Embed Size (px)
Citation preview
Pc
LSS
a
ARRAA
KPPCP
1
t[a[welnIlbbtwSods
(
0d
Colloids and Surfaces A: Physicochem. Eng. Aspects 345 (2009) 173–181
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical andEngineering Aspects
journa l homepage: www.e lsev ier .com/ locate /co lsur fa
reparation and characterization of porous biodegradable microspheres used forontrolled protein delivery
in Sun, Shaobing Zhou ∗, Weijia Wang, Xiaohong Li, Jianxin Wang, Jie Wengchool of Materials Science and Engineering, Key Laboratory of Advanced Technologies of Material, Minister of Education,outhwest Jiaotong University, Chengdu 610031, Sichuan, PR China
r t i c l e i n f o
rticle history:eceived 6 March 2009eceived in revised form 28 April 2009ccepted 29 April 2009vailable online 7 May 2009
eywords:
a b s t r a c t
In this study, a new approach for protein delivery is introduced, involving biodegradable porous poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres blocked with calcium alginate hydrogel. Porous microsphereswere prepared by solvent evaporation method using sodium oleate as an extractable porogen. The resul-tant microspheres show porous surface and three-dimensional network structure with interconnectingpores. Human serum albumin (HSA) used as model protein, was adsorbed into micropores of porousmicrospheres by a solution immersing method. Thus, the harsh preparation conditions, such as the aque-
orous microspheresLGAalcium alginate hydrogelrotein delivery
ous/organic interface produced by a water-in-oil microemulsion and the acute shearing strength broughtby a high-speed homogenizer or a sonicater, inducing protein denaturation and aggregation could beavoided. Then, calcium alginate hydrogel was fabricated to block micropores of protein-loaded porousmicrospheres. A sustained protein release in vitro could be achieved in this way. Furthermore, it is note-worthy that the protein maintained its structural integrity after this preparation. The porous polymericmicrospheres may have great potential application for water-soluble drugs, such as protein, vaccine and
gene delivery.. Introduction
Biodegradable polymeric microspheres used as injectable pro-ein and peptide carriers have been researched for many years1–6]. Many methods for loading different proteins into biodegrad-ble polymeric microspheres have been described in some works7–10]. The most commonly used method encapsulating proteinsithin microspheres is double emulsion method based on solvent
vaporation [11–13]. However, inherent protein instability prob-ems, such as unfolding, covalent/non-covalent aggregation andon-specific adsorption, occurred during this formulation process.
t is because that the presence of the harsh preparation or formu-ation conditions, such as the aqueous/organic interface producedy a water-in-oil microemulsion and the acute shearing strengthrought by a high-speed homogenizer or a sonicater, caused pro-ein denaturation and aggregation. Therefore, some improvementsere carried out to avoid the aqueous/organic interface, such as
/O/W emulsion method and spray drying [14,15]. In our previ-us study, we also prepared core-coated microspheres for proteinelivery, which held more advantages, e.g. maintainment of thetructural integrity of protein and improvement of the drug loading
∗ Corresponding author. Tel.: +86 28 87634023; fax: +86 28 87634649.E-mail addresses: [email protected], [email protected]
S. Zhou).
927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2009.04.053
© 2009 Elsevier B.V. All rights reserved.
efficiency, compared with conventional drug delivery system [16].Though these fabrications could protect protein from inactivation,these were more difficulty to carry out.
Recently, a new formulation, porous microspheres showing thelarge porous surface and interconnecting pores interior, has drawnattention for protein delivery. Protein could be entrapped into poresof porous microspheres by a solution immersing method. Aque-ous/organic interface was avoided and protein biological activitycould be protected during loading process. It has been reportedthat porous microspheres could be used for micro-carrier suspen-sion culture of cells, as well as injection of the cell/microspheresconstructs into a tissue defect site [17,18]. Meanwhile, the porousmicrospheres with large size and low mass density have beenproven to be the ideal carriers for pulmonary drug delivery [19,20].
At present, the disadvantage of porous microspheres was mainlythat the protein release behavior was unsatisfactory. For example,the cumulative release of recombinant human growth hormone(rhGH) from the porous PLGA microspheres reached 100 wt.% inonly 1 day [21]. Many investigations have been done to achieveprotein sustained release [22–25]. In this study, a new approachis introduced to achieve sustained HSA release, which is based
on calcium alginate hydrogel. HSA was incorporated into porousmicrospheres by a simple solution immersing method without anyorganic solvent. Therefore, protein denaturation and aggregationhappened on aqueous/organic interface can be avoided in loadingprocess. Then, micropores were blocked by calcium alginate hydro-
1 ysicoc
gp
2
2
lc(woS(AcCuc
2
msw5sSo
74 L. Sun et al. / Colloids and Surfaces A: Ph
el, which was expected to inhibit a rapid protein release fromorous microspheres.
. Materials and methods
.1. Materials
The copolymer poly(d,l-lactic-co-glycolic acid) (PLGA) withactide/glycolide molar ratio of 75/25 was synthesized by poly-onsendation and the average molecular weight was Mw 12 kDaPDI 1.83) determined by gel permeation chromatography (GPC,aters 2695 and 2414, Milford, MA). Polyvinyl alcohol (PVA, Mn
f 130,000 g mol−1, degree of hydrolysis 88) was purchased fromhanghai Petrochemical Industry Company. Human serum albuminHSA) with the isoelectric point (IEP) of 4.7 was purchased fromventis Behring Gmbh (Germany). Sodium alginate and calciumhloride anhydrous were both purchased from Chengdu Kelonghemical Reagent Company (Sichuan, China). All the chemicalssed in this research were the analytical reagent grade from theommercial market without further purification.
.2. Fabrications of porous microspheres and HSA loading
Porous microspheres were prepared by double emulsionethod (W1/O/W2) based on solvent evaporation [26]. Firstly, a
odium oleate solution was prepared in 500 �l of distilled water,
hich was used as the W1 phase. 0.3 g PLGA was dissolved inml methylene chloride and emulsified with sodium oleate water-olution under stirring, formed the stable initial emulsion (W1/O).econdly, the resultant emulsion was added dropwise into 100 mlf a 3% PVA solution with a 21-gauge needle and emulsified for
Fig. 1. The inner structure of porous microspheres (A and B), and porous mi
hem. Eng. Aspects 345 (2009) 173–181
40 min at a predetermined speed using overhead stirrer, resultingin the formation of a water-in-oil-in-water emulsion (W1/O/W2).Thirdly, 50 ml of a 6% isopropanol solution was poured into the dou-ble emulsion to extract the organic solvent and then, stirring about2 h in fuming cupboard. After the evaporation of organic solvent,hardened microspheres were collected by centrifugation (AvantiTM J-301, BECKMAN COULTER, USA). The resultant microsphereswere rinsed with distilled water and collected by centrifugationthree times. Finally, lyophilized overnight and stored at 4 ◦C in adesiccator.
The porous PLGA microspheres loaded HSA by dipping porousmicrospheres (200 mg) in 1 ml of HSA solution and gently shakingfor 1 h. The HSA-loaded porous microspheres were recollected bycentrifugation. The concentration of HSA in the supernatant wasdetected by UV–visible spectrophotometer (UV-2550, Shimadzu,Japan). The loading amount of protein adsorbed into pores wascalculated using following equation:
q = (ci − cf )Vm
,
where ci is the initial HSA solution concentration and cf is the HSAconcentration of supernatant after adsorption. V is the total volumeof HSA solution. And m is the weight of porous microspheres addedin the HSA solution.
2.3. Preparation of calcium alginate hydrogel on/in the porous
microspheresPorous PLGA microspheres (200 mg) were dispersed in sodiumalginate solution. These porous PLGA microspheres adsorbingsodium alginate were recollected by centrifugation. The resultant
crospheres blocked by calcium alginate hydrogel (C) detected by TEM.
ysicoc
mospwtl
2
bs
Fs
L. Sun et al. / Colloids and Surfaces A: Ph
icrospheres were re-dispersed and added dropwise into 100 mlf calcium chloride solution (1.0%, w/v) with a 21-gauge needle,tirred for about 10 min. Calcium alginate hydrogel absorbed on/inorous microspheres was formed by cross-linking sodium alginateith Ca2+. Then, the resultant microspheres were rinsed with dis-
illed water and collected by centrifugation three times. Finally,yophilized overnight and stored at 4 ◦C in a desiccator.
.4. Characterizations of porous microspheres
The surface morphology of porous microspheres was examinedy scanning electron microscope (QUANTA 200, FEI, USA). Severalolution droplets containing microspheres were placed on the SEM
ig. 2. The surface morphology of microspheres with the weight ratio of sodium oleate/PLize of porous microspheres detected by particle size analyzer.
hem. Eng. Aspects 345 (2009) 173–181 175
sample stage. The microsphere samples were sputter coated withgold after lyophilized overnight.
To observe the three-dimensional network structure withinporous microspheres matrix, transmission electron microscopy(TEM) observation was performed with a HITACHI H-700H at theacceleration voltage of 150 kV. Samples were prepared by droppingthe microspheres suspension on a carbon-coated copper grid andthen air-dried before measurement.
The mean size and size distribution were determined by laserdiffraction particle size analyzer (LA-920, HORIBA, Japan). Micro-spheres were resuspended in distilled water by ultrasonic toprevent aggregation. Then, the resultant microspheres solution waspoured into the sample tank of particle size analyzer.
GA: 0%, 17%, 20%, 25%. (A) Surface morphological change detected by SEM. (B) Mean
1 ysicoc
2
timmld
crtt
Fp
76 L. Sun et al. / Colloids and Surfaces A: Ph
.5. In vitro degradation
Pre-weighed microspheres were placed individually in testubes containing 10 ml PBS (0.1 M, pH 7.4). The tubes were keptn a thermo-stated incubator (Haerbin Dongming Medical Equip-
ent Company) which was maintained at 37 ◦C and 100 cycles perinute. The degradation process was evaluated from the morpho-
ogical change of microspheres, the mass loss and the pH change ofegradation medium at predetermined intervals.
The degradation samples were rinsed with distilled water andollected by centrifugation at predetermined intervals. Then, theesultant samples were dried to constant weight. The degrada-ion medium was collected to characterize the pH change duringhe degradation process. The morphological change of microsphere
ig. 3. The surface morphology and mean size of microspheres varying the PVA solutionorous microspheres detected by particle size analyzer.
hem. Eng. Aspects 345 (2009) 173–181
surfaces can be characterized by SEM. Mass loss was detected bycomparing the weight remained with the initial weight. The pHchange was determined by detecting the pH value of degradationmedium at predetermined intervals with pH meter (Shanghai LeiciInstrument Company) at 25 ◦C.
2.6. In vitro protein release
Predetermined microspheres (200 mg) were suspended in a test
tube containing 15 ml PBS (0.1 M, pH 7.4). The test tubes were placedin a thermo-stated incubator and continuously agitated with thesame condition as mentioned in degradation test. At predeterminedintervals, 1.0 ml of release medium was collected by centrifugationand 1.0 ml of fresh PBS was added back to the test tube. The amountconcentration. (A) Surface morphological change detected by SEM. (B) Mean size of
ysicochem. Eng. Aspects 345 (2009) 173–181 177
oa
2
mMMd
3
3
p(ctbmpsTsshpecmo
psohlt
opFnsit1tiomipe
toviadaeo
L. Sun et al. / Colloids and Surfaces A: Ph
f HSA released was determined by UV–visible spectrophotometert absorbance of 278 nm.
.7. Structural integrity of HSA
Structural integrity of HSA released was detected by gel per-eation chromatography (GPC, waters 2695 and 2414, Milford,A) with an Ultrahydrogel 250 column (7.8 mm × 300 mm, Waters,ilford, MA), compared with native HSA. The mobile phase was
istilled water with the flow rate of 0.5 ml/min.
. Results and discussion
.1. Fabrication of porous microspheres
In this research, double emulsion method was chosen to prepareorous microspheres. The oil phase (O) was methylene chlorideMC) containing PLGA. PVA aqueous solution was used as the outerontinuous aqueous phase (W2). Sodium oleate, an anionic surfac-ant, was selected as porogen. Due to its high hydrophilic/lipophilicalance value (HLB = 18) [27], sodium oleate can be extracted fromicrospheres matrix easily and influx into PVA solution. When
reparing porous microspheres, sodium oleate solution was emul-ified with MC containing PLGA, forming a stabilizing emulsion.hen, sodium oleate/PLGA emulsion droplets were dispersed in PVAolution. After evaporation of MC, the primary emulsion dropletshrank and water permeated into [28]. Thus, phase separationappened to form a PLGA-rich phase and a sodium oleate-richhase in droplets [21]. Finally, the water-soluble sodium oleate wasffused into PVA solution, leaving PLGA matrix behind and the inter-onnecting pores were formed. After MC removed, porous PLGAicrospheres with interconnecting pores preoccupied by sodium
leate phase were formed.The three-dimensional network structure with interconnecting
ores was one of the most important properties of porous micro-pheres. Thus, TEM was carried out to observe the inner structuref microspheres. Shown in Fig. 1A and B, the porous microspheresave excellent spherical structure and many interconnecting pores
ike microsponge. These structures with high specific surfaces werehe desirable to loading maximum of HSA in the research.
The effects of sodium oleate/PLGA weight ratio, concentrationf PVA solution and agitating speed on morphological change oforous microspheres were investigated in this work. As shown inig. 2, the surface morphological of microspheres changed fromon-porous to porous structure with increasing the weight ratio ofodium oleate/PLGA. As the ratio of sodium oleate/PLGA increas-ng, the mean size of microspheres enlarged from 11.78 ± 1.52o 12.91 ± 2.10 �m. And, the surface pores size increased from.35 ± 0.59 to 1.53 ± 0.62 �m, detected by image software. While,here were more pores on microspheres surface as the ratio increas-ng. It was caused by increasing the ratio of the hydrophilic sodiumleate in primary emulsion droplets, which made more water per-eate into the droplets. While, more sodium oleate phase effused
nto the outer aqueous phase and formed interconnecting poresreoccupied by sodium oleate phase and the surface pores sizenlarged.
The porous microspheres prepared by varying the concentra-ion of PVA solution were shown in Fig. 3. Fixing the weight ratiof sodium oleate/PLGA at 25%, the concentration of PVA solutionaried from 2% to 5%. As the concentration of PVA solution increas-ng, the mean size decreased from 37.16 ± 15.86 to 4.26 ± 1.62 �m
nd the pore size decreased from 3.88 ± 0.97 to 1.17 ± 0.66 �m. Thisecrease was because that the viscosity of PVA solution increaseds the concentration increased, which affect the dispersibility ofmulsion droplets in the continuous phase and the efflux velocityf sodium oleate phase.Fig. 4. The effects of time and pH value on adsorbing efficiency at 4 ◦C.
The agitating speed of the dispersive process was a key factor toaffect the morphology of porous microspheres (data not shown). Itwas found that the high agitating speed would destroy the sphericalstructure of porous microspheres and formed sponge-like struc-tures with irregular shape. It was because that porous structurehad degraded the mechanical property of the microspheres sys-tem. The spherical structure of microspheres would be destroyedduring agitating processing, which could provide the shearing forceto destroy the system.
In conclusion, porous microspheres with a highly intercon-necting pores and large porous surface were prepared with thiscondition in further study: sodium oleate/PLGA weight ratio 25%,PVA solution concentration 3% and agitating speed 700 rpm.
3.2. Adsorption of HSA on/into porous microspheres
HSA was adsorbed on/into porous microspheres by a simplesolution immersing method. Any organic solvents were not neededin loading process. Therefore, protein denaturation and aggregationhappened on aqueous/organic interface could be avoided. Pro-tein adsorbing on biomaterials is a complicated physiochemicalprocess and has been widely researched. Electrostatic interaction,hydrophobic interaction and hydrogen bond have been proven toplay important roles in protein adsorption [29–34]. In this study, theeffect of adsorbing temperature, pH value of adsorbing medium andadsorbing time on HSA adsorption onto porous microspheres wereexamined. The effect of temperature on protein adsorption wasreported in previous research [35]. It indicated that the temperaturehad insignificant effects on HSA adsorbing efficiency, which was thesame as our research. Protein adsorbing was carried out at 4 ◦C inthis research, which was a mild surroundings for protein activity.Shown in Fig. 4, the adsorption amount of HSA almost reached thezenith and kept a stable value after 1 h adsorption. These indicatedthat the adsorption of HSA on porous microspheres was satura-tion after 1 h. Two adsorbing mediums with different pH value of4.5 and 7.4 were used to investigate HSA adsorption. The adsorb-ing efficiency was 48.2% and 65.7% with pH value of 7.4 and 4.5,respectively. It was found that the adsorbing efficiency in acetatebuffer (pH 4.5) was higher than that in the PBS (pH 7.4). It wasbecause that the surface charge characteristics of protein depend
on the pH value of solution. The isoelectric point (IEP) of HSA is4.7 [32]. Therefore, the zeta potential of HSA molecule was posi-tive below the IEP [36,37]. The electrostatic attractive interactionbetween PLGA porous microspheres and HSA molecule happenedat pH 4.5 in acetate buffer. These also indicated that the electrostatic
1 ysicoc
if
3
dsitIm
FS
78 L. Sun et al. / Colloids and Surfaces A: Ph
nteraction of microspheres and HSA molecule was the dominantorce in HSA adsorption.
.3. Preparation of calcium alginate hydrogel on/in porous surface
Protein release from polymeric microspheres was depending onegradation of polymers and the protein diffusion out from micro-
pheres [38]. Generally, these two ways happened at same timen release process. Therefore, the release curve was made up ofwo phases, the burst release followed by a stable release stage.n the burst release phase, the protein adsorbed on the surface oficrospheres diffused out quickly in first few hours. Burst release
ig. 5. The surface morphology and mean size of microspheres coated by different concenEM: (a) 0.5%, (b) 1%, (c) 1.5%, (d) 2%. (B) Mean size of porous microspheres detected by p
hem. Eng. Aspects 345 (2009) 173–181
was not beneficial to patients who needed long term treatments.The dominant release from porous microspheres was diffusion ofprotein from the network structure. Therefore, the most proteinadsorbed in porous microspheres released in first day as seen inFig. 9. To improve the release rate, the pores in porous micro-spheres would be blocked. In this work, calcium alginate hydrogelwas used to block the pores to inhibit protein diffusion. The surface
morphological changed from porous to non-porous structure withincreasing the concentration of sodium alginate solution shown inFig. 5. As the concentration of sodium alginate solution increased,the pores on microspheres surface decreased obviously, seen inFig. 5A. When the concentration of sodium alginate solution wastration of sodium alginate solution. (A) Surface morphological change detected byarticle size analyzer.
L. Sun et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 345 (2009) 173–181 179
F detecc
0gcbatsmpFcwsaamfd
3
ArlwoPdpwriaptid
Therefore, the pores on microspheres surface played a key role inrelease rate. Seen from Fig. 9, it can be found that 88.7% of HSA load-ing in the PM was released at first day. It was because that porousstructure made water penetrate into PM and thus made HSA diffuse
ig. 6. The morphological changes of microspheres during degradation processing,alcium alginate hydrogel (D–F) degradated for 1, 3, and 5 weeks, respectively.
.5%, the porous surface was not blocked by calcium alginate hydro-el well. While, when the concentration increased to 2%, few poresould be found on microspheres surface after cross-linking. It wasecause that the viscosity of sodium alginate solution increaseds the concentration increased. The higher the concentration was,he more the sodium alginate adsorbed on/into the porous micro-pheres. Therefore, the calcium alginate hydrogel blocked porousicrospheres well after crosslinked by calcium ion. Meanwhile,
orous microspheres size after blocked was investigated. Shown inig. 5B, the mean size of microspheres was affected slightly by theoncentration of sodium alginate solution. When the concentrationas varied at 0%, 0.5%, 1%, 1.5% and 2%, the mean size of micro-
pheres was 13.16 ± 3.28, 13.22 ± 3.22, 13.76 ± 3.36, 13.90 ± 3.33nd 13.93 ± 3.23 �m, respectively. It was indicated that the calciumlginate hydrogel was almost formed in network structure of porousicrospheres and there was little calcium alginate hydrogel on sur-
ace of porous microsheres. These also could be proven in Fig. 1C,etected by TEM.
.4. In vitro degradation and protein release
Porous microspheres blocked by calcium alginate hydrogel (PM-lg) were placed in PBS to investigate the degradation and protein
elease, porous PLGA microspheres (PM) as a control. The morpho-ogical change of PM and PM-Alg during the degradation process
as shown in Fig. 6. The surface morphology of PM in degradationf 1, 3, and 5 weeks were shown in Fig. 6A, B, and C, respectively. TheM lost their spherical structure in 1 week degradation. This fasteformation was due to the porous structure, which made waterenetrate into the microspheres matrix easily and the hydrolysisas the dominant way in polymer degradation. While, the PM-Alg
etained their spherical structures in 1 week degradation, shownn Fig. 6D. The PM-Alg collapsed and lost their spherical structure
fter 1 week degradation, shown in Fig. 6E. In conclusion, the mor-hological change of PM was faster than PM-Alg. It also indicatedhat the calcium alginate hydrogel blocked on porous microspheres,nhibited water penetrating into microspheres and delayed theeformation of microspheres. These also could be proven by theted by SEM. Porous PLGA microspheres (A–C) and porous microspheres blocked by
mass loss and the pH decrease curves. The pH value of degradationmedium decreased during the degradation process shown in Fig. 7.In 1 week degradation, the pH value decreased from 7.4 to 7.16 forPM and 7.24 for PM-Alg, respectively. It proved that the degradationof PM was faster than PM-Alg in first week. In 2 weeks degradation,there was little difference of pH value decreasing between PM andPM-Alg. The reason may be that both kinds of microspheres hadcollapsed and lost their spherical structure at this time. The cal-cium alginate hydrogel had lost the protective effect. Therefore, thedegradation rate of two tended to the same. The phenomenon couldalso be observed in the mass lose experience, as shown in Fig. 8.
The protein release curves from PM and PM-Alg were shown inFig. 9. The dominant way of protein release from PM was diffusion.
Fig. 7. The pH value of degradation medium versus incubation time.
180 L. Sun et al. / Colloids and Surfaces A: Physicoc
Fig. 8. The percent residual weight of PLGA microsphere containing HSA in PBS.
oCtt
Fs
Fig. 9. Percent release of HSA from microspheres incubation in PBS at 37 ◦C.
ut easily. Therefore, the burst release was obvious in the first day.ompared with PM, HSA released from PM-Alg was more persis-ent. At first day, only 29.36% HSA released. It clearly indicated thathe calcium alginate hydrogel blocked in the PM inhibited HSA dif-
ig. 10. The GPC results of native HSA (a) and HSA released after adsorbed in micro-pheres for 1 h (b), 2 h (c), 3 h (d), and 4 h (e).
[
hem. Eng. Aspects 345 (2009) 173–181
fusing out efficiently. It was also remarked that the protein releasefrom PM-Alg lasted more than 20 days.
To determine the structural integrity of HSA released, GPC wascarried out in this research. The GPC results of HSA adsorbed in PMfor different time were shown in Fig. 10. The peaks appeared on thecurves indicated the molecular weight of HSA. Seen from Fig. 10, themajor peaks of native HSA appeared at 15.239 and 17.125 min. TheGPC results of HSA adsorbed in PM for 1, 2, 3, and 4 h also showedthe peaks around 15 and 17 min. It may suggest that no remarkablechemical polymerization, non-covalent aggregation and molecularhydrolysis occurred during the adsorbing process.
4. Conclusions
A novel drug delivery system with porous microspheres blockedby calcium alginate hydrogel was designed. Porous PLGA micro-spheres were prepared by common solvent evaporation method.Sodium oleate was employed as a water-solubility porogen. HSAcould be adsorbed on/into the porous microspheres by immers-ing method. Thus, HSA was entrapped into polymeric matrix andits structural integrity was maintained due to unemploying anyorganic solvent during this preparing. In vitro protein releaseindicated that porous microspheres blocked by calcium alginatehydrogel were efficient to depress the burst release and prolongthe release period. Therefore, the blocked porous microspheres arepotential application as injectable depots for biomacromolecules,such as protein, vaccine and gene drug delivery systems.
Acknowledgements
This work was partially supported by National Natural Sci-ence Foundation of China (50773065), Programs for New CenturyExcellent Talents in university, Ministry of Education of China(NCET-07-0719) and Sichuan Prominent Young Talent Program(08ZQ026-040).
References
[1] K. Kim, D. Pack, Microspheres for drug delivery, in: M. Ferrari, A.P. Lee, L.J.Lee (Eds.), BioMEMS and Biomedical Nanotechnology, Springer, USA, 2006, pp.19–50.
[2] J.I. Lee, H.S. Yoo, Biodegradable microspheres containing poly(�-caprolactone)–pluronic block copolymers for temperature-responsiverelease of proteins, Colloid Surf. B: Biointerf. 61 (2008) 81–87.
[3] A. Jaklenec, E. Wan, M.E. Murray, E. Mathiowitz, Novel scaffolds fabricated fromprotein-loaded microspheres for tissue engineering, Biomaterials 29 (2008)185–192.
[4] A. Matsumoto, Y. Matsukawa, T. Suzuki, H. Yoshino, Drug release character-istics of multi-reservoir type microspheres with poly(dl-lactide-co-glycolide)and poly(dl-lactide), J. Control. Release 106 (2005) 172–180.
[5] F.L. Mi, S.S. Shyu, Y.M. Lin, Y.B. Wu, C.K. Peng, Chitin/PLGA blend microspheresas a biodegradable drug delivery system: a new delivery system for protein,Biomaterials 24 (2003) 5023–5036.
[6] J. Hanes, J.L. Cleland, R. Langer, New advances in microsphere-based single-dosevaccines, Adv. Drug Del. Rev. 28 (1997) 97–119.
[7] S. Freiberg, X.X. Zhu, Polymer microspheres for controlled drug release, Int. J.Pharm. 282 (2004) 1–18.
[8] H. Kawaguchi, Functional polymer microspheres, Prog. Polym. Sci. 25 (2000)1171–1210.
[9] W.J. Jia, Y.C. Gu, M.L. Gou, Preparation of biodegradable PCEC nanoparticles,Drug Deliv. 15 (2008) 409–416.
[10] W.J. Jia, J.G. Liu, Preparation, characterization, and optimization of pancreas-targeted 5-Fu loaded magnetic bovine serum albumin microspheres, J. DrugTarget. 15 (2007) 140–145.
[11] I.D. Rosca, F. Watari, M. Uo, Microparticle formation and its mechanism in sin-gle and double emulsion solvent evaporation, J. Control. Release 99 (2004)271–280.
12] S.H. Wang, L.C. Zhang, F. Lin, X.Y. Sa, J.B. Zuo, Q.X. Shao, Controlled release of lev-
onorgestrel from biodegradable poly(d,l-lactide-co-glycolide) microspheres:in vitro and in vivo studies, Int. J. Pharm. 301 (2005) 217–225.[13] H.K. Kim, T.G. Park, Comparative study on sustained release of human growthhormone from semi-crystalline poly(l-lactic acid) and amorphous poly(d,l-lactic-co-glycolic acid) microspheres: morphological effect on protein release,J. Control. Release 98 (2004) 115–125.
ysicoc
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
L. Sun et al. / Colloids and Surfaces A: Ph
14] M.L. Gou, Z.Y. Qian, Preparation and characterization of magnetic poly(-caprolactone)–poly(ethylene glycol)–poly(-caprolactone) microspheres, J.Mater. Sci. Mater. Med. 19 (2008) 1033–1041.
15] M.J. Blanco-Prieto, K. Besseghir, O. Zerbe, D. Andris, P. Orsolini, F. Heimgartner,In vitro and in vivo evaluation of a somatostatin analogue released from PLGAmicrospheres, J. Control. Release 67 (2000) 19–28.
16] T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, H. Yoshino, Protein encapsulationinto biodegradable microspheres by a novel S/O/W emulsion method usingpoly(ethylene glycol) as a protein micronization adjuvant, J. Control. Release69 (2000) 435–444.
17] S. Zhou, X. Deng, X. Li, Investigation on a novel core-coated microspheres pro-tein delivery system, J. Control. Release 75 (2001) 27–36.
18] T.K. Kim, J.J. Yoon, D.S. Lee, T.G. Park, Gas foamed open porous biodegradablepolymeric microspheres, Biomaterials 27 (2006) 152–159.
19] A. Nokhodchi, Factors affecting the morphology of benzoyl peroxidemicrosponges, Micron 38 (2007) 834–840.
20] H.J. Chung, T.G. Park, Surface engineered and drug releasing pre-fabricatedscaffolds for tissue engineering, Adv. Drug Deliv. Rev. 59 (2007) 249–262.
21] D.A. Edwards, J. Hanes, G. Caponetti, J. Hrkach, A. Ben-Jebria, M.L. Eskew,Large porous particles for pulmonary drug delivery, Science 276 (1997)1868–1871.
22] H.K. Kim, H.J. Chung, T.G. Park, Biodegradable polymeric microspheres with“open/closed” pores for sustained release of human growth hormone, J. Control.Release 112 (2006) 167–174.
23] D.A. Edwards, A. Ben-jebria, R. Langer, Recent advances in pulmonary drugdelivery using large, porous inhaled particles, J. Appl. Physiol. 85 (1998)379–385.
24] H.O. Alpar, S. Somavarapu, K.N. Atuah, V.W. Bramwell, Biodegradable mucoad-
hesive particulates for nasal and pulmonary antigen and DNA delivery, Adv.Drug Deliv. Rev. 57 (2005) 411–430.25] M.J. Kwon, J.H. Bae, J.J. Kim, K. Na, E.S. Lee, Long acting porous microparticle forpulmonary protein delivery, Int. J. Pharm. 333 (2007) 5–9.
26] S. Giovagnoli, P. Blasi, A. Schoubben, C. Rossi, M. Ricci, Preparation oflarge porous biodegradable microspheres by using a simple double-emulsion
[
[
hem. Eng. Aspects 345 (2009) 173–181 181
method for capreomycin sulfate pulmonary delivery, Int. J. Pharm. 333 (2007)103–111.
27] X. Deng, S. Zhou, X. Li, J. Zhao, M. Yuan, In vitro degradation and release pro-files for poly-dl-lactide-poly(ethylene glycol) microspheres containing humanserum albumin, J. Control. Release 71 (2001) 165–173.
28] J.J. Najera, Phase transition behaviour of sodium oleate aerosol particles, Atmos.Environ. 41 (2007) 1041–1052.
29] G. Crotts, T.G. Park, Preparation of porous and nonporous biodegradable poly-meric hollow microspheres, J. Control. Release 35 (1995) 91–105.
30] W. Li, S. Li, A study on the adsorption of bovine serum albumin onto electrostaticmicrospheres: role of surface groups, Colloid Surf. A: Physicochem. Eng. Aspects295 (2007) 159–164.
31] S.M. Butler, M.A. Tracy, R.D. Tilton, Adsorption of serum albumin to thin filmsof poly(lactide-coglycolide), J. Control. Release 58 (1999) 335–347.
32] F.Y. Oliva, L.B. Avalle, O.R. Cámara, C.P.D. Pauli, Adsorption of human serumalbumin (HSA) onto colloidal TiO2 particles, Part I, J. Colloid Interf. Sci. 261(2003) 299–311.
33] S. Patil, A. Sandberg, E. Heckert, W. Self, S. Seal, Protein adsorption and cellularuptake of cerium oxide nanoparticles as a function of zeta potential, Biomate-rials 28 (2007) 4600–4607.
34] M.L. Gou, M. Dai, X.Y. Li, Preparation of mannan-coated anionic PCL-PEG-PCLnanoparticles at one-step for bFGF antigen delivery to improve humoral immu-nity, Colloid Surf. B: Biointerf. 64 (2008) 135–139.
35] R.C. Eberhart, M.E. Lynch, F.H. Bilge, H.A. Arts, Effects of fluid shear and tem-perature on protein adsorption on teflon surfaces, Trans. Am. Soc. Artif. Intern.Organs 26 (1980) 185–193.
36] S.J. Fang, H. Kawaguchi, Colloidal properties and protein adsorption of ampho-teric microspheres, Colloid Surf. A: Physicochem. Eng. Aspects 211 (2002)
79–84.37] J.Y. Yoon, J.H. Lee, J.H. Kim, W.S. Kim, Separation of serum proteins with uncou-pled microsphere particles in a stirred cell, Colloid Surf. B: Biointerf. 10 (1998)365–377.
38] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science303 (2004) 1818–1822.
