One-step preparation of glycopeptide microspheres based on α-amino acid-N-carboxyanhydride polymerization using interfacial protocols

One-Step Preparation of Glycopeptide Microspheres Basedon a-Amino Acid-N-carboxyanhydride Polymerization UsingInterfacial Protocols

Jing Wang, Changsheng Liu, Ping Chi

Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Center for Biomedical Materialsof Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China

Received 14 May 2007; revised 3 February 2008; accepted 17 March 2008Published online 20 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31186

Abstract: A type of polysaccharide-polypeptide hybrid material, chitin derivative with

polypeptide side chains was prepared by the graft copolymerization of L-leucine N-

carboxyanhydride triggered by water-soluble chitin (WSC). The studies on surface tension

and aggregation demonstrated surface activity of WSC. Using this extraordinary property, the

microspheres were synchronously obtained via interfacial polymerization. The method

employed here to form the microspheres was in direct contrast to previous syntheses that used

either templating method or oil-in-water emulsion. The study provided a facile approach for

synthesizing microspheres with a variety of distinct polypeptide and regulated graft length,

which had mimetic structure of glycoconjugation in extracellular matrix. Furthermore, the

swelling capability of the microsphere in both acidic aqueous and organic solvents would give

promising application in drug delivery. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl

Biomater 89B: 45–54, 2009

Keywords: bioactive material; biomimetic; extracellular matrix; graft/copolymer

INTRODUCTION

Many strategies have attempted to develop temporary scaf-

folds for tissue engineering using biodegradable and bio-

compatible synthetic or natural polymers over the past

decade.1–4 Aliphatic polyesters such as polylactide (PLA),

polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA) are among the few synthetic polymers

that have been used widely in the tissue engineering. How-

ever, cell affinity toward synthetic polymers is generally

poor as a consequence of their low hydrophilicity and lack

of surface recognition sites, which affect cell suspension

penetrating into the scaffolds.5–8 Principally, the scaffold’s

design should mimic the structure and biological function

of native extracellular matrix (ECM), which provide me-

chanical support and regulate cell activities.9–12

ECM, a stable structural element surrounding the

cells,13–16 may provide environments for proper growth,

differentiation, and migration of cells, and serve as reser-

voir of regulatory molecules in tissues such as growth fac-

tors.17–20 The components of ECM can be subdivided into

four classes: glycopeptides, proteoglycans, elastins, and col-

lagens. Because of the complexity of the ECM, studying

cell behavior in ECM derivatives or even single component

of the ECM would make it difficult to decipher the impor-

tance of each factor involved.21–24

Since numerous cellular activation processes are initiated

by ECM ligands, novel methods for mimicking the natural

ECM are of great interest for improving the biocompatibility

or creating new biomaterials. However, the utility of collagen

and proteoglycans, such as condroitin sulfates and hyaluronic

acid, has been limited by extremely high price and poor me-

chanical properties. To establish an ideal scaffold mimicking

the natural ECM, it is desirable to develop a simple approach

to combine the advantages of both natural and synthetic poly-

mers, which could enhance affinities with cells and speed up

tissue regeneration, as well as substitutes of ECMs.

Polysaccharides are interesting materials as their carbo-

hydrate moieties interact with or are integral components

of many cell adhesion molecules and matrix glycopro-

teins.25,26 Chitin, a natural polysaccharide consisting of b-(1?4)-linked 2-acetamide-2-deoxy-D-glucopyranose, is one

of the most abundant and easily obtained biopolymers next

to cellulose. It is commonly found in the shells of crusta-

cean such as crab and shrimp, exoskeletons or cuticles of

many invertebrates, and in the cell walls of most fungi and

Correspondence to: C. Liu (e-mail: [email protected])Contract grant sponsor: National Science Fund for Distinguished Young Scholars

of China; Contract grant number: 20425621Contract grant sponsor: Major State Basic Research Program of China; Contract

grant number: 2005CCA01000Contract grant sponsor: Basic Research Foundation of Shanghai Science and

Technique Committee; Contract grant number: 05DJ14005Contract grant sponsor: Shanghai Leading Academic Discipline Project; Contract

grant number: B502

' 2008 Wiley Periodicals, Inc.

45

some algae. This polymer is known to be nontoxic, bio-

compatible, and enzymatically biodegradable.27 Further-

more, chitin has structural characteristics similar to

glycosaminoglycans (GAGs, main component of proteogly-

cans such as condroitin sulfates and hyaluronic acid), which

occurs ubiquitously within the ECM.28–30 So, it is an excel-

lent starting template for the design of ECM-like materials.

Kurita et al.31,32 has showed the possibility of preparing chi-

tin-protein complexes by the graft copolymerization of

amino acid N-carboxyanhydride (NCA) onto chitin. Ma and

coworkers33 also reported the synthesis of chitosan-g-poly(L-leucine). Nevertheless, the previous study focused on

the synthesis of the copolymer. The graft production had

comparatively poor solubility in most of the common sol-

vents, which hampered its further process and applications.

In this study, we have developed a simplified polysaccha-

ride-polypeptide system mimicking the glycoconjugations in

ECM, which allows us to modulate the components and

properties expediently. Water-soluble chitin (WSC) with

degree of acetylation close to 0.50 was selected as the poly-

saccharide backbone moiety and was found possessing par-

ticular surfactant activities. In the light of these findings, it

was interesting to investigate the applicability of preparing

the glycoconjugate polymeric microsphere in situ without

additional emulsion. To our knowledge, such microsphere

based upon the polysaccharide-polypeptide hybrid materials

has not yet been reported in literature. This work provided a

novel drug carrier and a unique method for preparing the

glycoconjugate microsphere.

MATERIALS AND METHODS

Materials

All reagents used were available from commercial sources.

Chitosan with average molecular weight (Mw) of 1.5 3 105

and 90% degree of deacetylation (DD) was from Shanghai

Kabo and was used for the preparation of graft copolymer.

L-leucine was purchased from Huamei Biochemical. Tetra-

hydrofuran (THF) and n-hexane were distilled from so-

dium/benzophenone in an inert atmosphere to remove

water before using. Ethyl acetate was distilled from P2O5.

All other solvents were of analytical grade and used with-

out further purification. Pyrene was purchased from Sigma

and used as received.

Synthesis of Chitin-graft-Polypeptide Microsphere

WSC was prepared according to the method reported.34 DD

was determined by using titration method. L-leucine NCA

was synthesized by treating L-leucine with triphosgene in

THF at 508C and recrystallized from THF/hexane.35 The

graft copolymerization of a-amino NCA onto chitin was

carried out in heterogeneous in a similar manner to that

described in the previous paper (Scheme 1).31,32 In brief,

0.1 g WSC was dissolved in 10-mL deionized water. A

given amount of the NCA dissolved in dry ethyl acetate

with various molar equivalent of NCA and O/W ratio was

transferred into aqueous WSC solution (0.1 g/10 mL) and

Scheme 1. Schematic illustration to show the reaction between water-soluble chitin and L-leucine

NCA.

46 WANG, LIU, AND CHI

Journal of Biomedical Materials Research Part B: Applied Biomaterials

subjected to the reaction at 08C for 2 h under Argon. After

that, the mixture was poured into 120-mL N,N-dimethylfor-

mamide (DMF). The resulting grafting copolymer was fil-

tered and washed with DMF/deionized water (10:1) for

purification. The resultant microparticles were then washed

with acetone in grades and collected after lyophilizing.

Cryobreaking of Hollow Microspheres

Cryobreaking technique was carried out under ultrasonifica-

tion in liquid nitrogen medium36 to obtain the broken vesi-

cle and details as follows. Microspheres dispersed in DMF

were frozen by immersing into liquid nitrogen medium and

then partially melted by naturally exposing to the air. At

this point, the microspheres were placed into an ultrasonifi-

cation chamber, and let the tip of the ultrasonicator touch

the solid/liquid interface. The process lasted for about

45–50 s. After that, the sample was ready for subsequent

lyophilization.

Surface Tension Measurement

A RADIAN 300 Processor Tensionmeter (Thermo Cahn.)

was used to determine the surface tension of chitosan and

WSC solutions at various concentrations with plate method

at 258C 6 0.18C. All of the solutions were kept at room

temperature for 1 day before surface tension measure-

ment.37,38

Fluorescence Spectroscopy

Pyrene was selected as a fluorescent probe to demonstrate

the presence of hydrophobic domains. A known volume of

pyrene in methanol was put into a series of flasks and

evaporated under an argon purge to remove the solvent.

Then, an aqueous WSC solution was added into the test

tube. The final concentration of pyrene was l 3 1026 mol/

L. The solutions were sonicated for 15 min in an ultrasonic

bath and then kept at room temperature for 2 h before

spectroscopic analysis. The steady-state fluorescence spec-

tra were carried out on a Cary Eclipse fluorescence spectro-

photometer (Varian, USA) at 258C. The excitation

wavelength of pyrene was set to 335 nm. The hydrophobic

index, I1/I3, was calculated as the ratio of the intensities at

the first (374 nm) and the third (385 nm) vibrational peak

in the pyrene emission spectra.39

Characterization

1H NMR spectra of graft copolymers were obtained with

an AM-400 spectrometer (Bruker) at room temperature,

using CF3COOD as solvent. The Fourier transform infrared

transmission spectra were recorded using an attenuated

total reflection (ATR) method in AVATAR360 spectrome-

ter (Nicolet). Microspheres were observed through TE2000-

U optical microscope with a CCD camera (Nicon). Mor-

phological investigations of microsphere were performed

with a JEOL-JSM-6360LV scanning electron microscope

(Japan), operating at an acceleration voltage of 15 kV. The

microparticles were lightly sputter-coated with a thin layer

of Au/Pd prior to the observation.

Swelling Behavior Studies

In the swelling experiment, the dried microparticles of

about 20 mg were placed in a cylindrical tube. One millili-

ter of deionized water, hydrochloric acid (pH 5 5, 3), and

DMF were added to soak the microparticles, respectively.

Then seal the tube with a lid to avoid volatilizing. After

sufficient time for achieving swelling equilibrium, the tube

was centrifuged and the supernatant was removed. The

swollen microparticles were collected and weighed quickly.

The swelling ratio (Q) was calculated with the following

formula:

Q ¼ W2 �W1

W1

3100%

where W1 is the weight of the dried microparticles, W2 is

the weight of the swollen microparticles. Each sample was

measured in triplicate.

RESULTS

Surface Tension and Aggregation Behaviorof Water-Soluble Chitin

The solubility of chitin is remarkably poorer because of the

high crystallization supported by hydrogen bonds. Through

acetylation, chitosan with DD close to 0.5 could dissolve in

water. WSC can be prepared via either alkaline treatment

of chitin or acetylation of chitosan; the latter was selected

in this experiment (Scheme 2).

The surface tension of the chitosan and WSC were

measured as a function of concentration at room tempera-

ture. As shown in Figure 1, chitosan (DD [ 90%) showed

higher values than those of the acetylated productions with

varying DD. Moreover, an increase in the concentration of

the solution resulted in a decrease in surface tension (c)due to the introduction of hydrophobic groups. In addition,

it could also be seen that the surface tension lessened with

the decrease of DD at the same concentration, because

there were more hydrophobic groups on the surface.

Scheme 2. Synthetic routes to WSC from chitosan.

47ONE-STEP PREPARATION OF GLYCOPEPTIDE MICROSPHERES


The aggregation behavior of WSC in aqueous solution

was monitored by fluorometry in the presence of pyrene as

a fluorescence probe (Figure 2). The change of the intensity

ratio (I1/I3) in the fluorescence emission spectra was meas-

ured as a function of polymer concentration, which was

shown in Figure 3. The ratio of the signals I1/I3 decreased

when the polymer concentration increased and reflected the

hydrophobicity of the systems. The results indicated that

the polymer began to form aggregates at dilute conditions

and the microenvironment around pyrene got less and less

polarity with increasing concentration.

Synthesis and Characterization ofChitin-g-Poly-L-leucine Microsphere

In this article, we reported the results of the highly efficient

graft copolymerization of L-leucine NCA onto WSC

according to the method of Kurita et al.31,32

The graft copolymerization was thus confirmed to pro-

ceed quite efficiently and the specialty of the graft poly-

merization was in accordance with the Kurita’s results. The

yield of the copolymer increased with an incremental

amount of NCA added, indicating the introduction of poly-

peptide side chains with increasing length. Table I listed

the results of the graft copolymerization. The average

degrees of polymerization (DPs) of the polypeptide side

chains were calculated from weight increases, as well as

the amount of peptide units per amino group in the graft

copolymers. The lengths of polypeptide side chains intro-

duced were tunable through varying the amount of NCA

fed-in.

The FT-IR spectrum of the final grafted product, chitin-

graft-poly(L-leucine), with different side-chain length (DP)

was shown in Figure 4. The disappearance of carbonyl an-

hydride peaks at 1855 and 1785 cm21 indicated the ring-

open polymerization of NCA occurring without original

monomer, while the stretch and dissymmetry stretch librat-

ing peaks at 1000–1100 and 1152 cm21 (C��O��C) still

existed, which represented polysaccharide segments of chi-

tosan. Compared to the spectrum of the WSC, an obvious

increase of C��H stretch libration at 2958 cm21 and bend

libration of methylene groups at 1465 cm21 could be

observed. Moreover, it was noteworthy that the absorbance

of the amide bands I (1650 cm21), II (1540 cm21), and III

(1314 cm21) increased distinctly and that of the complex

bands due to pyranose rings of chitin (1000–1150 cm21)

were weaker with an increase in the length of the polypep-

tide side chains. This is interpreted as a result of graft

copolymerization, proving that the chains of polypeptide

were grafted onto chitin successfully. Besides, incorpora-

tion of poly(L-leucine) led to small modifications, that is,

the amide bands of WSC were slightly shifted at lower fre-

quencies. This result implied that hydrogen bonding might

occur between chitin and polypeptide, which was consistent

with the reported results in those of chitosan composi-

tions.40,41 However, the poor solubility of the product in

Figure 1. Surface tension as a function of different concentrationwith varying DD. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]

Figure 2. Fluorescence emission spectra of pyrene as a function ofWSC concentration in H2O at 25 8C. (1) 0.0001 g/mL; (2) 0.001 g/

mL; (3) 0.005 g/mL; (4) 0.01 g/mL; (5) 0.015 g/mL. The excitation

wavelength was 335 nm, and the spectra were accumulated with an

integration of 5 s/nm. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

Figure 3. Intensity ratio (I1/I3) for pyrene in deionized water of theWSC (DD 5 45.78%) as a function of logarithm concentration.



organic solvents was a serious hindrance for the determina-

tion of absolute molecular weights.

Figure 5 displayed the 1H NMR spectrum of chitin-

graft-polypeptide in CF3COOD. From the NMR spectrum,

the peaks appearing at 0.9 and 1.6–1.7 ppm could be

assigned unambiguously to the proton of methyl and meth-

ylene on the anomeric carbon of leucine. The resonance at

2.3 ppm was easily assigned to the N-acetyl protons. Thebroad complex peaks around 4.1 ppm were assigned to the

H-1, H-3, H-4, H-5, H-6 of b-(1?4)-linked 2-amino-2-

deoxy-D-glucopyranose (GlcN) residue, and H-2 of 2-acet-

amido-2-deoxy-D-glucopyranose (GlcNAc) on the saccha-

ride cyclic unit, respectively. These broad peaks overlapped

with the chemical shift of the CH units on the end group

of poly(L-leucine), so that calculation of the graft length

from NMR became incapable. The intensity ratio of poly-

peptide with chitin segments increased along with the ele-

vation of NCA/Cs, thus indicating that the length of the

polypeptide side chains could be regulated easily by vary-

ing the amount of NCA under these conditions.

Optical images and scanning electron microscopy (SEM)

observations were used to characterize the morphology of

the chitin-g-poly-L-leucine microparticles prepared under

optimized condition. The particles were found to have reg-

ular spherical geometry, especially dispersed in ‘‘wet’’ con-

dition such as DMF directly after preparation [Figure 6(a)].

The ‘‘dried’’ microspheres partially collapsed and some had

a little burr attached on the surface, but retained the sphe-

ricity on the whole as the solvent been pumped away with

[Figure 6(b)]. Further observation through SEM [Figure

6(c)] showed quasispherical morphology with diameters

ranging from 100 to 400 lm, and the typical particle size

lied between 100 and 150 lm. After cryobreaking treat-

ment, the particles exhibited opening holes on their surfa-

ces, which indicated that they had a truly hollow structure,

TABLE I. Graft Copolymerization of L-leucine NCA onto Water-Soluble Chitin

Cs

Feeded (g)

NCA

Feeded (g)

Yield

(g)

NCA/Cs

(mol/mol)aNCA/NH2

(mol/mol)

DP of Polypeptide

Side ChainsbConversionc

(%)

0.1 0.1505 0.19 1.76 3.85 3.20 83.10

0.1 0.2240 0.25 2.62 5.73 5.25 91.78

0.1 0.3654 0.33 4.28 9.35 8.38 89.61

0.1 0.4122 0.3677 4.31 9.41 9.52 90.24

0.1 0.8256 0.6515 9.67 21.13 19.61 92.81

aWater-soluble chitin with DD 5 45.75% was used.b Average degree of polymerization, determined from weight increase.c Amount of NCA polymerized/amount of NCA added.

Figure 4. Infrared spectra of WSC, polypeptide, and chitin-g-poly-

peptide. (a) Water-soluble chitin; (b–d) chitosan-g-poly (L-Leucine)

(b: DP 5 3.2; c: DP 5 5.25; d: DP 5 8.38); (e) poly L-leucine. [Color

figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

Figure 5. 1HNMR spectra of chitin-g-poly (L-Leucine) (a) DP 5 3.20;(b) DP 5 8.38.



with a wall thickness of about 40–80 lm, further support-

ing the feasibility of forming capsule in situ through this

special procedure. Moreover, the resultant microparticle

showed a rough surface morphology [Figure 6(d)].

Swelling behavior of the microspheres was presented in

Figure 7. Obviously, the swelling ability in polar organic

solvent such as DMF was much higher than that in aqueous

medium. At the early stage of immersion in DMF, the

dried microparticles were quickly swollen due to very fast

solvent absorption. In addition, the swelling ability

decreased with increasing DP, which was imputed to the

effect of longer side chains of the polypeptide moiety.

DISCUSSION

Aggregate Formation and Surface Activity Propertyof Water-Soluble Chitin

Chitosan, a linear polysaccharide, is composed of random

repeating units of b-(1?4)-linked 2-amino-2-deoxy-D-glu-

copyranose and 2-acetamido-2-deoxy-D-glucopyranose. Par-

tially deacetylated chitosan with DD close to 0.50 was

found to be soluble in water. This water-soluble polysac-

charide has a great advantage as a starting material for

modifications, since the water solubility allows smooth

reactions under mild conditions.

Both the measurements of surface tension (c) and pyrene

fluorescence in aqueous solution were combined to verify

Figure 6. Representative optical and SEM images of chitin-g-poly L-leucine microspheres (a) opti-

cal microscope image of the freshly prepared microsphere particles dispersed in DMF; (b) opticalmicroscope image of microspheres after lyophilizing; (c) SEM image of the microspherical particles.

The inset showed enlarged view of an individual broken sphere, scale bar 5 100 lm; (d) surface

morphologies of the microspheres observed by SEM. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

Figure 7. Swelling behavior of the chitin-g-poly L-leucine micro-spheres in different media.



the surface activity property and aggregation formation of

WSC. As shown in Figure 1, chitosan alone had the higher

value of c due to the little hydrophobic group in the poly-

meric chain as well as the effect of its protonated amine

groups. After hydrophobically acetylating modification, the

amount of amino group was close to the acetyl group. The

resulting amphiphilic polysaccharide could concentrate on

the surface with their hydrophobic chains pointing to the

air while the hydrophilic backbones laid on the surface to

reduce surface tension. Herein, two phenomena can be dis-

tinguished in the acetylation: firstly, the N-acetylation of

the amine functions would disrupt the crystallization and

make for dissolubility; and secondly, due to the existence

of a sufficient number of acetylated residues which are

quite hydrophobic, there is a change in the chain organiza-

tion owing to intermolecular hydrogen bonding or hydro-

phobic interactions. Hence, the results demonstrated that

the introduction of hydrophobic acetyl groups not only

improved the solubility but also made the WSC become

amphiphilic polymers which could decrease the surface ten-

sion. Also, the surface tension was dependent of the DD,

the lower DD would benefit to hydrophobic formation.

However, the solubility would be interfered if DD was

lower than 40%.

Mun et al.42 have demonstrated the behavior of chitosan

in aqueous solution. At DD over 80% and neutralization,

chitosan exhibits the highest structural charge density dis-

tributed along the chain. In this region, chitosan displays

polyelectrolyte behavior related to long-distance intra and

intermolecular electrostatic interactions. For DD lower than

50%, the electrostatic interactions are essentially short-dis-

tance interactions. Then, hydrophobic interactions due to

the increase of the content in acetyl groups become pre-

dominant, which induces polymer chain associations as

revealed by the high values of the radius of gyration.

To confirm the existence of aggregation behavior of

WSC in solution, fluorescence measurement in the presence

of a specific probe, pyrene, was used to detect the emission

spectra in relation with the polarity of the environment.

Being a hydrophobic molecule with low aqueous solubility,

pyrene is expected to localize preferentially in the hydro-

phobic domains of amphipathic molecules. There are five

peaks in the emission spectra of pyrene, and the emission

intensities of the first peak (374 nm) and of the third peak

(385 nm) are sensitive to the microenvironment. Thus, the

intensity ratio of the emission at 374 and 385 nm (I1/I3)

has been used to monitor the change in environmental po-

larity of amphipathic molecules upon association in aque-

ous solution. A bigger I1/I3 value means a greater polarity

of the solution around pyrene.

The curves given in Figure 8 showed the evolution of

the aggregation behavior with the polymer concentration. It

showed that the hydrophobic domains were formed at very

low concentration. The I1/I3 values began to lower down

while the surface tension decreases, and after the surface

tension had reached an equilibrium value, the I1/I3 value

still decreased. This indicated that the polymers began to

form aggregates by their hydrophobic chains in solution

while some of them transferred to the air–water interface to

decrease the surface tension. After the surface adsorption

reached equilibrium, the micropolarity in the polymer

aggregates still got less and less polarity due to the hydro-

phobic core getting more and more compact. The aggre-

gates formation of the polymers was a process of gradually

compacted with hydrophobic association.43–45

Thus, it was evident that the acetylated-modified WSC

had surface activity and aggregation specialty similar to an

amphipathic molecule. Hydrophobically modified carboxy-

methylchitosan such as 2-hydroxyl-3-dodecanoxyl and pro-

pylcarboxymethylchitosans had shown self-aggregates in

aqueous solution.46,47 But only few studies focused on so-

lution behavior of such partially acetylated WSC, which

was thought short of hydrophobic chains counteracting the

hydrophilic groups. However, the results had clearly dem-

onstrated that through N-acetylating, the WSC could con-

centrate on the surface to decrease the surface tension, and

could associate with hydrophobic chains to form aggregates

in the solution. This suggested the feasibility of preparation

of microsphere concurrently in the copolymerization.

Formation of Chitin-g-Poly-L-leucine Microsphere

Kurita et al. has shown the possibility of preparing chitin-

protein hybrids by graft copolymerization of a-amino acid

NCA onto partially deacetylated chitin under heterogeneous

conditions.31,32,34 WSC was found to be very promising for

this sensitive reaction with the NCA since it made the reac-

tion occurring in solution. The fabricating of the capsules

through interfacial polymerization process was illustrated in

Scheme 3. Firstly, in the presence of the aqueous WSC so-

lution, leucine-NCA monomer in vinyl acetate was dis-

persed into droplets under agitation. The existence of

hydrophobic domains in the polysaccharide molecules

facilitated the adsorption onto the oil droplets to reduce the

Figure 8. I1/I3 value and surface tension of WSC (DD 5 45.78%) asa function of polymer concentration. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]



surface energy. The adsorbed chitin serves as a stabilizer to

effectively emulsify the oil phase to form oil-in-water (O/W) emulsion. During the reaction process, the amino

groups of chitin attacked the NCA smoothly and the ring-

opening polymerization would start at the interface of oil

droplet to give the graft copolymer with polypeptide side

chains and form closed shells around the droplets spontane-

ously. Although NCA was very susceptible to hydrolysis,

the graft polymerization was carried out at 08C to suppress

side reactions. The graft copolymers were neither water-

soluble nor oil-soluble (ethyl acetate as oil phase) and

hereby precipitated, resembling the crosslinked network

formation. As the polymerization continued, all the NCA

monomers were used up and the chitin-g-polypeptidemicrogel was formed with a quasispherical structure at the

oil/water interface attributed to the surface activity proper-

ties of the polysaccharide segment. Herein, the WSC con-

tributed the saccharide-moiety and initiated the graft

copolymerization of NCA as well. Notably, it served as

emulsion simultaneously. Consequently, crosslinked poly-

saccharide-polypeptide hybrid capsules would be obtained

after evaporation of the oil phase.

Kurita had confirmed that the hydrolysis of the NCA to

form amino acid and carbon dioxide was the only evident

side reaction and without any evidence of homopolypeptide

formation under the conditions used in the reaction.31,32

Accordingly, the resulting graft copolymers were purified

with DMF and water for further removing the remainder of

either homopolypeptide or the amino acid monomer as

byproducts as well as unreacted chitin.

The experiments indicated that the morphology of the

production obtained was governed by the polymerization

conditions. In particular, the ratio of NCA/Cs and the con-

centration of NCA monomer had a strong influence on the

resulting morphology and the wall thickness of the micro-

spheres. In higher ratio of NCA/Cs, the final product had a

thinner shell, and was not strong enough to be a hollow

sphere; plethoric twisted microspheres would be formed.

The appropriate agitation rate was beneficial for better scat-

tering, but the emulsion might be destroyed if too high

speed was used. The polydisperse size distribution of the

microsphere is mainly due to the inadequate emulsifying

capability of the WSC, because the hydrophobic segments

were not longer enough like the typical surfactants.48,49

Through the orthogonal test, the optimized condition was

obtained as following: NCA/NH2 5 8:1(mol/mol), concen-

tration of WSC 5 0.01 g/mL, O/W 5 0.8, agitation rate 51000 r/min.

Swelling Behavior of the Microspheres

Preliminary test of swelling behavior showed the chitin-g-poly-L-leucine microspheres could swell in both acidic so-

lution and organic solvent. Particularly when immersed in

DMF, the dried microspheres swollen quickly and dramati-

cally due to the very fast absorption. While at lower pH,

Scheme 3. Schematic procedure for the synthesis of glycoconjugate hollow microspheres via inter-

facial polymerization approach. [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]



the degree of ionization was enhanced due to the protona-

tion of the amino units on chitin and the swelling capability

increased. In addition, the swelling rate was dependent on

the graft length of the polypeptide moiety ascribed to the

hydrophobic side-chain aggregation and the stronger hydro-

gen bonds in the graft copolymers. And that, the polypep-

tide chains with low DPs might simply act to disturb the

contact arrangement of chitin molecules, leading to high

swelling ability. When the DP increased, the chains would

form ordered structures including the a-helix, resulting in

poor affinity for the solvent. The results indicated that the

swell behavior could be regulated through tailoring the ra-

tio of compositions. These properties might be beneficial

for some special applications such as drug release.50,51

CONCLUSIONS

In conclusion, we have demonstrated the preparation of

polysaccharide-polypeptide hybrid microspheres based on

chitin-graft-poly L-leucine via ring-opening polymerization

of NCA. The swelling behaviors of the microspheres corre-

spond closely to the length of polypeptide side chain,

which could be regulated by the amount of feeding. Nota-

bly, the interest arises from the possibility of preparing the

microspheres in situ without adscititious stabilizer, utilizing

the specific amphiphilic polysaccharide and the heterogene-

ous interfacial copolymerization. Therefore the risk of

potential side effects introduced by the emulsifier can be

neglected. In spite of the conventional interfacial method,

the alternative approach to the manufacturing of micro-

spheres exhibits several advantages over existing methods.

It is fast, facile, and economical, since it consists of a one-

step reaction, produced in only 2 h, and no sophisticated

technology is required. Furthermore, the possibility to pre-

pare the glycopeptide microspheres in situ with controllable

composition and graft length opens new opportunities for

construction of glycoconjugate biomaterials that is featured

with ECM-analogical structure. Owing to the crosslinking

in the process of graft polymerization, the particles are

very stable and shape-persistent. The strategy makes the

best of the poor dissolvability of the graft production,

breaking through its solubility obstacle. The tunable swel-

ling ability in both organic solvents and acidic aqueous sol-

utions is highly desirable for the encapsulation and release

of bioactive molecules. Based on the above results, we

therefore envisage that the glycopeptides microspheres

reported here will be promising as drug carriers. Our cur-

rent work is focused on the further investigation of degra-

dation behavior of the microspheres and employing for

drug delivery.

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One-step preparation of glycopeptide microspheres based on α-amino acid-N-carboxyanhydride polymerization using interfacial protocols