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In vivo biocompatibility and degradability of a novel

In vivo biocompatibility and degradability of a novel
In vivo biocompatibility and degradability of a novel

Biomaterials23(2002)181–191

In vivo biocompatibility and degradability of a novel

injectable-chitosan-based implant

Fwu-Long Mi a,Yu-Chiun Tan b,Hsiang-Fa Liang b,Hsing-Wen Sung b,*

a Di v ision of Chemistry,Department of Mathematics,Physics and Chemistry,Chinese Na v al Academy,669Jiun Shiaw Road,

Kaohsiun g813,Taiwan,ROC

b Department of Chemical En g ineerin g,National Tsin g Hua Uni v ersity,Hsinchu30013,Taiwan,ROC

Received10October2000;accepted23February2001

Abstract

A novel injectable-chitosan-based delivery system with low cytotoxicity was fabricated in the study.The chitosan microspheres with small particle size,low crystallinity and good sphericity were prepared by a spray-drying method followed by treating with a crosslinker.In the study,a naturally occurring crosslinking reagent(genipin),which has been used in herbal medicine and in the production of food dyes,was used to crosslink the chitosan microspheres.The glutaraldehyde-crosslinked counterparts were used as a control.Histological study of the genipin-crosslinked chitosan microspheres injected intramuscularly into the skeletal muscle of a rat model showed a less in?ammatory reaction than its glutaraldehyde-crosslinked counterparts.The results of the scanning electron microscopic examination indicated that the glutaraldehyde-crosslinked chitosan microspheres retrieved at12-week postoperatively were already degraded into a loose and porous structure.However,the degradation of the genipin-crosslinked chitosan microspheres was not signi?cant after20weeks of implantation.The results of the study demonstrated that the genipin-crosslinked chitosan microspheres have a superior biocompatibility and a slower degradation rate than the glutaraldehyde-crosslinked chitosan microspheres.Accordingly,the genipin-crosslinked chitosan microspheres may be a suitable polymeric carrier for long-acting injectable drug delivery.#2001Elsevier Science Ltd.All rights reserved.

Keywords:Chitosan;Microspheres;Genipin;Glutaraldehyde;Degradation;Biocompatibility

1.Introduction

A variety of synthetic or natural polymers have been employed as the controlled-release drug delivery system in humans[1].Among them,biodegradable polymers have become increasingly important in the development of implantable biomaterials and drug delivery devices [2–6].A major advantage of these systems is that an invasive technique such as surgery is not required for their removal.The degradation of polymeric materials could be achieved through the process of hydrolysis or enzyme-speci?c reaction.Among the enzymatically degradable polymers that have been developed and evaluated for drug delivery usage,chitosan is considered to be an excellent one.

Chitosan is a copolymer of glucosamine and N-acetylglucosamine derived from the natural polymer chitin,which is commercially available.It has been reported that chitosan is a potentially useful pharma-ceutical material owing to its good biocompatibility and low toxicity[7].For these reasons,chitosan has been used in many applications in the formulations employed in drug delivery[8,9].Since chitosan can be hydrolyzed by lysozyme,it is also one of the biodegradable polymers in nature.The degradation rate of chitosan can be controlled by changing its polymer composition (i.e.,the co-polymerization ratio of glucosamine to N-acetylglucosamine or the length of acyl side-chain on N-acetylglucosamine)and/or its molecular weight[10,11]. Furthermore,the degraded products of chitosan are nontoxic,nonimunogenic,and noncarcinogenic[12]. For most of the drug delivery applications the chitosan should be crosslinked due to its hydrophilic properties.Various crosslinking reagents have been used to crosslink chitosan gels.They are mostly synthetic

*Corresponding author.Tel.:+886-3-574-2504;fax:+886-3-572-

6832.

E-mail address:hwsung@https://www.doczj.com/doc/eb11145203.html,.tw(H.-W.Sung).

0142-9612/02/$-see front matter#2001Elsevier Science Ltd.All rights reserved. PII:S0142-9612(01)00094-1

chemicals such as epoxy compound and glutaraldehyde [13,14].Microencapsulation of drugs such as theophyl-line,cis-platinum(II)diammine dichloride(CDDP),and 5-?uorouracil,etc.within the glutaraldehyde-cross-linked chitosan microspheres have been investigated [15–19].Studies reported in recent years have shown that the glutaraldehyde-crosslinked chitosan micro-spheres have a long-acting biodegradable ability suitable for controlled-delivery of many drugs[20–22].However, these synthetic crosslinking reagents are all highly cytotoxic that may impair the biocompatibility of the crosslinked biomaterials[23,24].It is,therefore,desir-able to provide a crosslinking reagent suitable for use in biomedical applications,that is not cytotoxic and may form stable and biocompatible crosslinked products. Genipin can be obtained from its parent compound, geniposide,which may be isolated from the fruits of Gardenia jasminoides E llis as described in the literature [25,26].Genipin and its related iridoid glucosides extracted from the fruits of Gardenia jasminoides E llis have been used as an antiphlogistic and cholagogue in herbal medicine[27].Additionally,it has been reported that genipin can spontaneously react with amino acids or proteins to form dark blue pigments[28,29].In vitro evaluation of the cytotoxicity of genipin for biological tissue?xation has been investigated in a previous study [30].It was found that genipin is about5000–10,000 times less cytotoxic than glutaraldehyde.In addition,it was reported that the genipin-?xed tissue had a comparable mechanical strength and resistance against in vitro enzymatic degradation as the glutaraldehyde-?xed tissue[31].The results of these studies prompted us to evaluate the feasibility of using genipin to prepare biodegradable chitosan microspheres for long-acting drug delivery application.

Crosslinking of chitosan using glutaraldehyde makes it less susceptible to degradation by lysozyme[20,21]. Additionally,the glutaraldehyde-crosslinked chitosan microspheres have been shown to have the ability for long-acting delivery of drugs[20–22].However,bothth e cytotoxicity of the crosslinking reagent used and the degradation rate of the crosslinked microspheres should be taken into considerations in the clinical applications. It is,therefore,desirable to provide a nontoxic cross-linking reagent for the preparation of chitosan micro-spheres that can be used as a drug delivery system.

In the present study,a novel chitosan-based drug delivery system was fabricated.The chitosan solution was spray-dried and subsequently crosslinked by genipin to prepare the chitosan microspheres which can be injected intramuscularly.The objective of the study was to examine the biocompatibility of the genipin-cross-linked chitosan microspheres in a rat model.In addition, the degradation rate of these microspheres was investi-gated.The results of this study may help us to evaluate the feasibility of using the genipin-crosslinked micro-spheres as a long-acting biodegradable drug delivery system.

2.Materials and methods

2.1.Materials

Chitosan(M w:70,000)was purchased from Fluka Chemical Co.(Switzerland).The deacetylation degree of the chitosan used was approximately85%.Glutaralde-hyde was obtained from Fisher Chemical Co.(USA). Genipin(crystal-like powders,reagent grade)was obtained from Challenge Bioproducts Co.,Taiwan.All other reagents and solvents used were reagent grade. 2.2.Preparation of the spray-dried chitosan microspheres

Chitosan powder(3g)was dispersed in200ml of water containing0.5wt%acetic acid.The chitosan-powder contained solution then was stirred mechani-cally at600rpm until it was completely dissolved. Subsequently,the dissolved chitosan solution and air were fed and passed separately to the nozzle of a spray dryer(Buchi B-191,Switzerland),where they were mixed.The air caused the fed chitosan solution to break up into a spray at the outlet of the nozzle.After the evaporation of solvent,microspheres were formed and collected at a cyclone separator through which the air and the product passed after exiting the drying chamber.The inlet temperature was1408C,which was higher than the boiling point of the polymer solvent. The spraying rate of feed was20ml/min,which could insure a narrow dispersed size of the microspheres.The ?nally produced chitosan microspheres were collected from the cyclone of the spray dryer.

2.3.Crosslinkin g of the spray-dried chitosan microspheres

The spray-dried chitosan microspheres(0.15g)were dispersed into a0.44m aqueous glutaraldehyde or genipin solution(5ml)containing90%ethyl alcohol for crosslinking.The degrees of crosslinking for both the glutaraldehyde-and genipin-crosslinked chitosan micro-spheres were approximately30%(determined by ninhy-drin assay)by adjusting their crosslinking durations for 20min and3h,respectively.Fresh and the crosslinked chitosan microspheres then were soaked in a NaOH (1n)aqueous solution to remove the residue acetic acid in the spray-dried chitosan microspheres,followed by rinsing with aqueous ethyl alcohol solution(99.5%)to remove NaOH.The rinsed microspheres were vacuum-dried for24h to evaporate ethyl alcohol.Finally,the products were collected in a desiccator for further use.

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2.4.Morpholo g y of microspheres

Freshand th e crosslinked ch itosan microsph eres were sprinkled onto a double-sided adhesive tape and?xed to an aluminum stage.The microspheres then were sputter-coated withgold in a th ickness of500?10à8cm using a Hitachi coating unit(IB-2coater).Subsequently,the coated samples were examined using a Hitachi S-2300 scanning electron microscope.

2.5.X-ray analysis of microspheres

The crystallinity of the chitosan powder and the spray-dried chitosan microspheres were examined by the X-ray analysis(Shimadzu di?ractometer,XD-5,Japan). The X-ray di?raction of the samples in the2y range of 5–308was conducted at ambient temperature using Cu K a radiation generated at30kV and30mA.The scan rate was38(2y)/min.

2.6.Animal study

The in vivo biocompatibility and degradability of fresh and the glutaraldehyde-and genipin-crosslinked chitosan microspheres were examined by implanting the spheres in the skeletal muscle of Wistar rats via the intramuscular injection.The test microspheres were sterilized using an autoclaving method under1218C for30min.Subsequently,the sterilized microspheres (1–10m m),50mg,were suspended in2ml of physiolo-gical saline and injected into the skeletal muscle using an 18G needle.Eachanimal received two injections.Th e implanted microspheres along with their surrounding tissues were retrieved at3-day,1-,4-,12-,and20-week postoperatively.The retrieved samples then were pro-cessed for histological and scanning electron micro-scopic(SEM)examinations.2.7.Histolo g ical examination

The samples used for the histological examination were?xed in a10%phosphate-bu?ered formaldehyde solution for at least3days.The?xed samples were embedded in para?n,sectioned into a thickness of5m m, and then stained with hematoxylin and eosin(H&E). The stained sections of each test sample were examined using light microscopy(Nikon Microphoto-FXA)for tissue in?ammatory reaction and photographed with a 100ASA Kodachrome?lm.

2.8.Scannin g electron microscopic(SEM)examination The samples used for the SEM examination were?rst ?xed with2%glutaraldeh yde in0.1m of sodium cacodylate and then post-?xed in1%osmium tetroxide. Subsequently,the samples were dehydrated in a graded series of ethanol solutions,critical-point dried withcarbon dioxide,and spattered withgold ?lm.The examination was performed with a scann-ing electron microscope(Hitachi,Model S-800, Japan).

3.Results

3.1.Preparation of the chitosan microspheres

The chitosan microspheres prepared by the spray-drying method showed a good sphericity[Fig.1(a)–(c)]. The operation conditions of the spray-drying process did not induce a signi?cant change in the morphology of microspheres[Fig.1(a)–(c)].However,the particle-size distributions of the chitosan microspheres prepared by the spray-drying process were a?ected signi?cantly by the process conditions,such as the mass ratio of air-to-chitosan-solution,the relative velocity of air-to-chito-san-solution,and the viscosity of chitosan solution. As shown in Fig.2(a)and(b),the average size of

the Fig.1.Scanning electron micrographs of the spray-dried chitosan microspheres made under di?erent conditions:(a)inlet temperature1408C and air ?ow-rate600l/h;(b)inlet temperature1108C and air?ow-rate600l/h;(c)inlet temperature1408C and air?ow-rate200l/h.

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spray-dried chitosan microspheres decreased with an increase in the air ?ow-rate;however,it increased with increasing the viscosity of chitosan solution.Never-theless,the particle sizes of these chitosan microspheres were all less than 10m m.

The results of the X-ray analysis showed that the crystallinity of the spray-dried chitosan micro-spheres was signi?cantly lower than that of the original chitosan powders (Fig.3).This indicated that the spray-drying process might be a very fast phase-inversion process that can decrease the crystallinity of polymeric chitosan.

3.2.Biocompatibility of the implanted chitosan microspheres

Fig.4(a)–(c)shows photomicrographs of the tissues implanted with fresh and the glutaraldehyde-and genipin-crosslinked chitosan microspheres stained with H&E retrieved at 3-day postoperatively.As shown in the ?gure,the tissue implanted with the glutaraldehyde-crosslinked chitosan microspheres (in?ltrated with neutrophils and monocytes)had the most notable in?ammatory reaction among all studied groups.On the other hand,the degree in in?ammatory reaction observed for the tissue implanted with fresh chitosan microspheres was less than that implanted with the genipin-crosslinked chitosan microspheres.

At 1-week postoperatively,the degree in in?amma-tory reaction for the tissue implanted with fresh micro-spheres declined signi?cantly.However,the in?ammatory cells surrounding the tissues implanted with the glutaraldehyde-or genipin-crosslinked chitosan microspheres were still abundant,suggesting that the in?ammatory reactions persisted for these two cases.However,the in?ammatory reaction for the tissue surrounding with the genipin-crosslinked chitosan mi-crospheres was signi?cantly less than their glutaralde-hyde-crosslinked

counterparts.

Fig.2.E?ect of (a)air ?ow-rate and (b)viscosity on the particle size of chitiosan

microspheres.

Fig.3.Crystallinity analysis of the original chitosan powders and the spray-dried chitosan microspheres.

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At 4-week postoperatively,the degree in in?amma-tory reaction for the tissue implanted with fresh micro-spheres was less than those retrieved at 3-day and 1-week postoperatively.At this time,only a few

lymphocytes were observed surrounding the tissue implanted with fresh microspheres.However,the in?ammatory reactions for the tissues implanted with the glutaraldehyde-or genipin-crosslinked chitosan microspheres were still signi?cant.It was noted that the amount of lymphocytes present in the tissue implanted with the glutaraldehyde-crosslinked chitosan microspheres was more than that present with the genipin-crosslinked counterparts.Furthermore,giant cells were observed surrounding the tissue implanted with the glutaraldehyde-crosslinked chitosan micro-spheres.

At 12-week postoperatively,the in?ammatory cells surrounding the tissues implanted with fresh or the genipin-crosslinked chitosan microspheres had almost disappeared.However,there were still in?ammatory cells present in the tissue implanted with the glutar-aldehyde-crosslinked chitosan microspheres.At 20-week postoperatively,the in?ammatory reactions for the tissues implanted with fresh or the glutaraldehyde-or genipin-crosslinked chitosan microspheres were almost all eliminated [Fig.5(a)–(c)].

3.3.De g radability of the implanted chitosan microspheres

After implantation,the morphology of the implanted chitosan microspheres changed progressively with time and ?nally disintegrated [Figs.6(a)–(c),7(a)–(c),and 8(a)–(c)].At 3-day and 1-week postoperatively,fresh and the glutaraldehyde-and genipin-crosslinked chitosan microspheres remained in good sphericity as those observed before implantation [Fig.6(a)–(c)].At 4-week postoperatively,a porous structure was observed on the surface of fresh chitosan microspheres,suggesting that degradation started to occur at the outer layer of fresh microspheres [Fig.7(a)].At this moment,the crosslinked chitosan microspheres remained intact,with the exception of some shallow cavities present on the surfaces of the glutaraldehyde-crosslinked chitosan micriosphers [Fig.7(b)and (c)].This implied that the degradation rates of the glutaraldehyde-and genipin-crosslinked chitosan microspheres were slower than their fresh counterparts.

At 12-week postoperatively,freshch itosan micro-spheres were partially degraded into fragments,while the glutaraldehyde-crosslinked microspheres were de-graded into a loose and porous structure.In contrast,the degradation of the genipin-crosslinked chitosan microspheres was not signi?cant.At 20-week post-operatively,both fresh and the glutaraldehyde-cross-linked chitosan microspheres were severely degraded into fragments [Fig.8(a)and (b)].The degradation of the genipin-crosslinked chitosan microspheres retrieved at 20-week postoperatively was more notable as compared to that retrieved at 12-week

postoperatively

Fig.4.Photomicrographs of the tissues implanted with:(a)fresh chitosan microspheres;(b)the glutaraldehyde-crosslinked chitosan microspheres;(c)the genipin-crosslinked chitosan microspheres stained withh ematoxyline and eosin (original magni?cation ?200)retrieved at 3-day postoperatively.

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[Fig.8(c)].However,the degradation of the genipin-crosslinked microspheres was signi?cantly less than fresh and the glutaraldehyde-crosslinked counterparts.

4.Discussion

The spray-dried chitosan microspheres made in the study had a small particle size and a low crystallinity [Figs.1(a)–(c)and 3].Therefore,the chitosan micro-spheres prepared by the spray-drying method may be used as an intramuscularly injectable drug-delivery-carrier in the consideration of their particle size.The decreased crystallinity of the spray-dried chitosan microspheres may increase their hydration degree and enzymatic-degradation rate.Crosslinking of the spray-dried chitosan microspheres may limit the hydration and degradation characteristics of these microspheres in order to maintain their long-term release function.In our previous study,it was found that the cytotoxicity of genipin is signi?cantly lower than glutaraldehyde [31].The investigation of a genipin-?xed porcine pericardium implanted subcutaneously in a growing rat model also showed a less in?ammatory reaction as compared to its glutaraldehyde-?xed counterpart [32].These results prompted us to use genipin as a reagent to crosslink the chitosan microspheres.

In the study,the in vivo biocompatibility and degradability of fresh and the glutaraldehyde-and genipin-crosslinked chitosan microspheres were assessed in a rat model via the intramuscular injection.The degrees in in?ammatory reaction observed surrounding the tissues implanted with these test chitosan micro-spheres were distinct.It was found that the degree in in?ammatory reaction surrounding the tissue implanted with fresh chitosan microspheres was less than their crosslinked counterparts observed at 3-day postopera-tively [Fig.4(a)–(c)]and it started to subside at 1-week postoperatively.It was reported previously that im-plants of membranes prepared from the 65–80%deacetylated chitin induced an initially severe in?amma-tion that subsided with time.In contrast,tissue reaction to implant of membrane containing the 94%deacety-lated chitin was mild and was characterized by a ?brous connective tissue encapsulation [33].Additionally,it was found that the implantation of the chitosan–xanthan complex subcutaneously resulted in an acute in?amma-tion reaction at 1-week post implantation.The chit-osan–xanthan particles were surrounded and individually encapsulated by ?brous connective tissue that was in?ltrated by macrophages and ?broblasts after 8weeks of implanation [34].

A disadvantage of the chemically modi?ed implants is the potential toxic e?ects a recipient may be exposed to from its remaining residues.Studies have shown that subcutaneous collagen implants crosslinked by

glutar-

Fig.5.Photomicrographs of the tissues implanted with:(a)fresh chitosan microspheres;(b)the glutaraldehyde-crosslinked chitosan microspheres;(c)the genipin-crosslinked chitosan microspheres stained withh ematoxyline and eosin (original magni?cation ?200)retrieved at 20-week postoperatively.The in?ammatory cells surround-ing the tissues (indicated by ‘‘’’)implanted withfreshor th e glutaraldehyde-or genipin-crosslinked chitosan microspheres had almost disappeared at this time.

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aldehyde with a concentration equal to or less than 0.1%exhibited a benign tissue response as compared to implants crosslinked with0.1–1.0%glutaraldeh yde in a guinea pig model [35].Other studies also showed that the benign response could be found surrounding the tissues implanted with the chitosan microspheres cross-linked witha very low concentration of glutaraldeh yde [23,24].It was found in our study that the degree in in?ammatory reaction for the tissue implanted with the glutaraldehyde-crosslinked chitosan microspheres was signi?cantly greater than those implanted with fresh and the genipin-crosslinked chitosan microspheres [Fig.4(a)–(c)].It is speculated that the lower in?amma-tory reaction observed withth e genipin-crosslinked chitosan microspheres may be due to the lower toxicity

of its remaining residues as compared to the glutar-aldehyde-crosslinked chitosan microspheres.This ob-servation implied that the biocompatibility of the genipin-crosslinked chitosan microspheres is superior to their glutaraldehyde-crosslinked counterparts.

The results obtained in the SEM study showed that freshch itosan microsph eres were degraded quickly.At 12-week postoperatively,fresh chitosan microspheres were already degraded into fragments.It was reported that the degradation rate of chitosan decreased with increasing its deacetylation degree [36].Chitosan is a copolymer comprising N -glucosamine and N -acetylglu-cosamine units that can crystallize.

Membranes

Fig.6.Scanning electron micrographs of:(a)fresh chitosan microspheres;(b)the glutaraldehyde-crosslinked chitosan microspheres;(c)the genipin-crosslinked chitosan microspheres retrieved at 3-day

postoperatively.

Fig.7.Scanning electron micrographs of:(a)fresh chitosan microspheres;(b)the glutaraldehyde-crosslinked chitosan microspheres;(c)the genipin-crosslinked chitosan microspheres retrieved at 4-week postoperatively.

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prepared from chitosan with a deacetylation degree greater than 80%were degraded by lysozyme [37].It was shown that the active site of lysozyme consists of six subsites,which bind the N -acetylglucosamine residues of chitosan ([38],Fig.9(a)).It is known that crystallized region prohibits enzyme from permeation,while amor-phous region allows permeation of enzyme.The fast degradation rate observed for the spray-dried micro-spheres prepared in the study may be attributed to their low crystallinity.Crystallization of chitosan inhibits the permeation of lysozyme into the NAG domain,leading to the limitation of its degradation rate.

Crosslinking signi?cantly reduced the degradability of the chitosan microspheres [Figs.6(a)–(c),7(a)–(c),and 8(a)–(c)].Jameela et al.reported that the glutaralde-hyde-crosslinked chitosan microspheres implanted in vivo up to 3months were not degraded signi?cantly [20–22].It was found that the genipin-crosslinked chitosan microspheres had a slower degradation rate than their glutaraldehyde-crosslinked counterparts.At 20-week postoperatively,the degradation of the genipin-crosslinked chitosan microspheres was observed only on the surfaces of the microspheres [Fig.8(c)],while the glutaraldehyde-crosslinked microspheres were degraded severely [Fig.8(b)].

The mechanism of crosslinking of chitosan with glutaraldehyde has been discussed in detail previously [39].The bifunctional glutaraldehyde reacts with the amino groups of chitosan to form Schi?bases (–C ?N–linkage).Furthermore,glutaraldehyde may undergo an aldol condensation to polymerize [Fig.9(b)].The reac-tion mechanism of chitosan with genipin has been investigated by our group recently [40].It was found that genipin may undertake a ring-opening reaction to form an intermediate aldehyde group,due to the

nucleophilic attack by the amino groups in chitosan.A heterocyclic compound of the genipin-crosslinked chito-san is formed via a nucleophilic attack by the amino group on the ole?nic carbon atom at C-3of deoxylo-ganin aglycone,followed by the opening of the dihydropyran ring and attacked by the secondary amine group on the resulting aldehyde group.The genipin reacted with a nucleophilic reagent such as chitosan may further undergo polymerization to form oligomer in the crosslinked network [Fig.9(c)].

As shown in Fig.9(b)and (c),the genipin-crosslinked network may have a higher stereohindrance for the penetration of lysozyme than the glutaraldehyde-cross-linked network,due to the bulky heterocyclic-structure of genipin.The structure of stereohindrance may prevent lysozyme from binding to the substrate of the enzyme in chitosan.Therefore,the degradation rate of the genipin-crosslinked chitosan microspheres was sig-ni?cantly slower than its glutaraldehyde-crosslinked counterparts [Figs.6(a)–(c),7(a)–(c),and 8(a)–(c)].

5.Conclusions

In the study,a novel injectable-chitosan-microsphere crosslinked by genipin was prepared.It was found that the degree in in?ammatory reaction surrounding the tissue implanted with the genipin-crosslinked chitosan microspheres was less than that implanted with the glutaraldehyde-crosslinked chitosan microspheres.Ad-ditionally,the degradation rate of the genipin-cross-linked chitosan microspheres was signi?cantly slower than their glutaraldehyde-crosslinked counterparts.These results indicated that the genipin-crosslinked chitosan microspheres may be used as a

long-acting

Fig.8.Scanning electron micrographs of:(a)fresh chitosan microspheres;(b)the glutaraldehyde-crosslinked chitosan microspheres;(c)the genipin-crosslinked chitosan microspheres retrieved at 20-week postoperatively.

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Fig.9.Schematic illustrations of:(a)the binding substrate for lysozyme;and the presumable stereohindrance structures for the enzymatic degradation of (b)the glutaraldehyde-crosslinked network and (c)the genipin-crosslinked network.

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intramuscularly implantable drug-delivery-vehicle. The drug-released characteristics of the genipin-cross-linked chitosan microspheres are currently under investigation.

Acknowledgements

This work was supported by a grant from the National Science Council of Taiwan,Republic of China (NSC-89-2314-B-008-001-M08).

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想要刷这款手机,有几个要点需要注意: 1、由于这款手机是高通的处理器,使用高通模式进行刷机,所以,要使用win7/win8/win1064位的系统进行刷机,32位的系统是无法给这款手机刷机的。 2、刷机一定要找一根连接稳定的数据线,并且保证USB接口连接正常,否则手机连接不上电脑或者刷机过程中突然断开连接,那就很麻烦了。 3、这款手机是使用高通模式刷机,所以进入刷机模式之后,和整个刷机过程,手机都会全程黑屏,如果手机亮屏,说明操作不正确,刷机肯定失败! 4、这款手机刷机之后,开机按住电源键要多按一会儿,开机的时间也比较长,请一定要耐心等待! 注意了以上几个问题,这款手机还是比较容易刷机成功的。下面就是vivoY55A 的刷机视频教程: https://www.doczj.com/doc/eb11145203.html,/guide/ShiPinJiaoCheng/4307.html/?utm_sourc e=wenku

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再选择您要下载的包(优化版&官方原版&ROOT版:点击查看版本区别。小编建议选择官方原版。),点击“普通下载”,线刷宝便会自动把包下载到您的电脑上(无法下载?点我解决问题)。 2:安装驱动与手机解锁 在电脑上安装手机的驱动;>>查看驱动安装教程 手机尚未解除BL锁?>>查看解锁教程 驱动已安装、BL已解锁或无BL锁请跳过此步骤;

第三步:解析刷机包 打开线刷宝客户端——点击“一键刷机”——点击右下方“去救砖”——点击“选择本地ROM”,打开您刚刚下载的线刷包,线刷宝会自动开始解析: 第四步:手机进入刷机模式 线刷包解析完成后,按照线刷宝右边的提示操作手机,直到手机进入刷机模式(不知道怎么进?看这里!):

第五步:线刷宝自动刷机 手机进入刷机模式,并通过数据线连接电脑后,线刷宝会自动开始刷机: 刷机过程大约需要两三分钟的时间,然后会提示您刷机成功,您的爱机就OK啦!

步步高vivo S7-步步高vivo s7 root 刷机教程

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点击“点我进行ROOT”过一会会弹出一个窗口(如下图) 不要动手机,等待它自己重启后,点击“确定”,过一会会弹出一个和上面一样的窗口(如下图) 也不要动手机,再等待它自己重启后,再点击“确定”,过一会会弹出一个窗口(如下图)

看到这个后,点击“确定”,手机第三次重启后,在手机上发现了安装了superuser,re管理器和移动叔叔工具箱软件!那就表示你的手机root成功了!! 如果没有看见这几款软件,则表示没有root成功,多试一次,就会成功!! 一般只要在期间3次重启别乱动手机,基本上一次都能成功!! (看到这你就可以下载工具为你的小7root啦!!)

中国移动A1(M623C移动4G)刷机教程图解(可救砖)

中国移动A1(M623C/移动4G)刷机教程图解(可救砖) 中国移动A1(M623C/移动4G)搭载Qualcomm骁龙四核处理器,于2015年06月上市,主屏尺寸5英寸,操作系统Android4.4,内存1G。这款手机要怎么刷机呢?请看下面的教程。 (图1) 1:刷机准备 中国移动A1(M623C/移动4G)一部(电量在百分之20以上),数据线一条,原装数据线刷机比较稳定。 刷机工具:线刷宝下载 刷机包:中国移动A1(M623C/移动4G)刷机包 1、打开线刷宝,点击“线刷包”(如图2)——在左上角选择手机型号(中国移动-A1M623C),或者直接搜索A1M623C

(图2) 2、选择您要下载的包(优化版&官方原版&ROOT版:点击查看版本区别。小编建议选择官方原版。), 3、点击“普通下载”,线刷宝便会自动把包下载到您的电脑上(如图3)。 (图3)

2:解析刷机包 打开线刷宝客户端——点击“一键刷机”—点击“选择本地ROM”,打开您刚刚下载的线刷包,线刷宝会自动开始解析(如图4)。 (图4) 第三步:安装驱动 1、线刷宝在解包完成后,会自动跳转到刷机端口检测页面,在刷机端口检测页面(图5)点击“点击安装刷机驱动”, 2、在弹出的提示框中选择“全自动安装驱动”(图6),然后按照提示一步步安装即可。

(图5) (图6)

第四步:手机进入刷机模式 线刷包解析完成后,按照线刷宝右边的提示操作手机(图7),直到手机进入刷机模式(不知道怎么进?看这里!): (图7) 第五步:线刷宝自动刷机 手机进入刷机模式,并通过数据线连接电脑后,线刷宝会自动开始刷机:

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2)Android操作系统以42、4%的关注度成为20**年上半年中国用户最关注的智能操作系统。 3)智能手机市场上超七成的用户关注的手机价位处于1000元—3000元。 小米手机在这个时候发布,当然是十分应景的,在智能手机最受欢迎的时候推出,不论是硬件配置、操作系统还是销售价格,都是令人无可挑剔 2、小米手机与其他智能手机参数对比分析 小米手机与其他手机参数对比 从图片我们知道,小米手机的硬件配置处于绝对的中上等,而CPU更是超人一等,但它的售价却只有1999元,对比其他的手机,最便宜的LG也要2575元(水货,没有售后服务)。从性价比上来说,小米手机可以说是NO、1。 三、网络营销方案 1、信息发布

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2:解析刷机包 打开线刷宝客户端——点击“一键刷机”—点击“选择本地ROM”,打开您刚刚下载的线刷包,线刷宝会自动开始解析(如图4)。 (图4) 第三步:安装驱动 1、线刷宝在解包完成后,会自动跳转到刷机端口检测页面,在刷机端口检测页面(图5)点击“点击安装刷机驱动”, 2、在弹出的提示框中选择“全自动安装驱动”(图6),然后按照提示一步步安装即可。

步步高vivo Y18L(移动4G)刷机教程图解(可救砖)

步步高vivo Y18L(移动4G)刷机教程图解(可救砖) 步步高vivoY18L(移动4G)搭载高通骁龙400四核处理器,手机成砖的教程工具摆在面前都难以救回手机。步步高vivoY18L(移动4G)要怎么刷机呢?今天线刷宝小编给大家讲解一下关于步步高vivoY18L(移动4G)的图文刷机教程,线刷教程和救砖教程,一步搞定刷机失败问题,跟着小编一步步做,刷机Soeasy!步步高vivoY18L(移动4G)搭载高通骁龙400四核处理器,于2014年5月上市。时尚音乐4G手机,4.7英寸720P屏幕,4G网络发布初期产品,主打快速的网络下载速率。总体来说,分辩率一般,像素一般,CPU过低,机身内存小,音量小,电池不能拆,发热过快,性价比一般。这款手机要怎么刷机呢?请看下面的教程。 (图1) 1:刷机准备 步步高vivoY18L(移动4G)一部(电量在百分之20以上),数据线一条,原装数据线刷机比较稳定。 刷机工具:线刷宝下载

刷机包:步步高vivoY18L(移动4G)刷机包 1、打开线刷宝,点击“线刷包”(如图2)——在左上角选择手机型号(步步高-Y18L),或者直接搜索Y18L (图2) 2、选择您要下载的包(优化版&官方原版&ROOT版:点击查看版本区别。小编建议选择官方原版。), 3、点击“普通下载”,线刷宝便会自动把包下载到您的电脑上(如图3)。

(图3) 2:解析刷机包 打开线刷宝客户端——点击“一键刷机”—点击“选择本地ROM”,打开您刚刚下载的线刷包,线刷宝会自动开始解析(如图4)。 (图4) 第三步:安装驱动 1、线刷宝在解包完成后,会自动跳转到刷机端口检测页面,在刷机端口检测页面(图5)点击“点击安装刷机驱动”, 2、在弹出的提示框中选择“全自动安装驱动”(图6),然后按照提示一步步安装即可。

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