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                DOI: 10.1002/adma.200702663


                Monodisperse Chitosan Microspheres with Interesting
                Structures 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                      the so-called moderate absorption method to load these
                and other modern biotechnology approaches have lead to the                  molecules onto C-G microspheres. However, the loading of
                emergence of protein drugs as a very important class of                     drugs onto the surfaces of the C-G microspheres leads to an
                therapeutic agents.[1] Consequently, it is not surprising that              initial burst during drug release, which significantly hampers
                microsphere-based therapy has attracted a lot of recent                     the therapeutic effect of certain drugs in clinical applications.
                research attention. In this therapeutic approach, protein drugs             Furthermore, the loading efficiency is not usually adequate
                are protected from enzymatic digestion and precisely delivered              because of the compact structure of the C-G microspheres. In
                to specific lesion sites. Two important variables, the dosage of             addition, C-G microspheres prepared by conventional meth-
                medication and the release kinetics, can be finely tuned by                  ods have a broad size distribution, and it can be rather difficult
                adjusting the structure of the microspheres to achieve desired              to precisely tune the particle size during synthesis. As a result
                results.[2]                                                                 of this situation, the reproducibility of drug delivery experi-
                   Chitosan, the second-most abundant polysaccharide after                  ments tends to be low, leading to possible side-effects in
                cellulose, has been proposed as a potential candidate for                   therapeutic applications. All these shortcomings have thus far
                protein drug delivery applications because of its several                   precluded the application of C-G microspheres in practical
                outstanding characteristics such as non-toxicity, biocompat-                applications.
                ibility, biodegradability, mucus adhesion, and low cost.[3] Over               The requirements for drug-release behavior vary signifi-
                the last few decades, chitosan microspheres have always been                cantly from case to case in clinical therapy applications. The
                prepared by facile chemical crosslinking using glutaraldehyde               main challenge thus involves the preparation of monodisperse
                (C-G microspheres).[4] Typically, acidic water-soluble drugs                C-G microspheres with different structures that can provide
                are simply dispersed in chitosan solution and entrapped by an               the required drug release profiles for different clinical
                emulsion crosslinking process. However, some drugs, espe-                   applications.
                cially protein drugs, can severely lose their activity during this             In this study, monodisperse chitosan microspheres with
                process because of the reaction between free amino groups of                different structural properties have been successfully prepared
                the drugs and the aldehyde groups of the crosslinker.[5] To                 by the Shirasu Porous Glass (SPG) membrane emulsification
                maintain the activity of protein dugs, researchers have devised             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
                 [*]   Prof. G.-H. Ma, Dr. W. Wei, Dr. L.-Y. Wang, Dr. J. Wu, X. Hu         types of microspheres have been performed to verify the
                       Prof. Z.-G. Su
                       National Key Laboratory of Biochemical Engineering                   practicality of their use in clinical applications.
                       Institute of Process Engineering                                        In a previous report, we have demonstrated the synthesis of
                       Chinese Academy of Sciences                                          monodisperse C-G microspheres by SPG membrane emulsi-
                       100080 Beijing (P.R. China)
                                                                                            fication.[6] These microspheres show remarkable autofluor-
                       E-mail: ghma@home.ipe.ac.cn
                       Dr. W. Wei
                                                                                            escent properties that have been attributed to the n—p*
                       Graduate University of Chinese Academy of Sciences                   transitions of C – N bonds in the Schiff bases formed during the
                                                                                                             –
                       100049 Beijing (P.R. China)                                          crosslinking reaction. These traditional C-G microspheres are
                       Prof. L. Yuan                                                        solid spherical objects without any pores on their surface.
                       Medical and Healthy Analytical Center                                   The microspheres have been observed to rapidly collapse
                       Peking University Health Science Center
                       100083 Beijing (P.R. China)                                          in acetone or ethanol because of the instability of the
                       Dr. G. Hu                                                            Schiff base when only one crosslinking procedure based on
                       Unilever Research China                                              p-phthaldehyde is used to prepare the microspheres. However,
                       200233 Shanghai (P.R. China)                                         the mechanical integrity of the spherical structures is retained
                [**]   This work was supported by the National Nature Science Foundation    upon the addition of a second crosslinking reagent, glutar-
                       of China (20 536 050, 50 703 043), Chinese Academy of Sciences       aldehyde, as mentioned above (Fig. 1a). This two-step cross-
                       (KJCX2-YW-M02), and the Knowledge Innovation Program.
                       Supporting Information is available online from Wiley InterScience   linking process has enabled the successful preparation of C-PG
                       or from the author.                                                  microspheres. As shown in Figure 1b, after washing with


  2292                                                              ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim              Adv. Mater. 2008, 20, 2292–2296
                                                                                                                                                      COMMUNICATION
                                                                                                   Several reports have shown that
                                                                                                quaternized chitosan is non-toxic, and
                                                                                                can increase the permeability of intestinal
                                                                                                epithelia.[7] A chitosan derivative N-[(2-hydroxy-
                                                                                                3-trimethylammonium)propyl] chitosan
                                                                                                chloride (HTCC) with 60% substitution
                                                                                                has been obtained by reacting chitosan
                                                                                                with glycidyltrimethylammonium, as pre-
                                                                                                viously reported by Wu et al.[8] A mixture
                                                                                                of chitosan 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 more
                                                                                                easily on the surface than in the center
Figure 1. a) Schematic illustration of the preparation of C-PG microspheres. b) Laser scanning because of the steric hindrance from
confocal microscopy (LSCM) image of C-PG microspheres; the overlay is shown in yellow and scale
bars represent 20 mm. c) Profile of the fluorescence distribution of C-PG microspheres.           quaternized groups. Indeed, the deficien-
                                                                                                cies of this crosslinking reaction also lead
                                                                                                to the formation of pores on the micro-
acetone and ethanol, the C-PG microspheres have been                       sphere surface (Fig. 2b). The formation of this hollow porous
transformed into monodisperse hollow particles exhibiting                  structure has been found to be highly dependent upon the
strong fluorescent events at the surface. A relatively weaker               quaternized group content and the degree of crosslinking. A
signal has been detected from the center (Fig. 1c), indicating             higher degree of substitution with HTCC and a shorter
that the crosslinking reaction based on glutaraldehyde occurs              crosslinking time lead to the formation of a more obvious
predominantly on the surface because of the steric hindrance               hollow space with more pores on the surface (as shown in Fig.
from pre-crosslinked p-phthaldehyde. No phenyl signal has                  S3, Supporting Information).
been detected in the infrared analysis of these structures,                   A mixture of chitosan and HTCC (with a 1:1 mass ratio) has
confirming that p-phthaldehyde has been removed upon                        been used in a two-step crosslinking process to prepare CH-PG
washing with acetone and ethanol (Fig. S1, Supporting                      microspheres after washing with acetone and ethanol. No
Information shows the Fourier transform infrared (FTIR)                    cavities have been observed in the as-prepared microspheres,
spectroscopy analysis). A gradual growth of the wall thickness             but instead the presence of macroporous structures is clearly
of these hollow microspheres has been obtained by decreasing               revealed (Fig. 3). This macroporosity can also be modulated by
the p-phthaldehyde crosslinking time and increasing the                    varying the quaternized group content as well as the degree of
glutaraldehyde crosslinking time (Fig. S2, Supporting Infor-               crosslinking during the two crosslinking processes (Fig. S4,
mation).                                                                   Supporting Information). It is thought that the formation of




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 the
fluorescence distribution of CH-G microspheres. The scale bars represent 20 mm and 100 nm in (a) and (b), respectively.



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                                                                                                                    BSA into the lumen, but also creates a
                                                                                                                    higher surface area and therefore adds to
                                                                                                                    the number of sites available for BSA
                                                                                                                    loading by absorption.
                                                                                                                       Figure 5b displays in vitro release
                                                                                                                    patterns of BSA from microspheres pre-
                                                                                                                    pared by the different methods mentioned
                                                                                                                    above. Microspheres with different struc-
                                                                                                                    tures yield measurably different release
                                                                                                                    profiles. The CH-PG microspheres show
                Figure 3. a) LSCM and b) SEM images of macroporous CH-PG microspheres; the scale bars the highest initial burst because of the
                represent 20 and 1 mm, respectively. c,d) High-magnification images showing details of the inner large amount of BSA loaded in the
                structure and surface morphology of the microspheres; the scale bars represent 500 nm in both the
                figures.                                                                                             macroporous outer shell and channels,
                                                                                                                    which results in the rapid release of BSA
                                                                                                                    into the medium. Similarly, the traditional
                pores is a result of aggravating phase separation occurring                  C-G microspheres also show a high initial burst since most of
                during the first crosslinking reaction with p-phthaldehyde.[9]                the BSA is present on the particle surface. BSA release in
                   Table 1 shows characterization data for microspheres                      C-PG microspheres follows a triphase profile with 42% being
                prepared by different methods. As compared to traditional                    released in an initial burst, followed by a plateau for about 60 h,
                methods, use of the SPG membrane technique has enabled the                   and finally 85% release as a second burst. This interesting
                preparation of monodisperse microspheres with coefficient of                  result can be explained by the hollow structure of these
                variation (CV) values all below 15%. Figure 4 schematically                  microspheres. The first burst results in the release of BSA
                depicts the structure of these microspheres. CH-G and CH-PG                  loaded in the shell, whereas the second originates from the
                microspheres both show a high positive charge because of the                 release of BSA molecules present in the lumen, which migrate
                presence of the quaternized group in HTCC. The surface area                  outwards much slower than the BSA in the outer shell. In
                and pore diameter results are also consistent with the images                contrast to microspheres prepared by other methods, the initial
                obtained by laser scanning confocal microscopy (LSCM) and                    BSA burst has been restricted to less than 20% in CH-G
                scanning electron microscopy (SEM).                                          microspheres which is followed by a slower linear release
                   In our comparative study, we have focused on loading BSA                  profile. We propose that most of the BSA molecules enter the
                onto microspheres prepared by different methods. As shown in                 interior of the CH-G microspheres via pore channels, and their
                Figure 5a, CH-G microspheres yield the highest loading                       later release is driven by diffusion induced by the concentra-
                efficiency, reaching about 390 mg BSA for 106 microspheres.                   tion gradient.
                The loading efficiencies of CH-PG microspheres and C-PG                          In clinical therapy, it is always necessary to maintain protein
                microspheres are also higher than that of traditional C-G                    drugs (e.g., insulin, interferon) at a specific therapeutic serum
                microspheres.
                   A possible explanation for the very high loadings can be
                proposed based on the role of electrostatic effects. The
                isoelectric point of BSA is 4.7, and the BSA molecules are
                negatively charged in phosphate buffered saline (PBS). As a
                result, strong electrostatic interactions are established between
                BSA and microspheres containing positively charged HTCC,
                resulting in an enhanced loading efficiency. Furthermore,
                spheres with a hollow structure have a relatively greater
                amount of accessible space for BSA storage, as compared to
                their conventional solid counterparts. In addition, the porosity
                of the microsphere walls not only facilitates the migration of

                Table 1. Characterization data for microspheres prepared by different
                methods.

                Microspheres                 C-G        C-PG       CH-G         CH-PG

                Particle Size [mm]            7.3        7.6          7.4         7.7
                CV value [%]                 12.4       14.3         13.3        12.9
                Zeta potential [mV]           2.4        3.7         20.3        19.7
                                                                                          Figure 4. Schematic representation of BSA (green dots) loading patterns in
                Surface area [m2 gÀ1]        16         55          142          98
                                                                                          microspheres prepared by different methods: a) C-G microspheres, b) C-PG
                Mean pore diameter [nm]       2.1        4.3         16.7        73.2
                                                                                          microspheres, c) CH-G microspheres, and d) CH-PG microspheres.



  2294          www.advmat.de                              ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim                          Adv. Mater. 2008, 20, 2292–2296
                                                                                                                                                COMMUNICATION
                                                                      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 was
                                                                      purchased from Putian Zhongsheng Weiye (P.R. China). The SPG
                                                                      membranes were obtained from SPG Technology (Japan). KP-18C was
                                                                      purchased from Shin-Etsu Chemicals (Japan). PO-500 ((hexaglycerin
                                                                      penta)ester) was kindly provided by Sakamoto Yakuhin Kogyo
                                                                      (Japan). BSA, glutaraldehyde, and p-phthaldehyde were acquired
                                                                      from Sigma (Germany). All other materials were of analytical reagent
                                                                      grade. The ratio of amino and aldehyde groups was 1:1 during the
                                                                      crosslinking reaction and a crosslinking time of 1 h was used for all
                                                                      reactions unless otherwise specified.
                                                                         Preparation of C-G Microspheres: Monodisperse chitosan micro-
                                                                      spheres were prepared according to a method previously described by
                                                                      Wei et al. [6]. Surface treatement of the SPG membranes with KP-18C
                                                                      made the membranes relatively hydrophobic. Next, 2 wt% chitosan
                                                                      was 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 all
                                                                      experiments. First, the water phase was permeated through the
                                                                      uniform pores of the SPG membrane into the oil phase using the
                                                                      pressure of nitrogen gas to form a monodisperse water-in-oil (w/o)
                                                                      emulsion. Glutaraldehyde-saturated toluene (GST) was used as the
                                                                      crosslinking agent to solidify the chitosan droplets. Finally, the
                                                                      crosslinked microspheres were collected and washed twice with
                                                                      petroleum ether, acetone, and ethanol by centrifugation (3000g) and
                                                                      redispersion.
                                                                         Preparation of C-PG Microspheres: A monodisperse w/o emulsion
                                                                      was prepared as discussed above. The C-PG microspheres were
Figure 5. In vitro BSA a) loading efficiency and b) release profiles    prepared by sequentially adding two kinds of crosslinking agents, first
measured for the different types of microspheres.                     p-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
concentration.[10] CH-G microspheres with a minimal initial           chitosan, 2 wt% chitosan—HTCC mixed polymer (mass ratio of
burst and relatively well-controlled release are likely to be         1:1) was dissolved in 1 wt% aqueous acetic acid. This mixture
                                                                      constituted the water phase used to form w/o emulsions by the SPG
ideal carriers for these protein drugs. In contrast, short but        membrane emulsification technique mentioned above. Subsequently,
intensive administration of drugs is preferable for treating          GST was used to form CH-G microspheres, whereas the CH-PG
cancer, hepatitis, and systemic lupus erythenatosus (SLE).[11]        microspheres were prepared by the aforementioned two-step solidi-
CH-PG microspheres with a strong initial burst are much more          fication process also used to prepare C-PG microspheres. Finally, the
                                                                      samples were washed with acetone and ethanol prior to collection, as
suitable candidates for such pulsed therapy. In the context of
                                                                      also described above.
conventional vaccination, repeated injections at appropriately           In Vitro BSA Loading: The absorption medium was PBS (pH ¼ 7.4)
timed intervals are required for vaccines preventing hemor-           containing 1000 mg mLÀ1 BSA and 106 mLÀ1 microspheres. After 48 h,
rhagic fever, hepatitis B, and rabies. CH-PG microspheres with        the BSA-loaded microspheres were separated from the medium and
two distinct bursts also offer a promising approach to mimic          the presence of residual BSA in the medium was detected using a
                                                                      bicinchoninic acid (BCA) assay.
these repeated immunizations.[12] Overall, the distinctive               In Vitro BSA Release Study: The release medium was PBS
release profiles of these different types of microspheres reflect       containing 0.05% (w/v) sodium azide as a preserving agent and
the potential therapeutic applications that can be achieved           0.02% (v/v) polysorbate-80 as a dispersing agent. The concentration of
with these systems to fulfill various drug delivery requirements.      BSA-loaded microspheres was 106 mLÀ1. The release experiments
                                                                      were performed in a thermostatic shaker (37 8C, 120 rpm). After
   In addition to uniformity, the SPG membrane technique
                                                                      predetermined intervals, 0.5 mL of the supernatant was extracted and
also enables the preparation of microspheres with a specific           analyzed using the BCA assay. Subsequently, the same volume of fresh
particle size by appropriate choice of the membrane pore              PBS buffer was added into the release medium to top up to the original
size.[6] Therefore, the novel monodisperse chitosan micro-            volume (4 mL).
spheres with controllable size discussed here are promising              Characterization Methods: The structure of the C-PG microspheres
                                                                      was characterized by FTIR using a FTIR-400/600 instrument from
systems for achieving better reproducibility, more repeatable         Jasco. The mean size and zeta potential of the microspheres were
release behavior, higher bioavailability, and passive target-         determined using a Mastersizer 2000 laser diffractometer and zetasizer
ability. The tunability of microsphere structural properties          analyzer from Malvern Instruments. Brunauer—Emmett—Teller



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