Biomaterials 28 (2007) 2754–2762
Protein complexed with chondroitin sulfate
in poly(lactide-co-glycolide) microspheres
Eun Seong Lee
, Keun-Hong Park
, Dongmin Kang
, In Suh Park
, Hyo Young Min
Don Haeng Lee
, Sungwon Kim
, Jong Ho Kim
, Kun Na
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 421 Wakara Way Suite 315, Salt Lake City, UT 84108, USA
College of Medicine, Pochon CHA University, Cell and Gene Therapy Research Institute 605, Yeoksam 1-dong, Kangnam-gu, Seoul 135-081 l,
Republic of Korea
Korea Basic Science Institute, Chuncheon Center, 192-1 Hyoja2-dong, Chuncheon, Kangwon-do 200-701, Republic of Korea
Department of Pathology, Inha University College of Medicine, Incheon 402-751, Republic of Korea
Department of Internal Medicine, Inha University, Incheon 402-751, Republic of Korea
Center for Advanced Medical Education, Inha University College of Medicine by BK-21 Project, Incheon 400-712, Republic of Korea
Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea
Division of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea
Received 2 October 2006; accepted 23 January 2007
Available online 20 February 2007
Chondroitin sulfate (CsA) is an acidic mucopolysaccharide, which is able to form ionic complexes with positively charged proteins. In
this study, a protein–CsA complex was constructed to nano-sized particles. Zeta potential measurements revealed that a CsA-to-protein
fraction of greater than 0.1 results in a neutralization of the positive charge on lysozyme (Lys). Based on this preliminary study, we have
prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring Lys/CsA complexes via the multi-emulsion method. Protein stability
in the PLGA microspheres was preserved during both microsphere preparation and protein release. The proﬁles of Lys release from the
PLGA microspheres evidenced nearly zero-order kinetics, depending on the quantity of CsA. An in vivo ﬂuorescent image of
experimental mouse tissue showed that the PLGA microspheres with the Lys/CsA complex had released the entirety of their Lys without
no residual amount after 23 days, but microspheres without the complex harbored a great deal of residual Lys, which is attributable to its
degradation by acidic PLGA degradates. The tissue reaction evidenced by the PLGA microspheres stabilized with CsA showed minimal
foreign body reaction and little conﬁguration of immune cells including neutrophils and macrophages, but the reactions of the PLGA
microspheres without CsA were characterized by a relatively elevated inﬂammation. These results show that CsA is a viable candidate for
long-acting micro-particular protein delivery.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Chondroitin sulfate; Ionic complex; Protein stabilization; Poly(lactide-co-glycolide) microsphere
Over the past decades, a variety of investigations have
been attempted to replace the daily injections for protein
delivery, which are currently a source of signiﬁcant patient
discomfort. Protein depots, particularly protein-loaded
poly(lactide-co-glycolide) (PLGA) microspheres, have been
formulated in an attempt to maintain proteins’ biological
activity and structural integrity for a long period [1–3].
Despite such promising approaches, proteins encapsulated
into PLGA microspheres have often evidenced a reduction
in their bioactivity, which has proved a major hindrance to
the utilization of Food and Drug Administration (PDA)-
approved PLGA for protein delivery in clinical situations
PLGA microspheres are known to degrade in hydrolytic
atmospheres while passing through the random scissoring
of ester linkages [4–6]. This process liberates acidic
ARTICLE IN PRESS
0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +82 2 2164 4832; fax: +82 2 2164 4865.
E-mail address: email@example.com (K. Na).
Contributed equally to this work.