Adv. Mater. 2008, 20, 2292–2296

8/7/2019 Adv. Mater. 2008, 20, 2292–2296

http://slidepdf.com/reader/full/adv-mater-2008-20-22922296 1/5

DOI: 10.1002/adma.200702663

Monodisperse Chitosan Microspheres with InterestingStructures for Protein Drug Delivery**

By Wei Wei, Lan Yuan, Gang Hu, Lian-Yan Wang, Jie Wu, Xue Hu, Zhi-Guo Su, and

Guang-Hui Ma*

The rapid development of DNA-recombinant techniques

and other modern biotechnology approaches have lead to the

emergence of protein drugs as a very important class of

therapeutic agents.[1] Consequently, it is not surprising that

microsphere-based therapy has attracted a lot of recent

research attention. In this therapeutic approach, protein drugs

are protected from enzymatic digestion and precisely delivered

to specific lesion sites. Two important variables, the dosage of medication and the release kinetics, can be finely tuned by

adjusting the structure of the microspheres to achieve desired

results.[2]

Chitosan, the second-most abundant polysaccharide after

cellulose, has been proposed as a potential candidate for

protein drug delivery applications because of its several

outstanding characteristics such as non-toxicity, biocompat-

ibility, biodegradability, mucus adhesion, and low cost.[3] Over

the last few decades, chitosan microspheres have always been

prepared by facile chemical crosslinking using glutaraldehyde

(C-G microspheres).[4] Typically, acidic water-soluble drugs

are simply dispersed in chitosan solution and entrapped by an

emulsion crosslinking process. However, some drugs, espe-cially protein drugs, can severely lose their activity during this

process because of the reaction between free amino groups of

the drugs and the aldehyde groups of the crosslinker.[5] To

maintain the activity of protein dugs, researchers have devised

the so-called moderate absorption method to load these

molecules onto C-G microspheres. However, the loading of

drugs onto the surfaces of the C-G microspheres leads to an

initial burst during drug release, which significantly hampers

the therapeutic effect of certain drugs in clinical applications.

Furthermore, the loading efficiency is not usually adequate

because of the compact structure of the C-G microspheres. In

addition, C-G microspheres prepared by conventional meth-ods have a broad size distribution, and it can be rather difficult

to precisely tune the particle size during synthesis. As a result

of this situation, the reproducibility of drug delivery experi-

ments tends to be low, leading to possible side-effects in

therapeutic applications. All these shortcomings have thus far

precluded the application of C-G microspheres in practical

applications.

The requirements for drug-release behavior vary signifi-

cantly from case to case in clinical therapy applications. The

main challenge thus involves the preparation of monodisperse

C-G microspheres with different structures that can provide

the required drug release profiles for different clinical

applications.In this study, monodisperse chitosan microspheres with

different structural properties have been successfully prepared

by the Shirasu Porous Glass (SPG) membrane emulsification

method. Bovine serum albumin (BSA) has been used as a

prototype and loaded onto four different types of micro-

spheres. In vitro studies of the release kinetics of different

types of microspheres have been performed to verify the

practicality of their use in clinical applications.

In a previous report, we have demonstrated the synthesis of

monodisperse C-G microspheres by SPG membrane emulsi-

fication.[6] These microspheres show remarkable autofluor-

escent properties that have been attributed to the np*

transitions of C––N bonds in the Schiff bases formed during thecrosslinking reaction. These traditional C-G microspheres are

solid spherical objects without any pores on their surface.

The microspheres have been observed to rapidly collapse

in acetone or ethanol because of the instability of the

Schiff base when only one crosslinking procedure based on

p-phthaldehyde is used to prepare the microspheres. However,

the mechanical integrity of the spherical structures is retained

upon the addition of a second crosslinking reagent, glutar-

aldehyde, as mentioned above (Fig. 1a). This two-step cross-

linking process has enabled the successful preparation of C-PG

microspheres. As shown in Figure 1b, after washing with

[*] Prof. G.-H. Ma, Dr. W. Wei, Dr. L.-Y. Wang, Dr. J. Wu, X. HuProf. Z.-G. SuNational Key Laboratory of Biochemical EngineeringInstitute of Process EngineeringChinese Academy of Sciences100080 Beijing (P.R. China)E-mail: [email protected]

Dr. W. Wei

Graduate University of Chinese Academy of Sciences100049 Beijing (P.R. China)

Prof. L. YuanMedical and Healthy Analytical CenterPeking University Health Science Center100083 Beijing (P.R. China)

Dr. G. HuUnilever Research China200233 Shanghai (P.R. China)

[**] This work was supported by the National Nature Science Foundationof China (20 536 050, 50 703 043), Chinese Academy of Sciences(KJCX2-YW-M02), and the Knowledge Innovation Program.Supporting Information is available online from Wiley InterScienceor from the author.

92 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2008, 20, 2292–2296

8/7/2019 Adv. Mater. 2008, 20, 2292–2296


COMMUNICATIO

N

acetone and ethanol, the C-PG microspheres have been

transformed into monodisperse hollow particles exhibiting

strong fluorescent events at the surface. A relatively weaker

signal has been detected from the center (Fig. 1c), indicating

that the crosslinking reaction based on glutaraldehyde occurs

predominantly on the surface because of the steric hindrance

from pre-crosslinked p-phthaldehyde. No phenyl signal has

been detected in the infrared analysis of these structures,

confirming that p-phthaldehyde has been removed upon

washing with acetone and ethanol (Fig. S1, Supporting

Information shows the Fourier transform infrared (FTIR)

spectroscopy analysis). A gradual growth of the wall thickness

of these hollow microspheres has been obtained by decreasing

the p-phthaldehyde crosslinking time and increasing the

glutaraldehyde crosslinking time (Fig. S2, Supporting Infor-

mation).

Several reports have shown that

quaternized chitosan is non-toxic, and

can increase the permeability of intestinal

epithelia.[7]A chitosan derivativeN -[(2-hydroxy-

3-trimethylammonium)propyl] chitosan

chloride (HTCC) with 60% substitution

has been obtained by reacting chitosanwith glycidyltrimethylammonium, as pre-

viously reported by Wu et al.[8] A mixture

ofchitosan and HTCC (in a 1:1 mass ratio)

has been used to prepare CH-G micro-

spheres by crosslinking with glutaralde-

hyde. The hollow structures obtained by

this method are shown in Figure 2a, and

are clearly somewhat smaller than the

structures shown in Figure 1b. The fluo-

rescence intensities have been observed to

gradually decrease from the surface to the

center in Figure 2c, which indicates that

the crosslinking reactions occur moreeasily on the surface than in the center

because of the steric hindrance from

quaternized groups. Indeed, the deficien-

cies of this crosslinking reaction also lead

to the formation of pores on the micro-

sphere surface (Fig. 2b). The formation of this hollow porous

structure has been found to be highly dependent upon the

quaternized group content and the degree of crosslinking. A

higher degree of substitution with HTCC and a shorter

crosslinking time lead to the formation of a more obvious

hollow space with more pores on the surface (as shown in Fig.

S3, Supporting Information).

A mixture of chitosan and HTCC (with a 1:1 mass ratio) has

been used in a two-step crosslinking process to prepare CH-PG

microspheres after washing with acetone and ethanol. No

cavities have been observed in the as-prepared microspheres,

but instead the presence of macroporous structures is clearly

revealed (Fig. 3). This macroporosity can also be modulated by

varying the quaternized group content as well as the degree of

crosslinking during the two crosslinking processes (Fig. S4,

Supporting Information). It is thought that the formation of

Figure 1. a) Schematic illustration of the preparation of C-PG microspheres. b) Laser scanningconfocal microscopy (LSCM) image of C-PG microspheres; the overlay is shown in yellow and scalebars represent 20mm. c) Profile of the fluorescence distribution of C-PG microspheres.

Figure 2. a) LSCM image of CH-G microspheres. b) Scanning electron microscopy (SEM) image of the surface of CH-G microspheres. c) Profile of thefluorescence distribution of CH-G microspheres. The scale bars represent 20mm and 100 nm in (a) and (b), respectively.

Adv. Mater. 2008, 20, 2292–2296 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advmat.de 229

8/7/2019 Adv. Mater. 2008, 20, 2292–2296


8/7/2019 Adv. Mater. 2008, 20, 2292–2296


COMMUNICATIO

N

concentration.[10] CH-G microspheres with a minimal initial

burst and relatively well-controlled release are likely to be

ideal carriers for these protein drugs. In contrast, short but

intensive administration of drugs is preferable for treating

cancer, hepatitis, and systemic lupus erythenatosus (SLE).[11]

CH-PG microspheres with a strong initial burst are much more

suitable candidates for such pulsed therapy. In the context of

conventional vaccination, repeated injections at appropriately

timed intervals are required for vaccines preventing hemor-

rhagic fever, hepatitis B, and rabies. CH-PG microspheres with

two distinct bursts also offer a promising approach to mimic

these repeated immunizations.[12] Overall, the distinctive

release profiles of these different types of microspheres reflect

the potential therapeutic applications that can be achieved

with these systems to fulfill various drug delivery requirements.

In addition to uniformity, the SPG membrane technique

also enables the preparation of microspheres with a specific

particle size by appropriate choice of the membrane pore

size.[6] Therefore, the novel monodisperse chitosan micro-

spheres with controllable size discussed here are promising

systems for achieving better reproducibility, more repeatable

release behavior, higher bioavailability, and passive target-

ability. The tunability of microsphere structural properties

such as surface charge, cavity size, and wall porosity enables

the modification of these systems to cater to specific

requirements for use as protein drug carriers.

Experimental

Materials: Chitosan with a molecular weight of 780 000 waspurchased from Putian Zhongsheng Weiye (P.R. China). The SPGmembranes were obtainedfrom SPGTechnology(Japan). KP-18C waspurchased from Shin-Etsu Chemicals (Japan). PO-500 ((hexaglycerinpenta)ester) was kindly provided by Sakamoto Yakuhin Kogyo(Japan). BSA, glutaraldehyde, and p-phthaldehyde were acquiredfrom Sigma (Germany). All other materials were of analytical reagentgrade. The ratio of amino and aldehyde groups was 1:1 during thecrosslinking reaction and a crosslinking time of 1 h was used for allreactions unless otherwise specified.

Preparation of C-G Microspheres: Monodisperse chitosan micro-spheres were prepared according to a method previously described byWei et al. [6]. Surface treatement of the SPG membranes with KP-18Cmade the membranes relatively hydrophobic. Next, 2 wt% chitosanwas dissolved in 1 wt% aqueous acetic acid, and this mixture was used

as the water phase. The oil phase was a 7:5 (v/v) mixture of liquid-paraffin/petroleum-ether containing 4 wt% PO-500 emulsifier.The volume ratio of water and oil phases was 1:10 (v/v%) in allexperiments. First, the water phase was permeated through theuniform pores of the SPG membrane into the oil phase using thepressure of nitrogen gas to form a monodisperse water-in-oil (w/o)emulsion. Glutaraldehyde-saturated toluene (GST) was used as thecrosslinking agent to solidify the chitosan droplets. Finally, thecrosslinked microspheres were collected and washed twice withpetroleum ether, acetone, and ethanol by centrifugation (3000g) andredispersion.

Preparation of C-PG Microspheres: A monodisperse w/o emulsionwas prepared as discussed above. The C-PG microspheres wereprepared by sequentially adding two kinds of crosslinking agents, firstp-phthaldehyde and then glutaraldehyde. After solidification, the

C-PG microcapsules were collected using the method described above.Preparation of CH-G and CH-PG Microspheres: Instead of chitosan, 2 wt% chitosanHTCC mixed polymer (mass ratio of 1:1) was dissolved in 1 wt% aqueous acetic acid. This mixtureconstituted the water phase used to form w/o emulsions by the SPGmembrane emulsification technique mentioned above. Subsequently,GST was used to form CH-G microspheres, whereas the CH-PGmicrospheres were prepared by the aforementioned two-step solidi-fication process also used to prepare C-PG microspheres. Finally, thesamples were washed with acetone and ethanol prior to collection, asalso described above.

In Vitro BSA Loading: The absorption medium was PBS (pH¼7.4)containing 1000mg mLÀ1 BSA and 106 mLÀ1 microspheres. After 48 h,the BSA-loaded microspheres were separated from the medium andthe presence of residual BSA in the medium was detected using abicinchoninic acid (BCA) assay.

In Vitro BSA Release Study: The release medium was PBScontaining 0.05% (w/v) sodium azide as a preserving agent and0.02% (v/v) polysorbate-80 as a dispersing agent. The concentration of BSA-loaded microspheres was 106 mLÀ1. The release experimentswere performed in a thermostatic shaker (378C, 120 rpm). Afterpredetermined intervals, 0.5 mL of the supernatant was extracted andanalyzed using the BCAassay. Subsequently, the same volume of freshPBS buffer was addedinto thereleasemedium to top up to theoriginalvolume (4 mL).

Characterization Methods: The structure of the C-PG microsphereswas characterized by FTIR using a FTIR-400/600 instrument fromJasco. The mean size and zeta potential of the microspheres weredetermined using a Mastersizer 2000 laser diffractometer and zetasizeranalyzer from Malvern Instruments. Brunauer EmmettTeller

Figure 5. In vitro BSA a) loading efficiency and b) release profilesmeasured for the different types of microspheres.

Adv. Mater. 2008, 20, 2292–2296 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advmat.de 229

8/7/2019 Adv. Mater. 2008, 20, 2292–2296


(BET) analysis was performed on an Autosorb-1 (Quantachrome)instrument. A JEM-6700F SEM from JEOL was used to observe theshape and surface features of the microspheres. The samples wereplaced on a metal stub andcoated with platinum under vacuum using aJFC-1600 ion sputter (JEOL) prior to observation. Suspensionscontaining microspheres in a Petri dish were also observed by LSCMusing a TCS SP2 instrument from Leica. The samples were excited at

488nm and two fluorescent images were obtained at 510–540nm(green) and 570–600nm (red).

Received: October 24, 2007

Revised: January 18, 2008

Published online: May 15, 2008

[1] J. E. Talmadge, Adv. Drug Delivery Rev. 1993, 10, 247.

[2] a) L. Pereswetoff-Morath, Adv. Drug Delivery Rev. 1998, 29, 185.b) S.

D. Putney, Curr. Opin. Chem. Biol. 1998, 2, 548.

[3] a) H. Zhang, I. A. Alsarra, S. H. Neau, Int. J. Pharm. 2002, 239, 197. b)

S. Nsereko, M. Amiji, Biomaterials 2002, 23, 2723. c) X. Y. Shi, T. W.

Tan, Biomaterials 2002, 23, 4469.

[4] a) S. R. Jameela, A. Jayakrishnan, Biomaterials 1995, 16, 769. b) M. C.

Gohel, M. N. Sheth, M. M. Patel, G. K. Jani, H. Patel, Ind. J. Pharm.

Sci. 1994, 56, 210. c) E. B. Denkbas, M. Seyyal, E. Piskin, Micro-

encapsulation1999, 16, 741. d) A. Berthold,K. Cremer, J. Kreuter, STP

Pharm. Sci. 1996, 6, 358.

[5] L.Y. Wang, Y.H. Gu, Q.Z. Zhou, G.H. Ma, Colloids Surf. B 2006, 50,

126.

[6] W.Wei, L.Y. Wang, L.Yuan,Q. Wei,X. D.Yang,Z. G.Su,G. H.Ma,

Adv. Funct. Mater. 2007, 17 , 3153.

[7] a) G. Sandri, S. Rossi, M. C. Bonferoni, Int. J. Pharm. 2005, 297 , 146.

b) J. H. Hamman, M. Stander, A. F. Kotze, Int. J. Pharm. 2002, 232,

235. c) M. Thanou, B. I. Florea, M. W. Langemeyer, Pharm. Res. 2000,

17 , 27.

[8] J. Wu, G. H. Ma, Z. G. Su, Int. J. Pharm. 2006, 315, 1.

[9] a) C. G. Gomez, L. C. Alvarez, M. C. Strumia, Polymer 2004, 45, 6189.

b) D. C. Sherrington, Chem. Commun. 1998, 2275.

[10] a) C. A. Gloff, L. Z. Benet, Adv. Drug Delivery Rev. 1990, 4, 359. b) J.

E. Talmadge, Adv. Drug Delivery Rev. 1993, 10, 247.

[11] a) R. Alexanian, B. S. Yap, G. P. Bodey, Blood 1983, 62, 572. b) S. K.

Sarin, B. S. Sandhu, B. C. Sharma, M. Jain, J. Singh, V. Malhotra, J.

Viral Hepatitis 2004, 11, 552. c) S.Kaur, A. J. Kanvar, Int. J. Dermatol.

1990, 29, 371.

[12] a) D. T. O’Hagan, M. Singh, J. B. Ulmer, Methods 2006, 4, 10. b) L.

Feng, X. R. Qi, X. J. Zhou, Y. Maitani, S. C. Wang, Y. Jiang,T. Nagai,

J. Controlled Release 2006, 112, 35. c) D. Shouval, J. Hepatol. 2003, 39,

S70. d) J. L. Cleland, Trends Biotechnol. 1999, 17 , 25. e) J. L. Imler,

Vaccine 1995, 13, 1143.

296 www.advmat.de ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2008, 20 , 2292–2296

Adv. Mater. 2008, 20, 2292–2296

Documents

Transcript of Adv. Mater. 2008, 20, 2292–2296