Biomaterials 25 (2004) 907–915

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Content

1. Intr

2. In v

2.1

2.2

3. Wa

3.1

3.2

4. Wa

4.1

4.2

5. Per

Referen

*Correspondin

University Schoo

E-mail addres

0142-9612/$ - see

doi:10.1016/S014

Review

N-succinyl-chitosan as a drug carrier: water-insoluble andwater-soluble conjugates

Yoshinori Kato*, Hiraku Onishi, Yoshiharu Machida

Department of Drug Delivery Research, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Received 21 February 2003; accepted 14 July 2003

Abstract

N-succinyl-chitosan (Suc-Chi) has favourable properties as a drug carrier such as biocompatibility, low toxicity and long-term

retention in the body. It was long retained in the systemic circulation after intravenous administration, and the plasma half-lives of

Suc-Chi (MW: 3.4� 105; succinylation degree: 0.81 mol/sugar unit; deacetylation degree: 1.0 mol/sugar unit) were ca. 100.3 h in

normal mice and 43 h in Sarcoma 180-bearing mice. The biodistribution of Suc-Chi into other tissues was trace apart from the

prostate and lymph nodes. The maximum tolerable dose for the intraperitoneal injection of Suc-Chi to mice was greater than 2 g/kg.

The water-insoluble and water-soluble conjugates could be prepared using a water-soluble carbodiimide and mitomycin C (MMC)

or using an activated ester of glutaric MMC. In vitro release characteristics of these conjugates showed similar patterns, i.e. a pH-

dependent manner, except that water-insoluble conjugates showed a slightly slower release of MMC than water-soluble ones. The

conjugates of MMC with Suc-Chi showed good antitumour activities against various tumours such as murine leukaemias (L1210

and P388), B16 melanoma, Sarcoma 180 solid tumour, a murine liver metastatic tumour (M5076) and a murine hepatic cell

carcinoma (MH134). This review summarizes the utilization of Suc-Chi as a drug carrier for macromolecular conjugates of MMC

and the therapeutic efficacy of the conjugates against various tumours.

r 2003 Elsevier Ltd. All rights reserved.

Keywords: N-succinyl-chitosan; Prodrug; Cancer chemotherapy; Biodistribution; Conjugates; Mitomycin C

s

oduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

ivo characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

. Biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

ter-insoluble N-succinyl-chitosan-drug conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

. Preparation and in vitro characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

. Antitumour activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 910

ter-soluble N-succinyl-chitosan-drug conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911

. Preparation and in vitro characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911

. Antitumour activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913

spective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

ces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

g author. Present address: MR Oncology Section, Division of MR Research, Department of Radiology, The Johns Hopkins

l of Medicine, 217 Traylor Bldg., 720 Rutland Ave., Baltimore, MD 21205, USA. Tel.: +1-410-955-6865; fax: +1-410-614-1948.

s: ykato@mri.jhu.edu, yk-no.1@jcom.home.ne.jp (Y. Kato).

front matter r 2003 Elsevier Ltd. All rights reserved.

2-9612(03)00598-2

ARTICLE IN PRESSY. Kato et al. / Biomaterials 25 (2004) 907–915908

1. Introduction

Chitosan and its derivatives draw attention as a drugdelivery vehicle [1]. They were much studied asmicroparticles, colonic delivery, bioadhesive polymerand the like as referenced by Illum [2]. We focused on N-succinyl-chitosan (Suc-Chi), which was obtained byintroduction of succinyl groups into chitosan N-terminalof the glucosamine units (Fig. 1). Succinylation degreeof Suc-Chi could be easily modified by changingreaction conditions using succinic anhydride [3].Furthermore, the molecular weight of Suc-Chi wasinexpensively reduced using hydrochloric acid [4].Although Suc-Chi was initially developed as wounddressing materials [5], it is currently also applied ascosmetic materials (Moistfine liquids) [6]. New wounddressings composed of Suc-Chi and gelatin were alsodeveloped [7]. In addition, water-soluble chitin andchitosan including Suc-Chi were applied for a patent asa treatment of arthritis in Japan [8]. Suc-Chi has uniquecharacteristics in vitro and in vivo due to many carboxylgroups. For example, ordinary chitosan can be dissolvedin acidic water but not in alkaline, whereas highlysuccinylated Suc-Chi (degree of succinylation: >0.65)exhibits the opposite behaviour [3]. Suc-Chi can easilyreact with many kinds of agents due to having –NH2

and –COOH groups in its structure (Fig. 1). It isvaluable for the drug carrier to readily prepare itsconjugates with various drugs to avoid vexatiouscomplications. This review deals with the applicationof Suc-Chi as a carrier for water-insoluble and water-soluble drug conjugates in cancer chemotherapy.

2. In vivo characteristics

2.1. Biodistribution

Fig. 2 shows the plasma concentration profiles of Suc-Chi after i.v. administration to various mice. Suc-Chiwas retained in the body after i.v. administration,especially in the systemic circulation [9,10]. The plasmahalf-lives of Suc-Chi (MW: 3.4� 105; succinylationdegree: 0.81 mol/sugar unit; deacetylation degree:1.0 mol/sugar unit) were approximately 100.3 h innormal mice and 43 h in tumour-bearing mice [10],which were significantly higher than other macromole-cules reported to exhibit relatively long systemicretention [11–14]. In general, anionic charges are knownto prolong the half-life of polymers in the systemiccirculation due to their reduced interaction with bloodvessels and tissues, whereas cationic charges exhibit theopposite behaviour [11,13]. In addition, many carboxylgroups of Suc-Chi were responsible for the retention inthe blood stream. This long circulating effect contributesto the good antitumour efficacy of the conjugates as

described later. The biodistribution of Suc-Chi into theliver, kidney and spleen was below 5% of dose/g tissue[10], and greater than 10% of dose/g tissue of Suc-Chiwas transferred to the prostate and lymph nodes at 48 hpost-administration [15]. The biodistributions of Suc-Chi into the testes, preputial gland, intestine (smallintestine plus caecum), femoral muscle, backbone andperitoneum were less than 10% of dose/g tissue [15]. Inthe case of Suc-Chi with low (51%) or high (100%)degrees of succinylation, the retention of Suc-Chi in theplasma was not significantly different from that innormal mice [9,16]. For sarcoma 180-bearing mice, theeffect of succinylation degree on the plasma concentra-tion was notably observed (Fig. 2). The plasma half-lifeof Suc-Chi may be decreased when Suc-Chi with evenlower succinylation is administered due to attenuationof the anionic charge. Low excretion into urine was alsoobserved [9,10,16]. It is conceivable that long systemicretention is responsible for the high molecular weight,possession of many carboxylic groups and low excre-tion. As indicated in Fig. 2, the retention of Suc-Chi inthe blood stream declined when Suc-Chi was injectedinto tumour-bearing mice. Blood vessels surroundingthe tumour tissue are known to be dense and showarchitectural incompleteness, and the lymphatic systemis defective [17,18]. Macromolecular carriers and nano-particules are transferred to the tumour tissue from thesystemic circulation and accumulate there with time,which is known as enhanced permeability and retention(EPR) effects [19–22]. Similar to other carriers, Suc-Chiwas accumulated in the tumour with time [9,10,16].

In contrast, the introduction of lactose to Suc-Chi byreductive amination conferred a liver-targeting ability innormal or tumour-bearing mice [23,24]. As is wellknown, the liver parenchymal cells have asialoglycopro-tein receptors, which specifically recognize the galactosemoiety [25,26], and this receptor has been utilized as auseful site for liver targeting in many studies [27–34]. Asexpected, lactosaminated Suc-Chi (Lac-Suc-Chi) (degreeof lactosamination: 30.1% (mol/sugar unit)) was rapidlydistributed to the liver and reached 22.3% of the dose at1 h post-injection to normal mice [23]. It reached themaximum of 23.0% of the dose at 8 h after injection,and then gradually reduced to 10.4% of the dose at 48 hpost-injection. This phenomenon produced the reduc-tion in the plasma level of Suc-Chi (Fig. 2). Thedistribution of Lac-Suc-Chi to the liver is inferior tothat of other glycosylated macromolecules, i.e. accumu-lation extents of galactosylated poly-l-lysine and poly-l-glutamic acid in the liver were ca. 70% and 50% of thedose at 60 min post-injection, respectively [33,34]. Thedistribution of Lac-Suc-Chi to other tissues appeared tobe suppressed compared with the above-referencedcarriers. It is conceivable that the low distribution ofintact Suc-Chi into the liver leads to low accumulationof Lac-Suc-Chi because of its negative charge. In

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Fig. 2. Plasma concentration of fluorescently labelled N-succinyl-chitosan after i.v. administration as a function of time. Open and closed symbols

represent normal and tumour-bearing mice, respectively.

Fig. 1. Synthetic examples for binding of N-succinyl-chitosan to various agents. Numerals in bracket are the reference numbers.

Y. Kato et al. / Biomaterials 25 (2004) 907–915 909

addition, Suc-Chi showed long-term retention in theliver. This was suggested to be due to the biologicalstability of Suc-Chi, while proteins are cleared rapidlydue to lysosomal degradation [35–37]. These propertieswere implied to result in a long half-life of the drug inthe liver. Although galactose receptors are known toexist not only on the liver parenchymal cells but also onprostate and testes [38], the concentration of Lac-Suc-Chi distributed to the prostate and the testes was notmarked [15]. The prostate localization of Lac-Suc-Chimight not be selective because the distribution into theprostate was observed not only for Lac-Suc-Chi but alsofor Suc-Chi.

2.2. Toxicity

Song et al. [39] and Onishi et al. [40] reported theacute toxicity of Suc-Chi in mice. The tolerable singledose for i.p. injection of Suc-Chi to mice was 2000 mg/kg due to the high viscosity of the sample solution. Allmice (six animals) were alive greater than 35 days post-

administration; therefore, the maximum tolerable dosefor the i.p. injection of Suc-Chi was determined to begreater than 2000 mg/kg. Izume [6] summarized thetoxicity of Suc-Chi including a skin sensitization study,temporal skin irritation study, ophthalmic sensitizationtest, mutagenicity test and patch test for humans; everystudy and test showed low toxicity of Suc-Chi.

3. Water-insoluble N-succinyl-chitosan-drug conjugates

3.1. Preparation and in vitro characteristics

Mitomycin C (MMC) is widely used anticancer agent.Anticancer activity of MMC has the tendency to reduceby chemical modification of the aziridine ring, one of theactive groups, and its effect is manifested as thesubstituent becomes larger [41]. Song et al. [42] herefromattempted the preparation of the conjugates of MMCwith Suc-Chi. MMC, Suc-Chi (MW: 3.0� 105; succiny-lation degree: 0.72 mol/sugar unit) and water-soluble

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Fig. 3. Synthetic approach for water-insoluble and water-soluble conjugates of MMC with N-succinyl-chitosan.

Fig. 4. In vitro release characteristics of water-insoluble and water-

soluble conjugates of MMC with 1. Circles: 1/15m phosphate buffer

(pH 9); squares and crosses: 1/15m phosphate buffer (pH 7.4);

triangles: 1/15m phosphate buffer (pH 6.2 (open symbols) or 5 (closed

symbols)). Broken lines indicate the release pattern of water-insoluble

conjugates at pH 7.4. Broken line alone (no symbol) represents the

MMC release from the cross-linking conjugate microparticles. Crosses,

closed and open symbols represent Suc-Chi-MMC, Suc-Chi-glu-MMC

and Suc(II)-Chi-MMC, respectively.

Y. Kato et al. / Biomaterials 25 (2004) 907–915910

carbodiimide (EDC) [43] were mixed in water for 45 minafter adjusting to pH 5.0, because the MMC content ofthe conjugates was lowered with the increase in the pHin the reaction medium. After that, the precipitate wasisolated by filtration, washed with water and dried invacuo. The resulting solid was used as Suc-Chi-MMC(MMC content: 12%). In addition, the preparation ofmicroparticles was attempted [44,45]. Onishi et al. [45]prepared the cross-linked conjugate microparticles ofSuc-Chi with MMC having an adequate size for livertargeting (0.2–3 mm) (Fig. 3).

The release characteristics of Suc-Chi-MMC in vitroare shown in Fig. 4. Suc-Chi-MMC exhibited mono-exponential liberation of MMC at pH 7.4 [40,42,46].Prolongation of the drug release was achieved atphysiological pH. Fig. 4 also shows the release ofMMC from the cross-linked conjugate microparticles ofSuc-Chi with MMC. These microparticles exhibited afaster release as compared with Suc-Chi-MMC. Thesedifferences may be attributed to the stiffness of theparticles (tight or loose) rather than the binding betweenSuc-Chi and MMC, because the liberation of MMCfrom the conjugates of MMC with Suc-Chi depends onthe pH in the medium [47–49].

3.2. Antitumour activities

In the case of Suc-Chi-MMC, the suspensions wereadministered subcutaneously, intratumourally or intra-peritoneally after the conjugates were homogenized in

saline. Fig. 5 shows tumour inhibitory effects of Suc-Chi-MMC against Sarcoma 180 solid tumour and B16melanoma. Suc-Chi-MMC suppressed the growth ofboth tumours as well as MMC alone [47,50]. Toxic side

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Fig. 5. Tumour growth inhibitory effects of Suc-Chi-MMC against Sarcoma 180 solid tumour (A, B) and B16 melanoma (C). At 7 d (A, B) or 6 d (C)

after s.c. inoculation, suspension of Suc-Chi-MMC was injected subcutaneously (A), intratumourally (B) and intraperitoneally (C). Crosses, open

and closed symbols represent control, MMC and Suc-Chi-MMC, respectively. Triangles, squares and circles indicate 5, 10 and 15 mg eq. MMC/kg,

respectively. Tumour size was estimated by the equation of k � a � b � c (cm3), where k; a; b and c represents constant, tumour length, width and

height, respectively.

Y. Kato et al. / Biomaterials 25 (2004) 907–915 911

effects were lowered in Suc-Chi-MMC with eachadministration route (s.c., i.t. or i.p.).

The therapeutic efficacy of Suc-Chi-MMC againstL1210- or P388-bearing mice is summarized in Table 1.Suc-Chi-MMC exhibited good antitumour effectsagainst both tumours and adverse effects were observedto be lowered [39,40,46,50]. It is suggested that theseeffective findings were achieved by virtue of thepreparation of prodrug and the sustained release.However, Suc-Chi-MMC is restricted to the injectionroute due to its particle diameter (1–9 mm) [44,46]. It isnecessary to decrease the particle size of Suc-Chi-MMCor to prepare water-soluble conjugates. It is worthnoting the future outcome of the antitumour activities ofcross-linking conjugate microparticles showing differentrelease profiles since those antitumour activities are notclarified.

4. Water-soluble N-succinyl-chitosan-drug conjugates

4.1. Preparation and in vitro characteristics

The conjugate prepared by the direct coupling ofMMC with Suc-Chi using EDC was obtained as a soliddue to cross-linking among or within the polymer

supports, as described above. A carboxyl group-activated ester of glutaric mitomycin C (MMC-glu-OSU) was utilized in the conjugation reaction as amethod to avoid the formation of the insoluble productof the conjugate (Fig. 2) [48,51]. Shortly, MMC-glu-OSU in N ;N 0-dimethylformamide was mixed with asolution of Suc-Chi in purified water, and was stirred at4�C overnight after adjusting to pH 6.0. The MMCcontent of Suc-Chi-glu-MMC conjugates was ca. 1.3%(w/w). It was difficult to obtain high MMC-loadingconjugates although the reaction between Suc-Chi andMMC-glu-OSU produced water-soluble conjugates.That is why water-soluble conjugates of MMC withSuc-Chi were further examined. The procedure, basedon a method described by Song et al. [42], produced awater-insoluble conjugate because of cross-linking with-in or among the polymer supports (Fig. 2). Since thecross-linking was considered to be formed betweenamino groups and carboxyl groups of Suc-Chi, in-creased N-succinylation of Suc-Chi was expected toreduce the cross-linking. Moreover, a small amount ofEDC was employed for the purpose of the suppressionof the condensation reaction between amino groups andcarboxyl groups of Suc-Chi. In addition, a reduction ofthe reaction time was also applied to suppress the cross-linking. Thus, the reaction scheme described in Fig. 2

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Table 1

Therapeutic efficacy of Suc-Chi-MMC, Suc-Chi-glu-MMC and Suc(II)-Chi-MMC conjugates against tumour-bearing mice

Types of conjugates Materials Tumour cells Routea Scheduleb ILSc (%) Survivors at 60 dd

5 mg/kg at 1 d 45.3 0/6

L1210 i.p.–i.p. 10 mg/kg at 1 d 43.2 0/6

20 mg/kg at 1 d 64.5 0/6

Water-insoluble Suc-Chi-MMC

5 mg/kg at 2 h 142.5 0/6

P388 i.p.–i.p. 20 mg/kg at 1 d 119.7 1/6

5 mg/kg at 1,6,11 d 137.4 0/6

1 mg/kg at 1 d 6.8 0/4

2.5 mg/kg at 1 d 27.3 0/4

Suc-Chi-glu-MMC P388 i.p.–i.p. 5 mg/kg at 1 d 50.5 0/4

10 mg/kg at 1 d 63.6 0/4

20 mg/kg at 1 d 73.9 0/4

Water-soluble

4 mg/kg at 7 d 54.8 0/5

Suc(II)-Chi-MMC M5076 i.v.–i.v. 4 mg/kg at 3 d >97.3 1/5

4 mg/kg at 3,4,5,6 d >192.5 3/6

i.p.–i.p. 4 mg/kg at 3,4,5,6 d >151.1 4/5

MH134 i.p.–i.p. 4 mg/kg at 3,4,5,6 d �5.08 0/5

a (Inoculation route of the tumour cells)–(administration route of the drug).b Administration day or hour post-inoculation. The dose of MMC conjugates was expressed in terms of amount of parent MMC.c ILS means the increase in life span. ILS ¼ ðT=C � 1Þ � 100ð%Þ:d The number of survivors in each group at 60 d post-inoculation.

Fig. 6. Tumour growth inhibitory effects of Suc-Chi-glu-MMC (A) and Suc(II)-Chi-MMC (B, C) against Sarcoma 180 solid tumour. Crosses

represent control group. (A) At 4 d after s.c. inoculation, Suc-Chi-glu-MMC was intravenously (open circles) or intratumourally (open triangles)

injected at a dose of 2.5 mg eq. MMC/kg. (B, C) At 5 d (open squares) or at 5, 9 and 13 d (closed squares) after s.c. inoculation, Suc(II)-Chi-MMC

was intravenously (B) or intraperitoneally (C) injected at a dose of 10 mg eq. MMC/kg. �Four mice died ðn ¼ 1Þ:

Y. Kato et al. / Biomaterials 25 (2004) 907–915912

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Table 2

Therapeutic efficacy of MMC-loading various conjugates against tumour-bearing mice

Materials Tumour cells Route Schedule ILS (%) Ref.

5 mg/kg at 1 d 41.7

10 mg/kg at 1 d 55.3

i.p.–i.p.

2.5 mg/kg at 1,4,7 d 49.9

L1210

5 mg/kg at 1,4,7 d 49.9

5 mg/kg at 1 d 12.8

i.v.–i.v.

10 mg/kg at 1 d 23.1 [54]

Dextran-MMC (T-70a)

5 mg/kg at 1 d 83.0

i.p.–i.p.

10 mg/kg at 1 d 113.9

P388

i.p.–i.v. 10 mg/kg at 1 d 14.8

i.v.–i.v. 10 mg/kg at 1 d 25.0

B16 i.p.–i.p. 10 mg/kg at 1 d 75.2

(T-10a) 10 mg/kg at 1 d 64.5

B16 i.p.–i.p.

(T-500a) 10 mg/kg at 1 d >105.1

[55]

5 mg/kg at 1 d >153.2

PGAb-MMC B16 i.p.–i.p.

10 mg/kg at 1 d 94.4

BSAb-MMC B16 i.p.–i.p. 5 mg/kg at 1 d 13.3

5 mg/kg at 1 d 47.7

Glycol-chitosan-glu-MMC P388 i.p.–i.p. [51]

10 mg/kg at 1 d 68.2

a T-70: molecular weight (MW)=70,000; T-10: MW=10,000; T-500: MW=500,000.b PGA: poly-l-glutamic acid (MW=14,000); BSA: bovine serum albumin (MW=66,000).

Y. Kato et al. / Biomaterials 25 (2004) 907–915 913

was proposed for preparation of water-soluble conju-gate of MMC with Suc-Chi by EDC coupling. Theresultant water-soluble conjugates were termed here asSuc(II)-Chi-MMC. Suc(II)-Chi-MMC afforded highMMC content (12%) which is comparable to Suc-Chi-MMC.

In vitro release of MMC from the water-solubleconjugates is shown in Fig. 4. The release of MMCdepended on the pH in the medium, i.e. the release fromthe conjugates increased with the rise of pH in themedium [47–49]. Suc-Chi-glu-MMC and Suc(II)-Chi-MMC showed a similar release rate at each pH. Bothwater-soluble conjugates exhibited a faster release thanwater-insoluble Suc-Chi-MMC at pH 7.4. It may beproposed as an interpretation that the drug moleculesare not only bound to Suc-Chi but also encapsulated inthe precipitates due to tight cross-linking in the case ofwater-insoluble conjugates. The cross-linking conjugatemicroparticles prepared by Onishi et al. [45] improvedthis hyperslow release at physiological pH.

4.2. Antitumour activities

Fig. 6 illustrates the tumour growth inhibitory effectsof the water-soluble conjugates of MMC with Suc-Chiagainst Sarcoma 180 solid tumour. Growth inhibitoryeffects of Suc-Chi-glu-MMC were inferior to those ofSuc(II)-Chi-MMC [16,49,51]. I.p. administration ofSuc(II)-Chi-MMC showed a better therapeutic effectcompared with i.v. administration; however, i.p. admin-istration elicited a lethal toxic effect. This may be relatedto the greater peripheral localization or lower elimi-nation of the polymer support after i.p. administra-tion [16].

Table 1 also demonstrates the therapeutic efficacy ofSuc-Chi-glu-MMC and Suc(II)-Chi-MMC against tu-mour-bearing mice. The water-soluble conjugates ofMMC with Suc-Chi exhibited good therapeutic effectsagainst tumour-bearing mice with a few exceptions[24,51–53]. Especially, the antitumour activities ofSuc(II)-Chi-MMC against M5076 were markedly high.

ARTICLE IN PRESSY. Kato et al. / Biomaterials 25 (2004) 907–915914

Suc(II)-Chi-MMC showed the high therapeutic effica-cies via repeated administration in both the i.v.–i.v. andi.p.–i.p. systems. The uptake of Suc-Chi into M5076 wasconfirmed to be high by on in vivo i.p.–i.p. uptake studyand microscopy [53]. In contrast, little uptake of Suc-Chi into MH134 was observed. A therapeutic efficacyagainst MH134 was demonstrated when the conjugatesof MMC with Lac-Suc-Chi were administered intraper-itoneally (ILS: >31.4%) [53]. This finding was consis-tent with the results of the in vivo i.p.–i.p. uptake study,indicating that Lac-Suc-Chi showed better distributionto MH134 than Suc-Chi. MH134 cell line was derivedfrom mouse liver, i.e. hepatoma cells, and M5076 cellline was from murine histiocytoma cells. In other words,Lac-Suc-Chi bearing hepatocyte specific ligand wasselectively taken up into MH134 cells. The biodistribu-tions of Suc-Chi and its derivative reflected therapeuticefficacy. These findings suggest that we should takeadvantage of a suitable carrier in response to the tumourtype. In the i.v. administration of Suc-Chi-glu-MMC, nolethal toxicity was shown at 2.5 mg eq. MMC/kg, butlethal toxicity was exhibited at a higher dose. This wasprobably due to the side effect caused by the highviscosity of Suc-Chi-glu-MMC. A similar phenomenonwas observed in the case of Suc(II)-Chi-MMC at ahigher dose [49]. In addition, the therapeutic efficacy ofSuc-Chi-glu-MMC was inferior to that of Suc-Chi-MMC against P388-bearing mice. These findings implythat the low MMC content of Suc-Chi-glu-MMC wasresponsible for the lower therapeutic efficacy comparedwith Suc-Chi-MMC and Suc(II)-Chi-MMC.

Table 2 summarizes the therapeutic efficacy of MMC-loading various conjugates against L1210-, P388- orB16-bearing mice. Every MMC conjugate exhibitedgood antitumour activities against various tumours[51,54,55]. Water-insoluble and water-soluble conju-gates of MMC with Suc-Chi were confirmed to haveantitumour activities against various tumours compar-able to other MMC conjugates.

5. Perspective

Chitin, the fertile material of chitosan, is one of themost abundant polysaccharides in nature, second onlyto cellulose. Suc-Chi and its conjugates can be readilyprepared from chitosan and using MMC and EDC,respectively. Suc-Chi can function well as a drug carrierdue to long systemic retention, low toxicity andaccumulation in the tumour tissue, and the conjugateswith MMC showed good antitumour activities againstvarious tumours. The release characteristics may have tobe further controlled to manifest Suc-Chi’s maximumaptitude. Further, it is necessary to confirm whether theconjugates of other anticancer drugs with Suc-Chi can

be prepared effectively and show good antitumouractivities.

References

[1] Felt O, Buri P, Gurny R. A unique polysaccharide for drug

delivery. Drug Dev Ind Pharm 1998;24:979–93.

[2] Illum L. Chitosan and its use of as a pharmaceutical excipient.

Pharm Res 1998;15:1326–31.

[3] Yamaguchi R, Arai Y, Itoh T, Hirano S. Preparation of partially

N-succinylated chitosans and their cross-linked gels. Carbohydr

Res 1981;88:172–5.

[4] Kato Y, Onishi H, Machida Y. Depolymerization of N-succinyl-

chitosan by hydrochloric acid. Carbohydr Res 2002;337:561–4.

[5] Kuroyanagi Y, Shiraishi A, Shirasaki Y, Nakakita N, Yasutomi

Y, Takano Y, Shioya N. Development of a new wound dressing

with antimicrobial delivery capability. Wound Repair Regen

1994;2:122–9.

[6] Izume M. The application of chitin and chitosan to cosmetics.

Chitin Chitosan Res 1998;4:12–7.

[7] Tajima M, Izume M, Fukuhara T, Kimura T, Kuroyanagi Y.

Development of new wound dressing composed of N-succinyl

chitosan and gelatin. Seitai Zairyo 2000;18:220–6.

[8] Nakagawa A, Myata S, Shimozono J, Soejima Y, Saida M.

Water-soluble chitosan or chitin for treatment of arthritis. JP

Patent No. 06107551, 1994.

[9] Kamiyama K, Onishi H, Machida Y. Biodisposition character-

istics of N-succinyl-chitosan and glycol–chitosan in normal and

tumor-bearing mice. Biol Pharm Bull 1999;22:179–86.

[10] Kato Y, Onishi H, Machida Y. Evaluation of N-succinyl-chitosan

as a systemic long-circulating polymer. Biomaterials 2000;

21:1579–85.

[11] Takakura Y, Takagi A, Hashida M, Sezaki H. Disposition and

tumor localization of mitomycin C-dextran conjugates in mice.

Pharm Res 1987;4:293–300.

[12] Takakura Y, Fujita T, Hashida M, Sezaki H. Disposition

characteristics of macromolecules in tumor-bearing mice. Pharm

Res 1990;7:339–46.

[13] Tanaka T, Kaneo Y, Shiramoto S, Iguchi S. The disposition of

serum proteins as drug-carriers in mice bearing Sarcoma 180. Biol

Pharm Bull 1993;16:1270–5.

[14] Yamaoka T, Kuroda M, Tabata Y, Ikada Y. Body distribution of

dextran derivatives with electric charges after intravenous

administration. Int J Pharm 1995;113:149–57.

[15] Kato Y, Onishi H, Machida Y. Lactosaminated N-succinyl-

chitosan: preparation and biodistribution into the intestine, bone,

lymph nodes and male genital organs after i.v. administration.

Macromol Res, in press.

[16] Kato Y, Onishi H, Machida Y. Biological fate of highly

succinylated N-succinyl-chitosan and antitumor characteristics

of its water-soluble conjugate with mitomycin C at i.v. and i.p.

administration into tumor-bearing mice. Biol Pharm Bull

2000;23:1497–503.

[17] Peterson H-I. Vascular and extraavascular spaces in tumors:

tumor vascular permeability. In: Peterson H-I, editor. Tumor

blood circulation: angiogenesis, vascular morphology, and blood

flow of experimental and human tumors. Boca Raton, FL: CRC

Press; 1979. p. 77–85.

[18] Gerlowski LE, Jain RK. Microvascular permeability of normal

and neoplastic tissues. Microvasc Res 1986;31:288–305.

[19] Matsumura Y, Maeda H. A new concept for macromolecular

therapeutics in cancer chemotherapy: mechanism of tumoritropic

accumulation of proteins and the antitumor agent Smancs.

Cancer Res 1986;46:6387–92.

ARTICLE IN PRESSY. Kato et al. / Biomaterials 25 (2004) 907–915 915

[20] Maeda H, Matsumura Y. Tumoritropic and lymphotropic

principles of macromolecular drugs. Crit Rev Ther Drug Carrier

Syst 1989;6:193–210.

[21] Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular

permeability and the EPR effect in macromolecular therapeutics:

a review. J Control Release 2000;65:271–84.

[22] Maeda H. The enhanced permeability and retention (EPR) effect

in tumor vasculature: the key role of tumor-selective macro-

molecular drug targeting. Adv Enzyme Regul 2001;41:189–207.

[23] Kato Y, Onishi H, Machida Y. Biological characteristics of

lactosaminated N-succinyl-chitosan as a liver-specific drug carrier

in mice. J Control Release 2001;70:295–307.

[24] Kato Y, Onishi H, Machida Y. Lactosaminated and intact N-

succinyl-chitosans as drug carriers in liver metastasis. Int J Pharm

2001;226:93–106.

[25] Regoeczi E, Debanne MT, Hatton MWC, Koj A. Elimination of

asialofetuin and asialoorosomucoid by the intact rat—quantita-

tive aspects of the hepatic clearance mechanism. Biochim Biophys

Acta 1978;541:372–84.

[26] Ashwell G, Harford J. Carbohydrate-specific receptors of the

liver. Ann Rev Biochem 1982;51:531–54.

[27] Fiume L, Busi C, Mattioli A, Balboni PG, Barbanti-Brodana G.

Hepatocyte targeting of adenine-9-b-d-arabinofuranoside 50-

monophosphate (ara-AMP) coupled to lactosaminated albumin.

FEBS Lett 1981;129:261–4.

[28] Fiume L, Busi C, Mattioli A. Lactosaminated human serum

albumin as hepatotropic drug carrier rate of uptake by mouse

liver. FEBS Lett 1982;146:42–6.

[29] O’Hare KB, Hume IC, Scarlett L, Chytry V, Kopeckova P,

Kopecek J, Duncan R. Effect of galactose on interaction of N-(2-

hydroxypropyl) methacrylamide copolymers with hepatoma cells

in culture: preliminary application to an anticancer agent,

daunomycin. Hepatology 1989;10:207–14.

[30] Kaneo Y, Tanaka T, Iguchi S. Targeting of mitomycin C to the

liver by the use of asialofetuin as a carrier. Chem Pharm Bull

1991;39:999–1003.

[31] Pimm MV, Perkins AC, Strohalm J, Ulbrich K, Duncan R.

Gamma scintigraphy of a 123I-labelled N-(2-hydroxypropyl)-

methacrylamide copolymer–doxorubicin conjugate containing

galatosamine following intravenous administration to nude mice

bearing hepatic human colon carcinoma. J Drug Targeting

1996;3:385–90.

[32] Hashida M, Hirabayashi H, Nishikawa M, Takakura Y. Targeted

delivery of drugs and proteins to the liver via receptor-mediated

endocytosis. J Control Release 1997;46:129–37.

[33] Mahato RI, Takemura S, Akamatsu K, Nishikawa M, Takakura

Y, Hashida M. Physicochemical and disposition characteristics

of antisense oligonucleotides complexed with glycosylated

poly(l-lysine). Biochem Pharmacol 1997;53:887–95.

[34] Akamatsu K, Imai M, Yamasaki Y, Nishikawa M, Takakura Y,

Hashida M. Disposition characteristics of glycosylated

poly(amino acids) as liver cell-specific drug carrier. J Drug Tar-

geting 1998;6:229–39.

[35] Bridges K, Harford J, Ashwell G, Klausner RD. Fate of receptor

and ligand during endocytosis of asialoglyocoproteins by isolated

hepatocytes. Proc Natl Acad Sci USA 1982;79:350–4.

[36] Schwartz AL, Fridovich SE, Lodish HF. Kinetics of internaliza-

tion and recycling of the asialoglycoprotein receptor in a

hepatoma cell line. J Biol Chem 1982;257:4230–7.

[37] Harford J, Bridges K, Ashwell G, Klausner RD. Intracellular

dissociation of receptor-bound asialoglycoproteins in cultured

hepatocytes. J Biol Chem 1983;258:3191–7.

[38] Kierszenbaum AL, Rivkin E, Chang PL, Tres LL, Olsson CA.

Galactosyl receptor, a cell surface C-type lectin of normal and

tumoral prostate epithelial cells with binding affinity to endothe-

lial cells. The Prostate 2000;43:175–83.

[39] Song Y, Onishi H, Nagai T. Conjugate of mitomycin C with N-

succinyl-chitosan: in vitro drug release properties, toxicity and

antitumor activity. Int J Pharm 1993;98:121–30.

[40] Onishi H, Song Y, Ichikawa H, Machida Y, Nagai T. Macro-

molecular prodrugs of cytarabine and mitomycin C with chitosan,

N-succinyl-chitosan and 6-O-carboxymethyl-chitin as drug car-

riers. In: Karnicki ZS, Brzeski MM, Bykowski PJ, Wojtasz-Pajak

A, editors. Chitin World. Bremerhaven: Wirtschaftsverlag NW-

Verlag fur neue wissenschaft; 1994. p. 301–10.

[41] Shirahata K. Section VI: derivatives and activities. In: Taguchi T,

editor. Mitomycin. Basic studies—chapter 1: chemical. Tokyo:

Kyowa Kikaku Tsushin; 1984. p. 25–43.

[42] Song Y, Onishi H, Machida Y. Synthesis and drug-release

characteristics of the conjugates of mitomycin C with N-

succinyl-chitosan and carboxymethyl-chitin. Chem Pharm Bull

1992;40:2822–5.

[43] Sheehan JC, Hlavka JJ. The use of water-soluble and basic

carbodiimides in peptide synthesis. J Org Chem 1956;21:439–41.

[44] Song Y, Onishi H, Machida Y, Nagai T. Particle characteristics of

carboxymethyl-chitin–mitomycin C conjugate and N-succinyl-

chitosan–mitomycin C conjugate and their distribution and

histological effect on some tissues after intravenous administra-

tion. STP Pharm Sci 1995;5:162–70.

[45] Onishi H, Takahashi H, Yoshiyasu M, Machida Y. Preparation

and in vitro properties of N-succinyl-chitosan- or carboxymethyl-

chitin–mitomycin C conjugate microparticles with specified size.

Drug Dev Ind Pharm 2001;27:659–67.

[46] Onishi H, Nagai T, Machida Y. Application of chitin, chitosan,

and their derivatives to drug carriers for microparticulated or

conjugated drug delivery systems. In: Goosen MFA, editor.

Applications of chitin and chitosan. Part IV: medicine and

biotechnology. Lancaster: Technomic Publishing Co; 1997.

p. 205–31.

[47] Song Y, Onishi H, Machida Y, Nagai T. Drug release and

antitumor characteristics of N-succinyl-chitosan–mitomycin C as

an implant. J Control Release 1996;42:93–100.

[48] Sato M, Onishi H, Kitano M, Machida Y, Nagai T. Preparation

and drug release characteristics of the conjugates of mitomycin C

with glycol–chitosan and N-succinyl-chitosan. Biol Pharm Bull

1996;19:241–5.

[49] Kato Y, Onishi H, Machida Y. A novel water-soluble N-succinyl-

chitosan–mitomycin C conjugate prepared by direct carbodiimide

coupling: physicochemical properties, antitumor characteristics

and systemic retention. STP Pharm Sci 2000;10:133–42.

[50] Song Y, Onishi H, Nagai T. Pharmacokinetic characteristics and

antitumor activity of the N-succinyl-chitosan–mitomycin C

conjugate and the carboxymethyl-chitin–mitomycin C conjugate.

Biol Pharm Bull 1993;16:48–54.

[51] Sato M, Onishi H, Takahara J, Machida Y, Nagai T. In vivo drug

release and antitumor characteristics of water-soluble conjugates

of mitomycin C with glycol–chitosan and N-succinyl-chitosan.

Biol Pharm Bull 1996;19:1170–7.

[52] Kato Y, Onishi H, Machida Y. Efficacy of lactosaminated and

intact N-succinyl-chitosan–mitomycin C conjugates against M5076

liver metastatic cancer. J Pharm Pharmacol 2002;54:529–37.

[53] Kato Y, Onishi H, Machida Y. Tumor cell uptake of

lactosaminated and intact N-succinyl-chitosans and antitumour

effects of conjugates with mitomycin C. Anticancer Res

2002;22:2771–6.

[54] Hashida M, Kato A, Kojima T, Muranishi S, Sezaki H, Tanigawa

N, Satomura K, Hikasa Y. Antitumor activity of mitomycin

C–dextran conjugate against various murine tumors. Gann

1981;72:226–34.

[55] Kato A, Takakura Y, Hashida M, Kimura T, Sezaki H. Physico-

chemical and antitumor characteristics of high molecular weight

prodrugs of mitomycin C. Chem Pharm Bull 1982;30:2951–7.