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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 and other modern biotechnology approaches have lead to the emergence of protein drugs as a very important class of therapeutic agents. 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. 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, biocompatibility, biodegradability, mucus adhesion, and low cost. Over the last few decades, chitosan microspheres have always been prepared by facile chemical crosslinking using glutaraldehyde (C-G microspheres). Typically, acidic water-soluble drugs are simply dispersed in chitosan solution and entrapped by an emulsion crosslinking process. However, some drugs, especially 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. To maintain the activity of protein dugs, researchers have devised
Prof. G.-H. Ma, Dr. W. Wei, Dr. L.-Y. Wang, Dr. J. Wu, X. Hu Prof. Z.-G. Su National Key Laboratory of Biochemical Engineering Institute of Process Engineering Chinese Academy of Sciences 100080 Beijing (P.R. China) E-mail: email@example.com Dr. W. Wei Graduate University of Chinese Academy of Sciences 100049 Beijing (P.R. China) Prof. L. Yuan Medical and Healthy Analytical Center Peking University Health Science Center 100083 Beijing (P.R. China) Dr. G. Hu Unilever Research China 200233 Shanghai (P.R. China) This work was supported by the National Nature Science Foundation of 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 InterScience or from the author.
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 methods 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 experiments 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 signiﬁcantly 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 microspheres. 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 emulsiﬁcation. These microspheres show remarkable autoﬂuorescent properties that have been attributed to the n—p* transitions of C – N bonds in the Schiff bases formed during the – crosslinking 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, glutaraldehyde, as mentioned above (Fig. 1a). This two-step crosslinking process has enabled the successful preparation of C-PG microspheres. As shown in Figure 1b, after washing with
ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2008, 20, 2292–2296
the deﬁcienbars represent 20 mm. Supporting Information). 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. 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. Adv. 2292–2296 ß 2008 WILEY-VCH Verlag GmbH & Co. the C-PG microspheres have been sphere surface (Fig.advmat. No phenyl signal has S3. 3).COMMUNICATION 2293 Several reports have shown that quaternized chitosan is non-toxic. Supporting microspheres after washing with acetone and ethanol. S1. spectroscopy analysis). Supporting Inforcrosslinking during the two crosslinking processes (Fig. A mixture of chitosan and HTCC (in a 1:1 mass ratio) has been used to prepare CH-G microspheres by crosslinking with glutaraldehyde. A relatively weaker quaternized group content and the degree of crosslinking. c) Proﬁle of the ﬂuorescence distribution of C-PG microspheres. 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. The hollow structures obtained by this method are shown in Figure 2a. b) Scanning electron microscopy (SEM) image of the surface of CH-G microspheres. been detected in the infrared analysis of these structures. which indicates that the crosslinking reactions occur more easily on the surface than in the center Figure 1. Mater. c) Proﬁle of the ﬂuorescence distribution of CH-G microspheres. 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. and can increase the permeability of intestinal epithelia. Supporting Information). S2. respectively. 1c). the overlay is shown in yellow and scale quaternized groups. 2008.de . 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. Weinheim www. b) Laser scanning because of the steric hindrance from confocal microscopy (LSCM) image of C-PG microspheres. S4. as previously reported by Wu et al. 20. from pre-crosslinked p-phthaldehyde. A chitosan derivative N-[(2-hydroxy3-trimethylammonium)propyl] chitosan chloride (HTCC) with 60% substitution has been obtained by reacting chitosan with glycidyltrimethylammonium. a) Schematic illustration of the preparation of C-PG microspheres. A signal has been detected from the center (Fig. a) LSCM image of CH-G microspheres. No Information shows the Fourier transform infrared (FTIR) cavities have been observed in the as-prepared microspheres. cies of this crosslinking reaction also lead to the formation of pores on the microacetone and ethanol. KGaA. 2b). The scale bars represent 20 mm and 100 nm in (a) and (b). The ﬂuorescence intensities have been observed to gradually decrease from the surface to the center in Figure 2c. and are clearly somewhat smaller than the structures shown in Figure 1b. It is thought that the formation of Figure 2. mation). Indeed.
and the BSA molecules are negatively charged in phosphate buffered saline (PBS).7 CH-PG 7.g. Figure 5b displays in vitro release patterns of BSA from microspheres prepared by the different methods mentioned above.d) High-magniﬁcation images showing details of the inner large amount of BSA loaded in the structure and surface morphology of the microspheres. a) LSCM and b) SEM images of macroporous CH-PG microspheres. respectively. followed by a plateau for about 60 h. the porosity of the microsphere walls not only facilitates the migration of Table 1. We propose that most of the BSA molecules enter the onto microspheres prepared by different methods. A possible explanation for the very high loadings can be proposed based on the role of electrostatic effects. use of the SPG membrane technique has enabled the and ﬁnally 85% release as a second burst. In addition. ﬁgures. As compared to traditional released in an initial burst. as compared to their conventional solid counterparts. which migrate presence of the quaternized group in HTCC. whereas the second originates from the microspheres both show a high positive charge because of the release of BSA molecules present in the lumen. Figure 4 schematically microspheres. Weinheim Adv.6 14. and their Figure 5a. As shown in interior of the CH-G microspheres via pore channels. The loading efﬁciencies of CH-PG microspheres and C-PG In clinical therapy. In and pore diameter results are also consistent with the images contrast to microspheres prepared by other methods.. Schematic representation of BSA (green dots) loading patterns in microspheres prepared by different methods: a) C-G microspheres. Characterization data for microspheres prepared by different methods.7 98 73.7 12. resulting in an enhanced loading efﬁciency.2 Figure 4. the BSA is present on the particle surface.4 2. Mater. 2294 www.4 13.de ß 2008 WILEY-VCH Verlag GmbH & Co.7 55 4. strong electrostatic interactions are established between BSA and microspheres containing positively charged HTCC. the scale bars the highest initial burst because of the represent 20 and 1 mm. Furthermore. spheres with a hollow structure have a relatively greater amount of accessible space for BSA storage. 2008. reaching about 390 mg BSA for 106 microspheres. c) CH-G microspheres.3 3. microspheres which is followed by a slower linear release In our comparative study. 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). KGaA.4 16 2. The isoelectric point of BSA is 4. As a result. 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. BSA release in Table 1 shows characterization data for microspheres C-PG microspheres follows a triphase proﬁle with 42% being prepared by different methods.3 CH-G 7. tion gradient. and d) CH-PG microspheres. methods. 2292–2296 .9 19. we have focused on loading BSA proﬁle.COMMUNICATION BSA into the lumen.7. CH-G microspheres yield the highest loading later release is driven by diffusion induced by the concentraefﬁciency. Similarly. Microspheres with different structures yield measurably different release proﬁles. The ﬁrst burst results in the release of BSA depicts the structure of these microspheres.3 12. which results in the rapid release of BSA into the medium.3 142 16. CH-G and CH-PG loaded in the shell. the scale bars represent 500 nm in both the macroporous outer shell and channels.1 C-PG 7.advmat.3 20. The CH-PG microspheres show Figure 3. Microspheres Particle Size [mm] CV value [%] Zeta potential [mV] Surface area [m2 gÀ1] Mean pore diameter [nm] C-G 7. c. it is always necessary to maintain protein microspheres are also higher than that of traditional C-G drugs (e. b) C-PG microspheres. interferon) at a speciﬁc therapeutic serum microspheres. insulin. but also creates a higher surface area and therefore adds to the number of sites available for BSA loading by absorption. 20. 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%. The surface area outwards much slower than the BSA in the outer shell.
glutaraldehyde. The volume ratio of water and oil phases was 1:10 (v/v%) in all experiments. 2008. In vitro BSA a) loading efﬁciency and b) release proﬁles measured for the different types of microspheres. In Vitro BSA Loading: The absorption medium was PBS (pH ¼ 7. ﬁrst p-phthaldehyde and then glutaraldehyde. After solidiﬁcation. China). and rabies. 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. and passive targetability. 2 wt% chitosan was dissolved in 1 wt% aqueous acetic acid. cavity size. The tunability of microsphere structural properties Adv. Characterization Methods: The structure of the C-PG microspheres was characterized by FTIR using a FTIR-400/600 instrument from Jasco. Preparation of CH-G and CH-PG Microspheres: Instead of chitosan. KGaA. and p-phthaldehyde were acquired from Sigma (Germany). hepatitis. the BSA-loaded microspheres were separated from the medium and the presence of residual BSA in the medium was detected using a bicinchoninic acid (BCA) assay. CH-PG microspheres with two distinct bursts also offer a promising approach to mimic these repeated immunizations. and wall porosity enables the modiﬁcation of these systems to cater to speciﬁc requirements for use as protein drug carriers. Next.4) containing 1000 mg mLÀ1 BSA and 106 mLÀ1 microspheres. In contrast.R. In Vitro BSA Release Study: The release medium was PBS containing 0. the SPG membrane technique also enables the preparation of microspheres with a speciﬁc particle size by appropriate choice of the membrane pore size.de 2295 . the same volume of fresh PBS buffer was added into the release medium to top up to the original volume (4 mL). First. The C-PG microspheres were prepared by sequentially adding two kinds of crosslinking agents. In addition to uniformity.02% (v/v) polysorbate-80 as a dispersing agent. whereas the CH-PG microspheres were prepared by the aforementioned two-step solidiﬁcation process also used to prepare C-PG microspheres. 2292–2296 ß 2008 WILEY-VCH Verlag GmbH & Co. Brunauer—Emmett—Teller Figure 5. Finally. CH-PG microspheres with a strong initial burst are much more suitable candidates for such pulsed therapy. the crosslinked microspheres were collected and washed twice with petroleum ether. Subsequently. and this mixture was used as the water phase. and ethanol by centrifugation (3000g) and redispersion. After 48 h. acetone. The concentration of BSA-loaded microspheres was 106 mLÀ1. the C-PG microcapsules were collected using the method described above. the samples were washed with acetone and ethanol prior to collection. Overall. Experimental Materials: Chitosan with a molecular weight of 780 000 was purchased from Putian Zhongsheng Weiye (P. as also described above. Subsequently. Finally. Preparation of C-G Microspheres: Monodisperse chitosan microspheres were prepared according to a method previously described by Wei et al. 20. Glutaraldehyde-saturated toluene (GST) was used as the crosslinking agent to solidify the chitosan droplets. Surface treatement of the SPG membranes with KP-18C made the membranes relatively hydrophobic. CH-G microspheres with a minimal initial burst and relatively well-controlled release are likely to be ideal carriers for these protein drugs. All other materials were of analytical reagent grade. repeated injections at appropriately timed intervals are required for vaccines preventing hemorrhagic fever. The SPG membranes were obtained from SPG Technology (Japan). Weinheim www. higher bioavailability. PO-500 ((hexaglycerin penta)ester) was kindly provided by Sakamoto Yakuhin Kogyo (Japan). BSA. In the context of conventional vaccination. After predetermined intervals. the novel monodisperse chitosan microspheres with controllable size discussed here are promising systems for achieving better reproducibility. This mixture constituted the water phase used to form w/o emulsions by the SPG membrane emulsiﬁcation technique mentioned above. The mean size and zeta potential of the microspheres were determined using a Mastersizer 2000 laser diffractometer and zetasizer analyzer from Malvern Instruments. 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. Therefore. more repeatable release behavior. 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.advmat. Preparation of C-PG Microspheres: A monodisperse w/o emulsion was prepared as discussed above. 120 rpm). .COMMUNICATION such as surface charge.5 mL of the supernatant was extracted and analyzed using the BCA assay. short but intensive administration of drugs is preferable for treating cancer. 2 wt% chitosan—HTCC mixed polymer (mass ratio of 1:1) was dissolved in 1 wt% aqueous acetic acid. and systemic lupus erythenatosus (SLE).05% (w/v) sodium azide as a preserving agent and 0. Mater. hepatitis B. KP-18C was purchased from Shin-Etsu Chemicals (Japan). GST was used to form CH-G microspheres. The oil phase was a 7:5 (v/v) mixture of liquid-parafﬁn/petroleum-ether containing 4 wt% PO-500 emulsiﬁer. The release experiments were performed in a thermostatic shaker (37 8C. concentration. 0.
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