Biodegradation of ornithine-containing polylysine hydrogels of ornithine... · Biodegradation of...

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Biomaterials 19 (1998) 1855—1860

Biodegradation of ornithine-containing polylysine hydrogels

Kousaku Ohkawa!, Tomohiro Kitsuki!, Masato Amaike!,Hideki Saitoh", Hiroyuki Yamamoto!,*

!Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan"Experimental Farm, Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan

Received 17 July 1997; accepted 8 March 1998

Abstract

The degradation of the cross-linked cationic poly(amino acid)-glutaraldehyde (GA) hydrogels by two kinds of proteolytic enzymes,trypsin and Aspergillus Protease Type XXIII, and by seven species of soil filamentous fungi has been investigated using homo- andcopolypeptides of lysine (Lys) and ornithine (Orn). Trypsin degraded the hydrogels prepared from poly(Lys) and copoly(Lys Orn)s butnot poly(Orn), while Aspergillus protease degraded all of them. Degradation time of hydrogels by the two proteases became longerwith increasing Orn content in the gel. Seven species of soil filamentous fungi were cultured with hydrogels on Czapeck medium toevaluate the degree of microbial degradation of the hydrogels, and the three species of the fungi, Aspergillus oryzae, Penicilliumcitrinum and Curvularia sp., were grown in culture with an accompanying degradation of the gel matrix, while the other four species,Mucor sp., Rhizopus sp., Cladosporium sp., and ¹richoderma sp., were not. The degree of degradation of gel matrix with growth of thethree fungi became lower with increasing Orn content in the gel matrix. The results might offer some clues to the applications for thecontrolled biodegradation of cationic poly(amino acid) hydrogel by introduction of Orn, suggesting that unnatural amino acid resistshydrolysis by proteases or microorganisms. ( 1998 Published by Elsevier Science Ltd. All rights reserved

Keywords: Biodegradation; Cationic poly(amino acid); Protease; Microorganism; Regulation

1. Introduction

Polymeric substances of natural occurrence possess anattractive potential not only for application with theirfunctional properties but also with biodegradable mate-rial, which is environmentally harmless. One of currentrequisites in application of polymeric materials is notonly that they be stable in the circumstance but also thatthey be degraded finally by microorganisms or by in vivoenzymes. Development of a technology to regulate thebiodegradation of polymeric material, therefore, is ur-gently required.

Cross-linked biological polymers in watery systemshave long been an important class of materials, and areused in a diverse assortment of applications as hydrogelsincluding medical wound dressing. Progress continues indeveloping approaches to describe the molecular struc-ture of cross-linked polymers [1, 2].

We have investigated gel formation experiments ofwater-soluble biopolymers such as cationic polypeptides,chitosan and lignin using organic cross-linking agentssuch as dialdehydes [3—6], and also reported biodegrada-tion of cross-linked biopolymer hydrogels [7—9].

As shown in our previous study, PLL-GA gels weredigested by trypsin but not by chymotrypsin and papain[7]. Difference in the susceptibility towards proteolyticenzymes was explained by specificity of the three pro-teases towards the peptide bond to be cleaved. The PLO-GA gel, on the other hand, was not hydrolyzed by any ofthe three proteases, indicating their stability towardshydrolytic enzymes [7].

This report describes an approach for the controlledbiodegradation of cationic cross-linked lysine polypep-tide hydrogels using hydrolytic enzymes and microor-ganisms. From the results of our experiments, it wasinferred that controlled biodegradation could beachieved by incorporation of unnatural Orn residue, asa resistant factor against proteases, into the main chain ofPLL-GA gels.

0142-9612/98/$—See front matter ( 1998 Published by Elsevier Science Ltd. All rights reserved.PII S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 0 9 1 - X

Table 1Estimated DP and M

8of synthetic poly(amino acid)s

Poly(amino acid)s DP M8

PLL 640 133,200Copoly(Lys9 Orn1) 1230 254,200Copoly(Lys7 Orn3) 690 140,700Copoly(Lys1 Orn1) 530 106,600Copoly(Lys3 Orn7) 300 59,500Copoly(Lys1 Orn9) 230 45,000PLO 290 56,300

2. Materials and methods

2.1. Materials

Poly(L-lysine) (PLL), copoly(Lys9 Orn1), copoly(Lys7Orn3), copoly(Lys1 Orn1), copoly(Lys3 Orn7), copoly(Lys1 Orn9) and poly(L-ornithine) were synthesizedaccording to the amino acid N-carboxyanhydride pro-cedure followed by polycondensation (Table 1). The DPs(average degrees of polymerization) of the polypeptideswere estimated from viscometry as described in our pre-vious article [10].

Enzymes, trypsin (from porcine pancreas; EC 3.4.21.4;activity, 5200 USP units mg~1), papain (from Caricapapaya; EC 3.4.22.2; activity, over 3000 USP units mg~1)were purchased from Wako Pure Chemical Industries,Ltd. Protease type XXIII (from Aspergillus oryzae; activ-ity, approximately 4 units mg~1) was purchased fromSigma.

Microorganisms employed for the biodegradation ofpoly(amino acid) hydrogels were the following; Penicil-lium citrinum, A. oryzae, Mucor sp., Rhizopus sp., Clado-sporium sp., Curvularia sp., and ¹richoderma sp.

2.2. Preparation of hydrogels

1/5, 1/10, 1/15, or 1/30 equivalent molar of glutaral-dehyde (GA) towards amino groups of lysyl and/orornithyl residue was added to a solution of 10 mgml~1

poly(amino acid)s in distilled water in a glass tube, andthe mixture was allowed to stand for 10 h at room tem-perature. The diameter of the glass tube is 0.8 cm or0.8 mm for the experiment on biodegradation or swell-ing—shrinking of the hydrogels, respectively. When thehydrogel was prepared in the glass tube with diameter of0.8 cm, the hydrogel was molded into a disk. The gelformed was transferred onto the center of a petri dish andwashed with distilled water three times.

The degree of swelling was estimated from (d/d0)3,

where d0

or d is the diameter of the gel matrix in initia-tion or transition in swelling—shrinking experiment, re-spectively. Distilled water or 70% dioxane (1,4-) was usedas the respective solvent for swelling or shrinking.

2.3. Enzymatic degradation of hydrogels

1000units of trypsin, papain or protease type XXIII indistilled water solution (1 ml) were added onto the geldisk swollen with distilled water, and the gel was allowedto stand at 25°C. Hydrolysis of the hydrogel by pro-teolytic enzymes was observed during the transition ofthe gel matrix into a liquid state. The degradation timewas defined as the period required for the completetransition of the gels into liquid under observation by thenaked eye.

2.4. Microbial degradation of hydrogels

Microorganisms were added to poly(amino acid) hy-drogel disks immersed in Czapeck medium on petridishes via a platinum needle under aseptic conditions.The microorganisms attached onto the hydrogels werestatically cultured at 25°C. To keep the gels wet, about3ml of sterilized water per week was added to the me-dium. Biodegradation of poly(amino acid) hydrogels wasobserved as a collapse of the hydrogel disk accompany-ing the growth of microorganisms. To evaluate thegrowth of microorganisms, relative amounts of maturesporangia of the microorganisms, A. oryzae, P. citrinumand Curvularia sp., were estimated by imaging analysison each photograph of the culture, defining the area sizeof sporangia fraction measured on the culture withcopoly (Lys9 Orn1)-1/10GA hydrogel as 100%. The anal-ysis was performed on Kontron Electonik Imaging Sys-tem KS300 (Carl Zeiss Vision K.K.,Munchen, Germany).

3. Results and discussion

Estimated DP and molecular weight (M8) of the poly-

mers obtained are listed in Table 1. In an examination onthe condition for gelation of poly(amino acid)s, the molarequivalent of GA or the polymer concentration was inde-pendently varied at 1/5, 1/10, 1/15, and 1/30, or at 10 and20%, respectively. Gelation of all 10% poly(amino acid)solutions were observed by addition of GA above 1/15equivalent molar, while increased viscosity of polymersolutions in 1/30 equivalent molar GA was observed.When polymer concentration was raised to 20% gelationwas also observed even in 1/30 equivalent molar GA(data not shown). From these findings described above,10% poly(amino acid)-1/10 GA hydrogels were em-ployed in a series of experiments from the basis of therelatively high material strength in swelling state amongother gels prepared using different equivalent molar GA.

As an example, the swelling time course of copoly(Lys1Orn1)-1/10 GA gel was shown in Fig. 1. The gel exhibitedmostly reversible behavior to swell in distilled water atthe swelling ratio of 9.6 and to shrink in 70% dioxane atthat of 3.9.

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Fig. 2. Enzymatic degradation of copoly(Lys1 Orn1)-1/10 GA hydrogel by trypsin (1000 unit) at 0 (a), 90 (b), and 180 min (c), or by Aspergillus protease(1000 unit) at 0 (d), 45 (e), and 75 min (f ).

Fig. 1. Swelling time course of copoly(Lys1 Orn1)-1/10 GA gel. Openand closed circles represent the solvents to swell (distilled water) and toshrink (70% dioxane), respectively.

Table 2Degradation time of poly(amino acid)-1/10 GA hydrogels by pro-teolytic enzymes

Hydrogels Orn content Degradation time (min)(%)

Trypsin! Aspergillusprotease!

PLL-GA 0 150 30Copoly(Lys9 Orn1)-GA 10 150 45Copoly(Lys7 Orn3)-GA 30 165 60Copoly(Lys1 Orn1)-GA 50 180 75Copoly(Lys3 Orn7)-GA 70 225 90Copoly(Lys1 Orn9)-GA 90 300 120PLO-GA 100 ND" 180

! 1000 unit."Not degraded during the observation.

In order to discuss the properties of poly(amino acid)hydrogels in the biodegradation process, the gel prepara-tions were subjected to hydrolysis by two kinds of pro-tease, trypsin or Aspergillus protease type XXIII (Fig. 2).In tryptic digestion, the diameter of the disk ofcopoly(Lys1 Orn1)-1/10 GA gel was reduced, and theliquified fraction of the gel gradually spread around thedisk during the time course (Fig. 2a, b). After 180 min,the gel matrix was hydrolyzed completely intoa homogenous and transparent liquid. When Aspergillusprotease was used in the hydrolysis, it took 75 min for

complete degradation of copoly(Lys1 Orn1)-1/10 GA gel(Fig. 2d—f). This degradation time was less than half thatof trypsin, suggesting a higher susceptibility of thepoly(amino acid)-1/10 GA gels towards enzymatic degra-dation by Aspergillus protease than by trypsin.

Table 2 summarizes the complete degradation timeof a series of hydrogels prepared from PLL, copoly(Lys Orn) array or PLO. Degradation of PLL and

K. Ohkawa et al. / Biomaterials 19 (1998) 1855—1860 1857

Fig. 3. Microbial degradation of copoly(Lys1 Orn1)-1/10 GA hydrogel by A. oryzae at 0 (a), 7 (b), and 23 days (c) or by P. citrinum at 0 (d), 23 (e), and31days (f).

copoly(Lys9 Orn1)-1/10 GA gels by trypsin were com-pleted with the fastest degradation time being 150 minamong all gels, while those of copoly(Lys7 Orn3)-,copoly(Lys1 Orn1)-,copoly(Lys3 Orn7)- and copoly(Lys1Orn9)-1/10 GA gels were 165, 180, 225 and 300 min,respectively. Furthermore, the initial gel matrix of PLOremained unchanged during the time course of observa-tion, suggesting a very slow progression of hydrolysis.

Aspergillus protease digested all hydrogels (includingcross-linked PLO gel) faster than those in the case oftrypsin. PLL-1/10 GA gel was liquified at 30 min anddegradation time became about 15 min longer with everyincrease of 20% of Orn content in the hydrogels. EvenPLO-1/10 GA gel was digested within 180 min.

These results indicate that the degradation time inenzymatic digestion increased with increasing Orn con-tent in the polypeptide gels, suggesting that ornithineresidue confers resistance to hydrolysis by both pro-teases.

Aspergillus fungi produce at least four different types ofproteolytic enzymes, including alkaline serine protease[11], acidic aspartic protease [12], metalloproteinase[13] and carboxyl proteinase [14]. Among the hydrolyticenzymes, however, it is still unclear which protease isinvolved in the degradation of PLO-1/10 GA hydrogel,since there have been no attempts so far to examine anyhydrolysis of ornithyl peptide by proteases. In commer-

cial preparation of Aspergillus protease, however, theinvolvement of an exopeptidase capable to digest or-nithyl peptides has been suggested in the results.

Use of papain in the hydrolysis of Lys copolypeptidehydrogels resulted in no apparent change of initial gelmatrices were being observed (data not shown), sugges-ting that all of the gels prepared were not degradable bypapain.

Further to the observed higher efficiency of Asper-gillus protease to digest poly(amino acid)-GA hydrogels(Table 2), a series of the hydrogels prepared were sub-sequently subjected to microbial degradation in a culturewith seven species of soil fungi, P. citrinum, A. oryzae,Mucor sp., Rhizopus sp., Cladosporium sp., Curvularia sp.and ¹richoderma sp. As a preliminary attempt, each ofthe seven microorganisms was cultured individually un-der a condition in which poly(amino acid)-1/10 GA hy-drogels swollen with distilled water were provided as thesole nutrition. In this condition, however, all seven spe-cies of microorganisms examined have gradually becomeweak and finally died, indicating a requirement ofother nutrients or carbon sources (data not shown).However, when Czapeck medium was added to theculture system as additional nutrients, the micro-organisms survived.

Figure 3 shows the time course of growth of twomicroorganisms, A. oryzae and P. citrinum, in the culture

1858 K. Ohkawa et al. / Biomaterials 19 (1998) 1855—1860

Fig. 4. Effect of Orn content in copoly(Lys Orn)-1/10 GA hydrogel on microbial dagradation by A. oryzae (a, 10%; b, 50%; c, 90%), byP. citrinum (d, 10%; e, 50%; f, 90%), or by Curvularia sp. (g, 10%; h, 50%; i, 90%).

system with Czapeck medium. The degree of growth ofA. oryzae or P. citrinum is generally represented by theformation of black sporangia, which are seen as a darkishfraction of a colony or by increasing numbers of the bulkcolony in the photographs, respectively.

When A. oryzae was cultured with copoly(Lys1 Orn1)-1/10 GA gels on Czapeck medium, the gel matrix grad-ually began to collapse with the growth of the colonyafter three days (data not shown). The development ofbrawny or darkish sporangia was observed after aboutseven days (Fig. 3b), and in this time the gel matrix wasmostly liquidified. Further observation was done to con-firm complete degradation of the gel after 23 days(Fig. 3c). A similar result was obtained when P. citrinumwas cultured with the same hydrogel and medium(Fig. 3f). In this case, the period required for complete

liquidification of the gel was about 1.5 times longer thanthat in the case of A. oryzae. These results suggest thatthe productive secretions of hydrolytic enzymes, such asproteases [11—14], from microorganisms during theirgrowth caused the degradation of gel materials.

In order to examine the effect of the Orn content in thepoly(amino acid)-1/10 GA hydrogels on the degradationtime by the microorganisms, the hydrogels with differentOrn contents were introduced to a culture with sevenspecies of microorganisms. Fig. 4 summarizes the resultsof the 15—20 days culture of the three microorganisms, A.oryzae, P. citrinum or Curvularia sp. with three poly(aminoacid) hydrogels, copoly(Lys9 Orn1)-, copoly(Lys1 Orn1)-and copoly (Lys1 Orn9)-1/10 GA gels. The degree ofgrowth of A. oryzae was higher when it was cultured withhydrogel containing less Orn residues (Fig. 4a—c). The

K. Ohkawa et al. / Biomaterials 19 (1998) 1855—1860 1859

relative degree of growth of A. oryzae on the hydrogelsestimated from the amount of mature sporangia are 100,52 and 16%, for copoly(Lys9 Orn1)-copoly(Lys1 Orn1)-and copoly (Lys1 Orn9)-1/10 GA hydrogel, respectively.Similar results were obtained from the cases of P. cit-rinum and Curvularia sp. The order of the growth evalu-ated by relative amount of mature sporangia was 100 or100% for copoly(Lys9 Orn1)-1/10 GA (Fig. 4d, g)'43 or49% for copoly(Lys1 Orn1)-1/10 GA (Fig. 4e, h)'15or 39% for copoly(Lys1 Orn9)-1/10 GA (Fig. 4f, i)in P. citrinum or Curvularia sp., respectively, suggesting aresistant property of Orn residues to secreted proteasefrom the microorganisms, and against the growth of thethree fungi. The reason for the latter effect probably maybe that Orn residue, as an unnatural amino acid, is notintroduced into fungal intracellular metabolic system. Allof the other four microorganisms were not grown in thecultures with the polypeptide hydrogels.

Concerning the medical use of the polylysine hydro-gels, we have previously reported that PLL-GA hydrogelpossesses selective absorption ability towards anionicmolecules including benzoic acid and acidic amino acids[4, 5], suggesting its application as a carrier material forsustained release of anionic drugs in vivo. However, PLLhas a high activity to agglutinate the erythrocyte and toaccelerate the polymerization of fibrin, resulting in thestimulation of blood clot formation [15]. Therefore, it isdifficult to use the PLL hydrogel directly in blood circu-lation system in vivo.

On the other hand, it has been revealed that PLL withan appropriate molecular weight has an antineoplasmicactivity to prevent the growth of Ehrlich ascites tumor inmice [16]. Furthermore, polylysine-drug complexes, inwhich an antitumor agent methotrexate (MTX) wasbound covalently to amino groups of PLL through di-sulfide compounds as spacers, inhibited the growth andthe increase of cell numbers in culture of Chinese hamsterovary cell, while PDL (poly-D-lysine)-MTX complex didnot [17]. This was due to that lysosomal enzyme couldnot digest the PDL-MTX complex, resulting in sup-pression of the release of MTX from PDL after thecomplex was incorporated in the cell [17]. These suggestthat property of biodegradation of polylysine as a carrierof drug is a significant feature for the effectiveness as wellas the development of a novel method for appropriatecontrol of biodegradation of polylysine is required forimprovement of the strategy to deliver the drugs. Withregards to this, lysosomal enzymes seem to be counter-parts of the proteases, such as trypsin and Aspergillusprotease, which we have employed in the present study.The properties of Orn residues described here afforda possibility for application of cross-linked cationicpoly(amino acid) gel as a ‘controlled-biodegradable ma-terial’, together with our previous observations thatPLO-GA gels behave like PLL-GA gels in other proper-ties except for susceptibilities to biodegradation, such as

swelling—shrinking behavior [7] and selective absorptionability towards anionic molecules [4, 5].

A potential of Orn to control the biodegradation pro-cess was derived from this work, and suggest furtherapplications of other unnatural amino acids with lysine-related chemical structures, such as diaminobutyric, dia-minopropionic, or also diaminoheptanic acids.

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