air and 5%CO2.Filters were used for transepithelial electrical resis-tance measurements and transport experiments2123days after seeding.Transport experiments were carried out following proce-dures described previously(Sandri et al.,2007).One milliliter of FITC-BSA l
40、oaded non-modi?ed or alginate modi?ed TMC nanoparticle suspension at either5mg/ml or20mg/ml in HBSS (buffered with30mM HEPES,adjusted with0.1M NaOH to pH7.4)was applied at the apical side of the cell monolayers, and3ml of buffered HBSS was applied at the basolateral side of the monolayers.Filters we
41、re moved into fresh basolateral medium at1,2,3h,and all basolateral phases were collected. The permeated nanoparticles were assayed by measuring?uo-rescence as above mentioned.After3h,the cells were washed three times with HBSS.Thereafter,1ml of LY HBSS solution (25?g/ml)was added at apical side,and
42、3ml of fresh HBSS at basolateral side.One hour later,the permeated LY was assayed on a spectro?uorophotometer atex=433nm andem=541nm. Permeability of nanoparticles and LY was calculated using the following equation:Permeate ef?ciency(PE)=cumulative amount transportedinitial amount100%During the expe
43、riments,the transepithelial electrical resis-tance(TEER)of the monolayers was measured at0,0.5,1,2,3, 4h,using a Millicell ERS-meter(Millipore Corp.,USA).2.5.Immunization studies of urease loaded TMC nanoparticles in mice2.5.1.Animal immunizationFemale Kunming mice,68weeks old,were housed under path
44、ogen-free conditions with food and drink provided ad libitum.Mice were immunized as designed(Table1).All for-mulations were prepared and/or resuspended in HBSS.Groups of6mice were immunized3times on days0,21and42either by the oral route(i.g.)or by the subcutaneous route(s.c.).For mice treated with f
45、ormulation2and3,0.3ml of0.1M carbonate buffer(pH9.6)was administrated into the stomach0.5h before immunization to neutralize the pH.Three weeks after the?nal immunization,blood samples of mice were collected from orbit venous plexus and serum samples were obtained by centrifugation at10,000g and 4?C
46、 for10min.Thereafter,mice were sacri?ced and then gastric and intestinal secretions were collected.Gastric secre-tions were collected in cellulose wicks placed on intact gastric mucosa for1min.Then,wicks were moved into lavage medium (PBS pH7.4with0.2?M aminoethyl-benzene sulfonyl?u-oride and1?g/ml
47、aprotinin)and secretions were recovered from the wicks by centrifugation(Lee et al.,1999).Intesti-nal secretions were collected using a modi?ed procedure described by Vila et al(Vila et al.,2004).Lengths(10cm) of ileum from each mouse were sectioned longitudinally,and the luminal mucosa was carefull
48、y scraped into lavage medium, then mixed and centrifuged.Supernatants were used for investigation.2.5.2.Enzyme-linked immunosorbent assay(ELISA)The urease-speci?c IgG in serum and urease-speci?c IgA in gastric and intestinal secretions were examined by ELISA. For analysis of IgG,96-well plates were
49、coated overnight at 4?C with10?g urease in100?l of0.05M carbonate buffer (pH9.6)and blocked with200?l of1%(m/v)bovine serum albumin for1h at37?C.After washed three times with PBS-Tween(PBS containing0.05%Tween20,pH7.4),samples (serum diluted serially in twofold steps)were added to the plates at100?l/well,and incubated at37?