Poster presented at the 10th Conference of the Materials Research Society of Serbia - YUCOMAT 2008, Herceg Novi, Crna Gora, 8-12. septembar 2008

Ignjatović, Nenad

Jovanović, Ivana

Mitrić, Miodrag

Nedeljković, B.

Uskoković, Dragan

Škapin, Srečo Davor

Ignjatović, Nenad L.

Repository of the Vinča Nuclear Institute (VinaR)

Injectable poly-D,L-lactide (PDLLA) microspheres containing proteins or peptides as controlled release devices have been widely used for the treatment of human diseases and animal health. Fundamental  understanding of the relationship among the size of microspheres, encapsulation efficiency and protein release capacity are essential for the design of microsphere delivery systems [1,2]. The modified  precipitation method [3,4] is method of encapsulating hydrophilic drugs, especially protein and peptide ones, into microspheres. Since the release profiles of  proteins dominantly depend on the nature and  morphology of the polymer, drug distribution within microspheres and release temperature, the fabrication of microspheres with specific morphology and drug distribution is a challenge for chemical engineers [5]. PDLLA microspheres can protect proteins against  biological inactivation and can ensure their release for long time frames, and at specified time. Finally, the size of the particles can be used to passively target the delivery vehicles for uptake by specific types of cells, such as professional antigen-presenting cells, or to target specific tissues [6].PREPARATION AND CHARACTERIZATION OF POLY-DL,L-LACTIDE MICROSPHERES FOR CONTROLLED RELEASE OF HORSERADISH PEROXIDASE AS MODEL PROTEINI. Jovanović1, M. Mitrić2, B. Nedeljković3, S. Škapin4, N. Ignjatović1 and D. Uskoković11Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia, 2Institute for Nuclear Sciences Vinča, Belgrade, Serbia, 3Faculty of Technical Sciences, University of Čačak, Serbia, 4Jožef Stefan Institute, Ljubljana , Slovenia[1] Y. Ataman-Önal, S. Munier, A. Ganée, C. Terrat, P.-Y. Durand, N. Battail, F. Martinon, R. Le Grand, M.-H. Charles, T. Delair, B. Verrier, J. Control. Release 112 (2006) 175-185.[2] Y.-Y. Yang, T.-S. Chung, X.-L. Bai, W.-K. Chan, Chem. Eng. Sci. 55 (2000) 2223-2236.[3] M. Stevanović, J. Savić, B. Jordović, D. Uskoković, Colloids Surf., B 59 (2007) 215-223.[4] I. Jovanović, B. Jordović, M. Petković, N. Ignjatović, D. Uskoković, Microsc. Res. and Tech. 71 (2008) 86-92.[5] F. Mollica, M. Biondi, S. Muzzi, F. Ungaro, F. Quaglia, M. I. La Rotonda, P. A. Netti, J Mater Sci: Mater Med 19 (2008) 1587-1593.[6] M. Liu, Z. Zhou, X. Wang, J. Xu, K. Yang, Q. Cui, X. Chen, M. Cao, J. Weng, Q. Zhang, Polymer 48 (2007) 5767-5779.[7] N. Rizkalla, C. range, F.-X. Lacasse, P. Hildgen, J Microencapsul 23 (2006) 39-57.HRP-loaded PDLLA particles were  successfully obtained by precipitation  method. PDLLA-HRP particles, prepared by modified precipitation method, have perfectly spherical shape, smooth surface  and are non-agglomerated. The mean diameter of the particles is 530 nm, and  encapsulation efficiency is 46 %. The main advantage of this method is that it does not require an increase in temperature and,  therefore, may be useful when the heat- sensitive drugs, like proteins, are used. Poly-D,L-lactide with an average molecular weight of 50,000 g/m was purchased from Sigma (Sigma-Aldrich, Germany). Horseradish peroxidase (HRP) with molecular weight of 45 kDa was used as model protein. Microspheres were prepared by modified precipitation method [4]. Briefly, the method was performed as follows: 40 mg of commercial granules PDLLA (SIGMA- Aldrich, Germany) were dissolved in chlorophorm, and 1 mg of HRP was dissolved in 1 ml of water. This mixture was added to ethanol to form a dispersion. This dispersion was added dropwise to 20 ml PVA solution containing 5 wt% of  PVA while the mixture was stirred at 1200 rpm using magnetic stirrer.  After that, solution was homogenized 10 min on 25 000 rpm, centrifuged 2 hours on 4000 rpm, decanted and vacuum dried. Introduction Materials and MethodsThe aim of this study was to produce HRP loaded PDLLA spheres with the best properties for controlled and sustained delivery of HRP. The ability to control the size of PDLLA spheres with incorporated HRP, should facilitate the investigations of their scope for applicability in drug delivery [7]. The sphere preparation technique is so complex that it is very difficult to predict optimal conditions for a specific polymer. In practice, optimal conditions have to be empirically found for each polymer drug system. We have investigated the key parameters to fabricate PDLLA spheres containing HRP as a model protein using the modified precipitation method. Various factors that influence the size and morphology of particles, encapsulation efficiency and initial release were varied. From the FESEM images (Fig. 1.) we can see that all PDLLA-HRP particles, prepared by modified precipitation method, have perfectly spherical shape, smooth  surface and are non-agglomerated. The average diameter of the PDLLA- HRP spheres, prepared by precipitation method is 530 nm (Table 1.). The UV spectroscopy has been used to estimate encapsulation efficiency of HRP loaded PDLLA particles (Fig. 2.). From the spectrophotometric analysis (Fig. 2.), the encapsulation efficiency of 46% is calculated. Differential scanning calorimetry (DSC) (Fig. 3.) and X-ray diffraction (XRD) (Fig. 4.) were used to characterize the particles. ResultsYUCOMAT 2 008, HE RCEG NOVI 8.YUCOMAT 2 008, HE RCEG NOVI 8. -- 12 .09.2008 12.09 .200 8 ReferencesConclusionsFig. 1. FESEM images of HRP-loaded PDLLA spheresFig. 2. Spectrophotometric  analysis of PDLLA-HRP powder  prepared by precipitation methodFig. 3. DSC diagrams of  PDLLA powder and PDLLA-HRP powderFig. 4. XRD diagrams of PDLLA, HRP and PDLLA-HRP powderTable 1. Results of stereological analysis

Preparation and Characterization of Poly-D,L-Lactide Microspheres for Controlled Release of Horseradish Peroxidase as Model Protein

Serbian Academy of Science and Arts Digital Archive (DAIS)

English

VinaR - Repository of the Vinča Institute of Nuclear Sciences

Injectable
 
poly-D,L-lactide (PDLLA)
 
microspheres
 
containing proteins or peptides as controlled release 
devices have been widely used for the treatment of 
human diseases and animal health. Fundamental
 
 
understanding of the relationship among the size of 
microspheres, encapsulation efficiency and protein 
release capacity are essential for the design of 
microsphere
 
delivery systems [1,2]. The modified
 
 
precipitation method [3,4] is method of encapsulating 
hydrophilic drugs, especially protein and peptide ones, 
into microspheres. Since the release profiles of
 
 
proteins dominantly depend on the nature and
 
 
morphology of the polymer, drug distribution within 
microspheres
 
and release temperature, the fabrication 
of microspheres
 
with specific morphology and drug 
distribution is a challenge for chemical engineers [5]. 
PDLLA microspheres
 
can protect proteins against
 
 
biological inactivation and can ensure their release for 
long time frames, and at specified time. Finally, the 
size of the particles can be used to passively target 
the delivery vehicles for uptake by specific types of 
cells, such as professional antigen-presenting cells, or 
to target specific tissues [6].
PREPARATION AND CHARACTERIZATION OF POLY-DL,L-LACTIDE MICROSPHERES FOR 
CONTROLLED RELEASE OF HORSERADISH PEROXIDASE AS MODEL PROTEIN
I. Jovanović1, M. Mitrić2, B. Nedeljković3, S. Škapin4, N. Ignjatović1
 
and D. Uskoković1
1Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia, 2Institute for Nuclear Sciences Vinča, Belgrade, 
Serbia, 3Faculty of Technical Sciences, University of Čačak, Serbia, 4Jožef Stefan Institute, Ljubljana , Slovenia
[1] Y. Ataman-Önal, S. Munier, A. Ganée, C. Terrat, P.-Y. 
Durand, N. Battail, F. Martinon, R. Le Grand, M.-H. Charles, T. 
Delair, B. Verrier, J. Control. Release 112 (2006) 175-185.
[2] Y.-Y. Yang, T.-S. Chung, X.-L. Bai, W.-K. Chan, Chem. Eng. 
Sci. 55 (2000) 2223-2236.
[3] M. Stevanović, J. Savić, B. Jordović, D. Uskoković, Colloids 
Surf., B 59 (2007) 215-223.
[4] I. Jovanović, B. Jordović, M. Petković, N. Ignjatović, D. 
Uskoković, Microsc. Res. and Tech. 71 (2008) 86-92.
[5] F. Mollica, M. Biondi, S. Muzzi, F. Ungaro, F. Quaglia, M. I. 
La Rotonda, P. A. Netti, J Mater Sci: Mater Med 19 (2008) 
1587-1593.
[6] M. Liu, Z. Zhou, X. Wang, J. Xu, K. Yang, Q. Cui, X. Chen, 
M. Cao, J. Weng, Q. Zhang, Polymer 48 (2007) 5767-5779.
[7]
 
N. Rizkalla, C. range, F.-X. Lacasse, P. Hildgen, J 
Microencapsul
 
23 (2006) 39-57.
HRP-loaded PDLLA particles were
 
 
successfully obtained by precipitation
 
 
method. PDLLA-HRP particles, prepared by 
modified precipitation method, have 
perfectly spherical shape, smooth surface
 
 
and are non-agglomerated. The mean 
diameter of the particles is
 
530 nm, and
 
 
encapsulation efficiency is
 
46 %.
 
The main 
advantage of this method is that it does not 
require an increase in temperature and,
 
 
therefore, may be useful when the heat-
 
sensitive drugs, like proteins, are used. 
Poly-D,L-lactide
 
with an average molecular weight of 50,000 g/m
 
was purchased from Sigma 
(Sigma-Aldrich, Germany). Horseradish peroxidase
 
(HRP) with molecular weight of 45 kDa
 
was 
used as model protein. Microspheres
 
were prepared by modified precipitation method
 
[4]. 
Briefly, the method was performed as follows: 40 mg of commercial granules PDLLA (SIGMA-
 
Aldrich, Germany) were dissolved in chlorophorm, and 1 mg of HRP
 
was dissolved in 1 ml of 
water. This mixture was added to ethanol to form a dispersion. This dispersion was added 
dropwise
 
to 20 ml PVA
 
solution containing 5 wt% of  PVA while the mixture was stirred at 1200 
rpm using magnetic stirrer.  After that, solution was homogenized 10 min on 25 000 rpm, 
centrifuged 2 hours on 4000 rpm, decanted and vacuum dried. 
Introduction Materials and Methods
The aim of this study was to produce HRP loaded PDLLA spheres
 
with the best properties for controlled and sustained delivery of HRP. The 
ability to control the size of PDLLA spheres with incorporated HRP, should 
facilitate the investigations of their scope for
 
applicability in drug delivery 
[7].
 
The
 
sphere
 
preparation technique is so complex that it is very difficult 
to predict optimal conditions for a specific polymer. In practice, optimal 
conditions have to be empirically found for each polymer
 
drug system. We 
have investigated the key parameters to fabricate PDLLA spheres 
containing HRP as a model protein using the modified precipitation 
method. Various factors that influence the size and morphology of particles, 
encapsulation efficiency and initial release were varied.
 
From the FESEM 
images (Fig. 1.) we can see
 
that all PDLLA-HRP particles, prepared by 
modified precipitation method, have perfectly spherical shape, smooth
 
 
surface and are non-agglomerated. The average diameter of the PDLLA-
 
HRP spheres, prepared by precipitation method is 530 nm (Table 1.). The 
UV spectroscopy has been used to estimate encapsulation efficiency of 
HRP loaded PDLLA particles (Fig. 2.). From the spectrophotometric
 
analysis (Fig. 2.), the encapsulation efficiency of 46% is calculated. 
Differential scanning calorimetry (DSC)
 
(Fig. 3.) and X-ray diffraction (XRD)
 
(Fig. 4.) were used to characterize the particles. 
Results
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ReferencesConclusions
Fig. 1.
 
FESEM images of HRP-loaded PDLLA spheres
Fig. 2. Spectrophotometric  analysis of PDLLA-HRP powder  prepared by precipitation method
Fig. 3. DSC diagrams of  PDLLA powder 
and PDLLA-HRP powder
Fig. 4. XRD diagrams of PDLLA, HRP and PDLLA-HRP powder
Table 1. Results of stereological analysis


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Preparation and Characterization of Poly-D,L-Lactide Microspheres for Controlled Release of Horseradish Peroxidase as Model Protein

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