Hydrogel Microspheres: Influence of Chemical Composition on

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Langmuir 2004, 20, 9968-9977

Hydrogel Microspheres: Influence of Chemical Composition on Surface Morphology, Local Elastic Properties, and Bulk Mechanical Characteristics Malgorzata Lekka,†,| Dianelys Sainz-Serp,‡,| Andrzej J. Kulik,§ and Christine Wandrey*,‡ The Henryk Niewodniczanki Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Krako´ w, Poland, Swiss Federal Institute of Technology, Institute of Chemical Science and Engineering, CH-1015 Lausanne, Switzerland, and Swiss Federal Institute of Technology, Institute of Physics of Complex Matter, CH-1015 Lausanne, Switzerland Received June 29, 2004. In Final Form: August 27, 2004 Hydrogel microspheres, beads, and capsules of uniform size, differing in their chemical composition, have been prepared by electrostatic complex formation of sodium alginate with divalent cations and polycations. These have served as model spheres to study the influence of the chemical composition on both surface characteristics and bulk mechanical properties. Resistance to compression experiments yielding the compression work clearly identified differences as a function of the composition, with forces at maximal compression in the range of 34-455 mN. The suitability and informative value of atomic force microscopy have been confirmed for the case where surface characterization is performed in a liquid environment equivalent to physiological conditions. Surface imaging and mechanical response to indentation revealed different average surface roughness and Young’s moduli for all hydrogel types ranging from 0.9 to 14.4 nm and from 0.4 to 440 kPa, respectively. The hydrogels exhibited pure elastic behavior. Despite a relatively high standard deviation, resulting from both surface and batch heterogeneity, nonoverlapping ranges of Young’s moduli were reproducibly identified for the selected model spheres. The findings indicate the reliability of contact mode atomic force microscopy to quantify local surface properties, which may have an impact on the biocompatibility of alginate-based hydrogel materials of different composition and conditions of preparation. Moreover, it seems that local elastic properties and bulk mechanical characteristics are subject to analogous composition influences.

Introduction Hydrogels are soft materials, which are composed of either covalently or electrostatically cross-linked networks containing a high percentage of water. The high water content renders them suitable for a variety of medical, biological, and biotechnological applications such as, for example, tissue engineering or immunoisolation.1,2 A particular application is in the production of hydrogel microspheres, which may serve as containers to release drugs or even products of encapsulated living cells. The most demanded characteristics for such microspheres are adjustable mechanical stability and/or durability, adequate permeability, and biocompatibility.3 Polyelectrolyte hydrogel formation with sodium alginate as the preferred polyanion has become one of the most frequently used procedures to produce microspheres.4 Sodium alginate has the ability to form hydrogels by * To whom correspondence should be addressed. Mail: EPFLISIC-LBCh, CH-1015 Lausanne, Switzerland. Phone: +41-21-693 36 72. Fax: +41-21-693 60 30. E-mail: [email protected]. † The Henryk Niewodniczanki Institute of Nuclear Physics, Polish Academy of Sciences. ‡ Swiss Federal Institute of Technology, Institute of Chemical Science and Engineering. § Swiss Federal Institute of Technology, Institute of Physics of Complex Matter. | The first two authors have contributed equally to this work. (1) Hoffmann, A. S. Adv. Drug Delivery Rev. 2002, 43, 3-12. (2) Kishida, A.; Ikada, Y. In Polymeric Biomaterials, 2nd ed; Dumitriu, S., Ed.; Marcel Dekker: New York, 2002; pp 133-145. (3) King, S. R.; Dorian, R.; Storrs, R. W. Graft 2001, 4, 491-499. (4) Tønnesen, H. H.; Karlsen, J. Drug Dev. Ind. Pharm. 2002, 28, 621-630.

electrostatic interaction with divalent counterions and polycations.5 These may be binary complex beads consisting of alginate cross-linked with calcium or barium, multicomponent spheres, such as cell-containing capsules surrounded by alginate/polycation complex membranes, or even capsules with semipermeable networks built from polyanion and polycation mixtures in one or more reaction steps. A broad variety of microspheres, beads, and capsules, differing in size, alginate type, composition, and properties is presently under investigation. In addition to general physicochemical characterization, in vitro and in vivo studies aim at optimizing the material suited to fulfill the requirements of bioartificial organs. Nevertheless, an optimized material has yet to be developed.6 The application of semipermeable hydrogel microspheres as immunoprotecting materials, in particular for cell transplantation and bioartificial organs, requires a careful design based on fundamental knowledge of how component characteristics and preparation conditions affect the final properties. Since electrostatic complex formation is a spontaneous process, being subject to kinetic and thermodynamic control, pliability is restricted to some extent. Moreover, electrostatically cross-linked hydrogels can be considered as dynamic or environment-responsive materials, which can alter bulk and transport properties due to variations in composition and modifications to medium parameters and process conditions. Whereas (5) Dumitriu, S. In Polymeric Biomaterials, 2nd ed; Dumitriu, S., Ed.; Marcel Dekker: New York, 2002; pp 1-62. (6) Orive, G.; Hernandez, R. M.; Gascon, A. R.; Calafiore, R.; Chang, T. M. S.; de Vos, P.; Hortelano, G.; Hunkeler, D.; Lacik, I.; Shapiro, A. M. J.; Pedraz, J. L. Nat. Med. 2003, 9, 104-107.

10.1021/la048389h CCC: $27.50 © 2004 American Chemical Society Published on Web 10/09/2004

Hydrogel Microspheres

mechanical and transport properties can be measured directly and characterized by physical parameters, biocompatibility is difficult to define precisely. In the case of microspheres, the chemical composition and the purity of the material are subject to intense research concerning biocompatibility. Relatively less attention has been directed to surface properties, in particular correlations with microsphere chemistry and preparation. There is some evidence that not only the purity and the chemical composition of the microsphere material determine biocompatibility but also the physical surface properties will have an impact on the body response to injection or transplantation.7,8 During recent years, atomic force microscopy (AFM) has advanced to become a useful tool not only for recording topographical images of a variety of surfaces but also for studying local mechanical properties including adhesion, friction, or stiffness.9 Moreover, it has rapidly become one of the most widely used techniques to study mechanical properties of soft samples such as living cells with Young’s modulus values in the range of 1-200 kPa.10-12 However, relatively few studies have been performed on polymeric hydrogel microspheres. The majority of the reports describe the influence of the cross-linking procedure on the surface topography13 or how the ratio of antigelling and gelling cations affects surface roughness.14 Recently, the surface morphology of selected microcapsules has been correlated directly to biocompatibility, with improved compatibility being found for smoother and flawless surfaces. However, in this work elastic properties have not been considered.7 The goal of the research presented here is to show, quantitatively, how chemical composition and preparation conditions affect the mechanical bulk and surface properties of alginate-based microspheres. The focus is on microsphere composition and preparation conditions, which are considered as suitable for future applications, preferably in the biomedical field. The first part of the paper is dedicated to evaluate the suitability of AFM for studies of alginate microspheres. Studies on microspheres, beads, and capsules, prepared under different conditions and with different composition, served to screen the possible variation width of the surface properties including roughness and local elastic properties. Furthermore, the study aimed at obtaining quantitative information on whether reproducibility and batch homogeneity are sufficient to define composition and process related characteristics. Moreover, it had to be discovered if AFM is suitable for distinguishing between different types of microsphere in which alginate is the main component. The final part of the paper reports the results of a more systematic and comprehensive study, which included, in addition to AFM measurements, compression experiments on a variety of different alginate-based beads, capsules, (7) Bu¨nger, C. M.; Gerlach, C.; Freier, T.; Schmitz, K. P.; Pilz, M.; Werner, C.; Jonas, L.; Schareck, W.; Hopt, U. T.; de Vos, P. J. Biomed. Mater. Res. 2003, 67A, 1219-1227. (8) Lacik, I.; Anilkumar A. V.; Wang T. G. J. Microencapsulation 2001, 18, 479-490. (9) Radmacher, M. IEEE Eng. Med. Biol. 1997, 16, 47-57. (10) Lekka, M.; Laidler, P.; Ignacak, J.; Lekki, J.; Struszczyk, H.; Stachura, Z.; Hrynkiewicz, A. Z. Biochim. Biophys. Acta, Mol. Cell Res. 2001, 1540, 127-136. (11) Vinckier, A.; Semenza, G. FEBS Lett. 1998, 430, 12-16. (12) Weisenhorn, A.; Khorsandit, M.; Kasas, S.; Gotzost, V.; Butt, H.-J. Nanotechnology 1993, 4, 106-113. (13) Zimmermann, H.; Hillga¨rtner, M.; Manz, B.; Feilen, P.; Brunnemeier, F.; Leinfelder, U.; Weber, M.; Cramer, H.; Schneider, S.; Hendrich, C.; Volke, F.; Zimmermann, U. Biomaterials 2003, 24, 20832096. (14) Xu, K.; Hercules, D. M.; Lacik, I.; Wang, T. G. J. Biomed. Mater. Res. 1998, 41, 461-467.

Langmuir, Vol. 20, No. 23, 2004 9969 Table 1. Polyanion Characteristics: Intrinsic Viscosity, [η], in 0.1 m NaCl, and Ratio of Guluronic to Mannuronic Chain Units, G/M polyanion

[η] (mL/g)

G/M ratio

polyanion

[η] (mL/g)

SA1 SA2 SA3

807 952 1414

0.67a ≈0.67b ≈0.67b

SA4 SA5 SCS

866 511 552

a

G/M ratio ≈1b 0.69a

Determined by 1H NMR. b Determined by FTIR. Table 2. Microsphere Preparation

microsphere type

composition

B1 B2 B3 B4 B5 C1 C2 C3 C4

SA1/Ba SA1/Ba SA2/Ba SA3/Ba SA1/Ca SA4+SCS/Ca/PMCG SA4+SCS/Ca/PMCG/SA5 SA4+SCS/Ca/PMCG/SCS SA1/Ca/PLL

a

polyanion concentration ηa in PBS (wt %) (mPa s) 1.2 1.7 1.5 0.9 1.7 1.5 (1:1) 1.5 (1:1)/0.15 1.5 (1:1)/0.15 1.7

215 553 549 570 553 90 90 90 553

Dynamic viscosity of the polyanion solutions.

and coated capsules. This serves to define relationships between microsphere materials and production conditions on one hand and bulk and local elastic properties on the other hand. Experimental Section Materials. Five sodium alginates (SAs) differing in mass and/ or slightly in composition have been selected as polyanions (PAs) in this study: SA1, Keltone HV (Kelco, Chemical Co., San Diego, CA, batch no. 61650A); SA2 and SA3, NSPL and NSPH2 (Kuraray Medical, Inc., Japan, batches no. 24494 and 74621); SA4, UPMVG (Endocrine Transplant, Inc., New York, batch no. 701-256-09); and SA5, Keltone LV (Kelco Chemical Co., batch no. 49198A). Sodium cellulose sulfate (SCS) (Acros Organics, Geel, Belgium, batch no. A013801301) possessing a degree of substitution DS ) 2.4 ( 0.2 was used as a second PA. All polyanions have been characterized by their intrinsic viscosity in 0.1 M NaCl at 20 °C, which is directly related to the molar mass. 1H NMR spectroscopy and FTIR spectroscopy served to determine the content of guluronic and mannuronic acid.15,16 The macromolecular characteristics of all PAs are summarized in Table 1. Poly(methylene-co-guanidine)hydrochloride (PMCG) and poly(L-lysine) (PLL) were used as polycations (PCs). PMCG (Scientific Polymer Products, Inc., Ontario, NY) was purchased as 35 wt % aqueous solution. For PLL (Sigma-Aldrich Co, St. Louis, MO), a molar mass of 30 000 g/mol was communicated by the supplier. NaCl, CaCl2, BaCl2 (Fluka, Switzerland), and phosphate buffered saline (PBS) were of analytical grade. Bead and Capsule Preparation. Four types of Ba-alginate and one type of Ca-alginate microbeads, B1-B5 in Table 2, were prepared by atomizing the polyanion solutions of SA1, SA2, or SA3 with a coaxial-air-flow encapsulator.17 The droplets were gelled for 3 min in a bath containing 1.5 wt % BaCl2 or 2.2 wt % CaCl2 in 0.9 wt % NaCl (37 °C). Following gelation, the Babeads were washed five times whereas the Ca-beads were washed three times in 0.9 wt % NaCl and ultimately stored in their washing solutions at 4 °C. Microcapsules, C1-C4, were produced by multistep processes. Briefly, a 1.5 wt % polyanion solution consisting of SA/SCS (1:1) was gelled with 2.2 wt % CaCl2 in 0.9 wt % NaCl (20 °C) and subsequently transferred into 1.2 wt % PMCG (C1) to form within 45 s the capsule membrane.17,18 The capsules were then rinsed (15) Grasdalen, H. Carbohydr. Res. 1983, 118, 255-260. (16) Dupuy, B.; Arien, A. J. Biotechnol. 1994, 22, 71-84. (17) Ceausoglu, I.; Hunkeler, D. J. Microencapsulation 2002, 19, 725. (18) Wandrey, C.; Espinosa, D.; Rehor, A.; Hunkeler, D. J. Microencapsulation 2003, 20, 597-611.

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Lekka et al. available M5, designated as AFM1 below (Park Scientific Instruments, Switzerland), and one built for the purpose, designated as AFM2 below (Institute of Nuclear Physics, Krakow, Poland), both equipped with a “liquid cell”. Commercially available unsharpened and gold-coated Si3N4 cantilevers (Atos GmbH, Germany) with spring constants of 0.03 and 0.1 N/m and tip radii of 50 nm were used. The topography of microsphere surfaces was recorded by AFM working in contact mode at forces ranging from 0.1 to 0.5 nN and scan rates of 0.8 and 1 Hz. Five microspheres per type were randomly selected from one batch for scanning. Each sphere was evaluated in triplicate. The microspheres were localized using an optical microscope (Nikon SMZ 800, Nikon, Japan). Topographic images and force curves were recorded around the central part of the spheres immersed in 0.9 wt % NaCl solution at room temperature. The average surface roughness was calculated according to Joergensen:19

Sa ) Figure 1. AFM experimental setup. The microspheres were placed in 25 conical wells of a polycarbonate plate and covered by a poly(ethylene terephthalate) plate with conical holes. During the measurements in a “liquid cell”, the microsphere surfaces were in contact with the buffer solution. four times with 0.9 wt % NaCl. For the preparation of the C2 and C3 capsules, additional coating was performed using 0.15 wt % SA5 in 0.9 wt % NaCl or 0.15 wt % SCS in 0.9 wt % NaCl for 10 min under gentle mixing. C4 capsules were prepared from B5 type beads by additional reaction in 0.1 wt % PLL in 0.9 wt % NaCl (pH ) 7.4) for 10 min. Details of the microsphere preparation including the composition of each sphere type and the concentration and dynamic viscosity of the polyanion solutions are summarized in Table 2. The selected concentrations and conditions have been identified as being in a range suitable for medical applications. To exclude size effects, process conditions were selected to ensure a narrow size distribution and allow comparative discussions. In particular, the viscosity of the PA solution had to be adjusted by modifying the PA concentration. An exception was the PA solution for the preparation of B1. In this case, the investigation of the influence of concentration was achieved by comparing B1 and B2. Microsphere components, which originate from the same solution and which participate in gel network formation, are denoted by a plus (+). On the other hand, components derived from different solutions are denoted by a slash (/). For example, SA4+SCS/Ca/PMCG/SA5 represents preparation of beads by dropping a mixture of SA4+SCS into CaCl2, subsequent preparation of capsules by membrane formation in a PMCG solution, and, finally, coating in SA5 solution. Size and Membrane Thickness of Microspheres. Twenty spheres were randomly selected from batches produced in triplicate, to determine the size/size distribution, sphericity, and membrane thickness by optical microscopy. Experimental Setup for AFM Measurements. The experimental setup for atomic force microscopy measurements is demonstrated in Figure 1. To fix the microspheres, a support consisting of two plates was designed and constructed. The lower plate (polycarbonate, 1 mm thickness) with 25 trapezoid-shaped wells in the central region avoids horizontal movement of the microspheres. Vertical movement was limited by the upper plate, poly(ethylene terephthalate) (PET) of 100 µm thickness, possessing trapezoidshaped holes fitting to the wells of the lower plate. The dimensions for the wells were 785 µm (lower diameter), 865 µm (upper diameter), and 656 µm (well depth) and for the holes 580 µm (lower diameter) and 560 µm (upper diameter). All of them were manufactured with a precision of (5 µm. Such mechanical fixation completely avoided surface modifications due to the use of additives associated with chemical or electrostatic fixation. Atomic Force Microscopy. Imaging and measurement of the local elastic properties of selected microspheres were performed using two atomic force microscopes, a commercially

1

N

N

∑∑|z(i,j)|

(1)

N2 i)1 j)1

assuming that AFM scanning provides a set of N × N topographic data z(x1; yi) where xi and yi are plane coordinates. The following software was used: AFM32 for data acquisition and image analysis, FORCE for force curve analysis (with AFM2), and scanning Probe Image Processor, SPIP (demo version, http:// www.imagemet.com, with AFM1). Hertz Model and Young’s Modulus Determination. The model applied for the determination of the Young’s modulus is based on Sneddon’s modification of the Hertzian contact mechanics.20 It describes the case of the elastic half-space pushed by a hard axisymmetric rigid indenter with the relationship between loading force and the indentation depending on the tip geometry. There are two geometries frequently used for the calculation of the Young’s modulus values when determined from AFM measurements. These geometries assume either a conical or paraboloidal tip shape. For a paraboloidal tip with a radius of curvature R, the relationship between the loading force F and the indentation ∆z is

4 F ) xRE′∆z1.5 3

(2)

For a conical tip with an angle R, it becomes

F)

2E′ (∆z)2 π tan R

(3)

where E′ is a reduced Young’s modulus value of the tip-sample system: 2

1 - µtip 1 - µsample 1 ) + E′ Etip Esample

2

(4)

where Etip and Esample are Young’s modulus of both the tip and the cell, and µtip and µsample are Poisson ratios ranging from 0 to 0.5. Taking into account that Etip . Esample, one finally obtains for the reduced Young’s modulus

E′ )

Esample 1 - µsample2

(5)

Force Curves. Force curves reflecting the interaction between the probing tip and the microsphere surface were collected using the laser deflection technique. To obtain the force versus indentation curves, a calibration procedure described by Lekka et al. was employed.21 For this purpose, curves were first recorded on the hard surface of the polycarbonate plate. On such a nondeformable surface, the cantilever deflection is proportional (19) Joergensen, J. F. Nanotechnology 1993, 4, 152-158. (20) Sneddon, I. Int. Eng. Sci. 1965, 3, 47-57. (21) Lekka, M.; Lekki, J.; Marszanek, M.; Golonka, P.; Stachura, Z.; Cleff, B.; Hrynkiewicz, A. Z. Appl. Surf. Sci. 1999, 141, 345-349.

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Table 3. Types of Microspheres and Their Components and Preparation Processa anionic components

cationic components

coating

type SA1 SA2 SA3 SA4 SCS Ba Ca PLL PMCG SA5 SCS B1 B2 B3 B4 B5

c1 c2 c2

Two-Component Microspheres X X X c4 X X

C4

c2

Three-Component Microspheres X X

C1 C2 C3 a

c3

Multicomponent Microspheres c3 X X c3 X X c3 X X

X X X

microsphere sizea (µm)

ASRa (nm)

YMb (kPa)

B1 B2 B3 B4 B5 C1 C2 C3 C4

870 ( 20 825 ( 39 830 ( 23 825 ( 27 425 ( 15* 860 ( 17 850 ( 13 830 ( 13 850 ( 25

1.9 ( 0.4 2.0 ( 0.4 1.7 ( 0.2 1.6 ( 0.1 0.93 ( 0.02 14.4 ( 1.4 7.3 ( 0.4 7.4 ( 0.5 1.2 ( 0.3

4.5 ( 0.8 6.9 ( 1.4 17.7 ( 2.9 15.1 ( 2.4 0.4 ( 0.2 178 ( 88 250 ( 68 438 ( 150 1.0 ( 0.4

a

X

to the relative sample position resulting in a linear slope in the section of the calibration curve for which the tip and the sample are in contact. When soft samples such as the hydrogel microspheres investigated here are indented by the AFM tip, the dependence of the cantilever deflection on the relative sample position is not linear. More precisely, the deflection is smaller than that obtained on nondeformable samples. This difference corresponds to the indentation depth (compare Figure 6). Multiplying the cantilever deflection with the value of the spring constant yields the force scale. Finally, the comparison of the force curves obtained for the nondeformable material and for the hydrogel material delivers a force versus indentation curve used to determine Young’s modulus values.11,22 Mechanical Stability Measurements. The mechanical properties of beads and capsules were evaluated by determining the resistance to compression while compressing individual microspheres between two parallel plates. The measurements were performed with a Texture Analyzer (TA-2xi, Stable Micro Systems, Godalming, U.K.) equipped with a force transducer characterized by a resolution of 1 mN. One bead or capsule was placed on a microscope slide. Then the probe was moved with a constant speed of 0.4 mm s-1 toward the sphere until the distance travelled had reached 99% of the initial distance between probe and slide. From these curves, the values of the force, F, at discrete displacements, 40, 60, 65, 75, 85, 92, 94, and 96%, and covering the range of bursting, were extracted and served to calculate the compression work Wb according to

∫F ds

microsphere type

(Standard error. b (Standard deviation.

X

Concentrations: c1 ) 1.2%; c2 ) 1.7%; c3 ) 1.5%; c4 ) 0.9%.

Wb )

Table 4. Average Surface Roughness (ASR) and Young’s Modulus (YM) of Alginate Microspheres

(6)

where ds denotes the distance until maximum compression or “bursting point”. The mechanical tests were conducted on a series of 10 beads or capsules per batch from 3 batches of each microsphere model type; that is, 30 spheres in total contributed to the average result. Further experimental details on the mechanical testing have been published elsewhere.23

Results and Discussion Table 3 presents a summary of the specific microsphere compositions as well as their preparation processes. The parameter modification was restricted to conditions and compositions that are relevant to the intended application of such microspheres in the biomedical field. This concerns the selection of the alginate type with G/M e 1, the variation of the alginate concentration in the range of 0.9-1.7 wt %, the restriction to the two bivalent cations calcium and barium, as well as the selection of the second PA and the two polycations PLL and PMCG. (22) Capella, B.; Dietler, G. Surf. Sci. Rep. 1999, 34, 1-104. (23) Rehor, A.; Canaple, L.; Thang, Z.; Hunkeler, D. J. Biomater. Sci., Polym. Ed. 2001, 12, 157-170.

From some studies, it may be concluded that alginates with higher mannuronic content are more suited for cell encapsulation.24,25 Moreover, M-alginate forms under comparable preparation conditions denser and more elastic gel networks.26 When transplanted into the portal vein, Ba-alginate beads performed better than Ca-alginate beads.27 Therefore, the primary focus for the twocomponent microspheres was to study the influence of the alginate molar mass and concentration on the properties of Ba-alginate beads. In the case of the multicomponent microspheres, uncoated and coated capsules have been compared. The coating PA was either SA or SCS. The multicomponent capsule SA+SCS/Ca/PMCG was proposed by several groups as advantageous for biotechnological and medical applications.28-30 Despite the restrictions above, the overall purpose of the study was to identify general tendencies of the pliability of the surface properties of hydrogel microspheres. The results will be presented, analyzed, and discussed in detail in the subsequent paragraphs. Size and Shape of Microspheres. The size of the microspheres was determined to be in the range of 825870 µm, with the exception of B5, for AFM studies. For mechanical testing, the size was in the range of 860-990 µm for all microspheres, with a maximum batch heterogeneity of 50 µm, that is, sufficiently narrow to exclude significant size effects (compare Tables 4 and 5).23 To avoid long storage periods, separate batches have been prepared for AFM and compression studies. Figure 2 presents examples of beads and capsules and demonstrates the spherical shape and the narrow size distribution. Topography and Roughness. AFM images were taken from all nine microsphere types, beads B1-B5 and capsules C1- C4, with their compositions and preparation described in Table 2. Figure 3 presents six selected AFM images, which are characteristic for all microsphere types studied herein. Differences in the gray scale reflect the surface morphology. Image A in Figure 3 is representative for all Ba-alginate beads. Such a “spongelike” image was recorded independent of the alginate molar mass and concentration. (24) Stabler, C.; Wilks, K.; Sambanis, A.; Constandinidis, I. Biomaterials 2001, 22, 1301-1310. (25) Simpson, N. E.; Stabler, C. L.; Simpson, C. P.; Sambanis, A.; Constandinidis, I. Biomaterials 2004, 2603-2610. (26) Strand, B. L.; Morch, Y. A.; Syvertsen, K. R.; Espevik, T.; SkjåkBræk, G. J. Biomed. Mater. Res. A 2003, 64A, 540-550. (27) Toso, C.; Mathe, Z.; Morel, Ph.; Oberholzer, J.; Bosco, D.; Sainz Vidal, D.; Hunkeler, D.; Buhler, L. H.; Wandrey, C.; Berney, T. Cell Transplant., in press. (28) Bartkowiak, A.; Canaple, L.; Ceausoglu, I.; Nurdin, N.; Renken, A.; Rindisbacher, L.; Wandrey, C.; Desvergne, B.; Hunkeler, D. Ann. N.Y. Acad. Sci. 1999, 875, 135-145. (29) Lacik, I.; Brissova, M.; Anilkumar, A. V.; Powers, A. C.; Wang, T. J. Biomed. Mater. Res. 1998, 39, 52-60. (30) Hearn, E.; Neufeld, R. J. Process Biochem. 2000, 35, 12531260.

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Figure 2. Photomicrographs of microspheres demonstrating the uniform size and shape. Left: SA3/Ba beads (B4) of 825 ( 27 µm. Right: SA4+SCS/Ca/PMCG/SA5 capsules (C2) of 850 ( 13 µm, with a membrane thickness of 35 µm.

Figure 4. Surface profiles of microspheres corresponding to the surface characteristics along the white dashed lines in Figure 3. For better visualization, the profiles were shifted vertically: (B1) 60 nm, (B2) 40 nm, (B3) 25 nm, (B4) 20 nm, (B5) 15 nm, and (C4) 5 nm in Figure 4a; (C1) 266 nm, (C2) 150 nm, and (C3) 55 nm in Figure 4b. (Note the different height scales in panels a and b.)

Figure 3. Selected AFM images (10 × 10 µm) of microsphere surfaces: (A) SA/Ba, (B) SA/Ca, (C) SA/Ca/PLL, (D) SA+SCS/ Ca/PMCG, (E) SA+SCS/Ca/PMCG/SA, and (F) SA+SCS/Ca/ PMCG/SCS. The white dashed lines correspond to the surface profiles in Figure 4.

Therefore, only one image is shown. The height distribution appears randomly without any structural specifics in the submicron range. Ca-alginate bead images such as that shown in photo B of Figure 3 differ remarkably from those of Ba-alginate beads. No spongelike surface features were observed. When the Ca-alginate beads reacted in a subsequent step with PLL forming the microcapsule C4, the calcium ions were replaced in a thin network layer by PLL. The corresponding surface image (image C in Figure 3) changed into a cloudier one.

Images D-F in the right column of Figure 3 are representative of alginate multicomponent capsules, uncoated or coated with either SA5 or SCS. The gray scale indicates higher surface roughness compared to images A-C. Moreover, some structuring seems to exist in images D and F. The images represent exclusively the PA/PC complexes. It can be assumed that the macromolecules have replaced the divalent cations in the outer layer. Several procedures are possible to evaluate the surface roughness based on AFM images. Two were selected and will be presented below. Surface Profiles. To demonstrate principal differences, surface profiles taken along the white dashed lines in Figure 3 are plotted in Figure 4. Appropriate profiles from images not shown in Figure 3 have been added for B2B4. For better visualization, the profiles of the beads are plotted with a higher resolution in Figure 4a. The profiles of capsules possessing much rougher surfaces are separately shown in Figure 4b. Different morphologies are visible from these randomly selected profiles clearly indicating the lowest roughness and the highest homogeneity for the SA1/Ca two-component bead (B5) but the highest roughness for the SA4+SCS/Ca/PMCG multicomponent capsules (C1). Despite the same image type for B1-B4, the randomly selected profiles in Figure 4a reveal slight differences concerning the smoothness of the Ba-alginate beads. If the Ca-alginate bead was coated with PLL, the smoothness did not change significantly. It is visible from the profile in Figure 4b that the capsule C1 (image D in Figure 3) possesses the roughest surface. Coating with alginate SA5 seems to smooth the surface with a less structured surface resulting afterward. By contrast, coating with SCS seems to modify the surface structure to a more fine-structured one. This becomes obvious from both image F in Figure 3 and the profile in Figure 4b. Although images D-F in Figure 3 are most representative of the three capsule types, a low percentage of deviating spheres were found in identical batches. The

Hydrogel Microspheres

Figure 5. Surface profiles demonstrating the batch heterogeneity. (a) C1, (b) C2 (C1 coated with SA), (c) C3 (C1 coated with SCS). For better visualization, the profiles were shifted vertically. From top down: (a) 1150 nm, 970 nm, 850 nm; (b) 695 nm, 560 nm, 435 nm; (c) 220 nm, 60 nm.

profiles in Figure 5 are intended to demonstrate such fluctuations of roughness profiles, being determined on different capsules in the same batch. Despite the heterogeneity, general differences between the three types are obvious: the highest heterogeneity for the uncoated capsule C1 (Figure 5a), a smoother and less structured surface after coating with SA5 (Figure 5b), and a finer, although with slight capsule-to-capsule variation, structure by SCS coating (Figure 5c). Average Surface Roughness. As a second evaluation procedure, a more detailed analysis of the surface roughness has considered a multitude of measurements performed in triplicate on five microspheres of each type. The resulting mean values and the standard errors of the average surface roughness (ASR) are summarized in Table 4. These have been calculated from a scan area of 10 × 10 µm. Such an evaluation provides more reliable data; however, specific fluctuations are hard to identify or may even be overlooked. These calculated values indicate the lowest average surface roughness for the SA1/Ca bead and the highest one for the SA4+SCS/Ca/PMCG capsule. This is qualitatively in agreement with the conclusions from the random profiles in Figure 4. Slightly lower averages than for all other beads were calculated for B5 and C4. Considering the higher standard error for B1 and B2, the ASR may be concluded as in the same range for all Baalginate beads. The relatively high standard error reflects the heterogeneity. Consequently, the conclusions from random profiles in Figure 4a, that B2 has a higher roughness than B1, cannot be confirmed. This example demonstrates the necessity to consider both surface profiles and average surface roughness for the surface morphology analysis. Significantly higher ASR values were obtained for all PMCG-containing alginate capsules. Moreover, for the C1 capsules approximately double the average roughness value was determined compared to the coated capsules C2 and C3. In addition, a remarkably lower standard error was calculated for C2 and C3. This agrees with the conclusions from the random profiles in Figures 4 and 5.

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Figure 6. Force curves for two types of microspheres: (a) SA1/ Ba bead and (b) SA4+SCS/Ca/PMCG/SA5 capsule. The calibration curve taken on the polycarbonate plate is added to each graph for comparison. (All curves were slightly shifted vertically by the same value in order to visualize the baseline, e.g., the section of the curve where the tip-sample interaction was not detectable.) It is obvious that the same force causes a deeper indentation on the bead surface than on the capsule surface.

The observed heterogeneity of the surface roughness may serve to explain the large standard deviation of Young’s modulus values, as will be discussed in detail below. Nevertheless, it has to be emphasized that there is no clear correlation between surface roughness and Young’s modulus. Young’s Modulus. Typical force curves, obtained on both hard and soft surfaces with polycarbonate assumed as nondeformable, are plotted in Figure 6. Furthermore, Figure 6 serves to illustrate how the force-indentation curves are used for the Young’s modulus determination described in the preceding Experimental Section. Depending on the microsphere type, the force curves exhibit differently pronounced nonlinearity. This is demonstrated in Figure 6 comparing, for example, a SA1/Ba microbead and a SA4+SCS/Ca/PMCG/SA5 microcapsule. The bead is more compliant because the same loading force produces a larger indentation depth. Analysis of the experimental force-indentation curves indicates almost pure elastic behavior for all microspheres, beads, and capsules, since both loading and unloading curves overlap in Figure 6. No adhesion during unloading was observed. This makes such hydrogels an ideal material since they represent an infinitely elastic half space indented by an axisymmetric rigid indenter. In this case, the AFM cantilever, manufactured of Si3N4 and possessing a Young’s modulus value of 150 GPa, can be considered as such an indenter.22 Model Selection. Further model evaluation involved the fitting of force-versus-indentation curves, for either paraboloidal or conical tip shape, to two models. The fits were performed based on the assumption that the interdependence between the force and the indentation reflects the geometry of the measurement with a rigid

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Figure 7. Force versus indentation values (0) fitted to two model approaches assuming two different shapes of the AFM tip, either paraboloidal (s) or conical (- - -). The experimental data for B1 reveal a better fit for the paraboloidal approach.

AFM tip indenting a sphere considered as an elastic halfspace. Rigid means, in this case, nondeformable if compared with microspheres. The correlation with the appropriate model curve, calculated as either y ) ax1.5 with χ2 ) 0.02 for a paraboloidal geometry or y ) ax2 with χ2 ) 0.22 for a conical geometry, was decisive for the identification of ultimate model suitability (Figure 7). For the majority of microspheres, a paraboloidal tip shape of the AFM tip was more applicable. Only for the SA1/Ca/PLL capsules was the force versus indentation dependence better in accordance with the model assuming a conical shape of the probing tip. With the additional assumption that all microspheres consist of deformable but incompressible material, the Young’s modulus was calculated for a Poisson ratio of 0.5. The weighted averages for each microsphere type calculated from the experimental findings of five randomly selected beads or capsules are summarized in Table 4 together with the appropriate sphere size and average surface roughness. Young’s Modulus Values. The Young’s modulus values cover a wide range revealing differences according to which the microspheres may be distinguished into four groups: (B5 e C4) < (B1 ≈ B2) < (B4 ≈ B3) , (C1 e C2 e C3) with Young’s modulus ranges that do not overlap [(0.21.4 kPa) < (3.7-8.3 kPa) < (12.7-20.6 kPa) < (90-530 kPa)] if the standard deviations are considered for the comparison. Moreover, this order corresponds, approxi-

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mately, to values in the following ranges: below 2.5 kPa for C4, between 2.5 and 7.0 kPa for B1, between 10 and 24 kPa for B3, and between 50 and 400 kPa for C2. These ranges were concluded from a more detailed data analysis supported by histograms (Figure 8). The histograms demonstrate the variation of Young’s modulus reflecting the surface heterogeneity on a single sphere as well as the batch heterogeneity since the frequency versus Young’s modulus was calculated from experiments analyzing five spheres for each case. Furthermore, the maxima in the histograms of B1, B3, C2, and C4 are closely located to the positions of the Young’s modulus average values in Table 4. Microsphere versus Batch Homogeneity. Comparison of the histograms with data sets taken from discrete spheres suggests that broad distributions such as from the histogram of the SA4+SCS/Ca/PMCG microcapsule C1 (histogram not shown here) probably result from batch heterogeneity of these bead types rather than from the surface heterogeneity of one bead. This is qualitatively supported by the surface profiles in Figure 5a. This may serve as an additional indicator for the conclusion of batch heterogeneity of the noncoated PMCG capsules. The range of Young’s modulus fluctuations on one discrete microsphere is demonstrated in Figure 9 for one randomly selected sphere for which the appropriate values recorded along the white lines in Figure 9a are plotted in the graph of Figure 9b. The fluctuations are within the range in the histogram of the SA4+SCS/Ca/PMCG/SA5 microcapsule, Figure 8d, though not all values appear with the same frequency. In addition, the profiles in Figures 4b and 5b reveal sections of varying roughness along a line. An irregular surface may be concluded from the data evaluation for this capsule type rather than additional batch heterogeneity since the standard deviation is relatively low in comparison to the other two PMCG capsule types. The software of the AFM2 allowed recording force curves in defined square areas, of which the size may be chosen. In this study, 256 force curves were recorded on an area of about 25 µm2 corresponding to a map of 16 × 16 experimental points as demonstrated in Figure 10. Comparing the SA1/Ba (B1) and SA2/Ba (B3) beads, a more homogeneous surface is obvious for the beads B1 prepared from SA1, the alginate with a lower molar mass

Figure 8. Distributions of Young’s modulus (YM) representing four groups of microspheres. (a) C4: YM < 2.5 kPa. (b) B1: 2.5 < YM < 7.0 kPa. (c) B3: 10 < YM < 24 kPa. (d) C2: 50 < YM < 400 kPa.

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Langmuir, Vol. 20, No. 23, 2004 9975 Table 5. Force, F, and Work, Wb, at Maximal Compression, MCa microsphere type

SR (µm)

B1 B2 B3 B4 B5 C1 C2 C3

940-990 880-900 960-995 900-920 880-906 900-930 860-900 880-920

MTR (µm) MC (%)

55 35 40

96 96 96 96 94B 96B 92B 96B

F (mN)

Wb (J × 105)

34 ( 1.5 52 ( 6 190 ( 2 83 ( 6 67 ( 5 455 ( 50 157 ( 17 348 ( 16

0.15 ( 0.004 0.29 ( 0.06 1.36 ( 0.05 0.46 ( 0.04 0.52 ( 0.03 2.66 ( 0.6 1.19 ( 0.1 2.67 ( 0.06

a SR, average size range of microspheres; MTR, average membrane thickness range; B, bursting point. In the case of capsules and B5 beads, the maximal compression percent is that obtained at the bursting point B. A maximal compression of 96% corresponding to the bursting points of C1 and C3 was considered as the maximal compression percentage for all Ba-alginate beads, in the absence of a bursting point of these beads.

Figure 9. Young’s modulus (b) recorded at distances of 1 µm on the surface of a SA4+SCS/Ca/PMCG/SA5 microcapsule (C2) along the white lines of the AFM image a.

than SA2. This concerns both the absolute value of Young’s modulus within the range of 2-5 kPa and the local homogeneity indicated by relatively large areas of the same gray scale value. Beads B3 containing SA2 are characterized by higher Young’s modulus values covering a broader range within 11-23 kPa. In addition, a locally more heterogeneous surface may be suggested from gray scale fluctuations of neighboring experimental points in Figure 10b. This evaluation supports the conclusions from the profiles and correlates with the conclusions from the histograms. “Bulk” Mechanical Properties. For microbeads and microcapsules possessing a similar size of approximately

900 µm, the resistance to compression has been investigated. In general, different behavior was observed for beads B1-B4 and capsules C1-C3. Compression Profiles. While for B1-B4 the force monotonically increased until maximal compression at 99% of the bead diameter, a bursting point has been identified for B5 at 94% of the bead diameter. Such a bursting point was detected for all capsules in the range of 92-96% of their diameter, however, at remarkably higher forces than for the Ca-alginate beads. The SA1/ Ca/PLL capsules were not investigated by this method because of their extremely low resistance to compression of less than 10 mN. Figure 11 represents the three different types of compression profiles. The bursting points at 92% for C2 and at 94% for B5 are clearly visible while the force continuously increases for B2. The resistance of all three types is comparable until approximately 50% compression; thereafter the resistance increases in the order C2 > B5 > B2. However, from all force values, which are summarized in Table 5, it is visible that the force for the other Ba-beads, B3 and B4 prepared from higher molar mass alginates (SA2 and SA3), is superior to the force estimated for B2 and even B5. Also, the forces for C1 and C3 exceed that of C2 considerably. These experimental findings will be analyzed in more detail. Compression Force. Clearly, the lowest force was applied to deform Ba-beads (B1). These have been prepared from a PA solution of 1.2 wt % SA1, the core alginate possessing

Figure 10. Determination of Young’s modulus distribution on the surface of two different types of barium alginate beads: left, B1; right, B3. (White squares indicate where no measurements have been performed. Note the different scales covering 3 and 12 kPa, respectively.)

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Figure 11. Force-distance curves for uniaxial compression to hydrogel microspheres: (2) C2 (SA4+SCS/Ca/PMCG/SA5), (b) B5 (SA1/Ca), and (4) B2 (SA1/Ba). B indicates the bursting point at approximately 150 mN for C2 and 60 mN for B5.

the lowest molar mass. Increasing the concentration of the same alginate to 1.7 wt % (B2) creates more resistance to deformation resulting from a more dense gel network. Interestingly, Ca-beads (B5) were found to be less compressible than Ba-beads (B2) of the same size prepared from the same SA1 solution. Moreover, B5 exhibited a bursting point. All Ba-beads did not burst even when compressed to 99% deformation. If the beads were prepared from SA of higher molar mass (B3 from SA2, B4 from SA3), much higher resistance was monitored even for lower SA concentrations, 1.5 wt % for B3 and 0.9 wt % for B4 compared to 1.7 wt % for B2. Despite the lower network density, the higher degree of chain entanglement for the longer chains seems to play a role. Nevertheless, the concentration influence seems to be more significant if the beads B3 and B4 are compared. For the selection of the experimental concentrations, the polyanion solution viscosity was given priority in order to ensure similar droplets and, consequently, similar bead size. Therefore, the comparison of molar mass and concentration influences is limited to a certain extent from these studies. Comparing the capsules, C2, the C1 capsule coated with SA5, exhibits the lowest value of compression force at the bursting point. The mechanical resistance of the C1 capsule significantly decreased by SA coating. This may result from membrane shrinking during the coating process. A correlation of the membrane volume with the mechanical resistance has been reported elsewhere.23 Coating with SCS causes less shrinking, and as a consequence, the decrease of the mechanical resistance is less pronounced than for SA5 coating. Moreover, the reduction of the membrane thickness suggests the replacement of the G-alginate by the M-alginate for which a higher degree of shrinking was reported for the complex formation.18 In general, the resistance to compression has been found for beads and capsules prepared under comparable conditions in the same order of magnitude by other authors.18,31,32 However, studies under identical conditions have not been reported. Compression Work. The compression work calculated according to eq 6 for the beads and capsules at various percentages of deformation is presented in Figures 12 and 13. This parameter considers the deformation behavior in more detail than the force at maximum compression. In general, a correlation of the compression force with the compression work is expected for the same hydrogel type with similar network structure and homogeneity. This is the case if Ba-beads are compared. However, deviations are clearly seen by comparing the Ba-bead B4 with the Ca-bead B5 and the capsules C1 with C3. Even though a higher force value was recorded for B4, the compression

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Figure 12. Compression work calculated at different percentages of compression of microbeads related to their initial diameter: (O) B1, (b) B2, (0) B4, (2) B5, and (9) B3. B indicates the bursting point for B5 beads.

Figure 13. Compression work calculated at different percentages of compression of microcapsules related to their initial diameter: (b) C1, (1) C2, and (9) C3. B indicates the bursting point for all capsules.

work was lower than that calculated for B5. This can be explained by the different force curve profiles obtained in each case. As the Wb plots in Figure 12 demonstrate, the difference is even more significant for the values calculated at different deformation. Coating the C1 capsule with SCS may strongly modify the composition of the hydrogel capsule membrane by replacing alginate. The modified composition probably compensates for the decrease in membrane dimension. This results in a similar compression work for C1 and C3 at maximum compression. Conclusions AFM surface imaging clearly revealed the influence of the composition of the microspheres on the surface morphology. No significant differences were found for Babeads when the alginate molar mass and/or concentration was varied. On the contrary, the chemical composition of the microspheres had a strong impact on their surface roughness, local elasticity, and homogeneity. Ba-beads had stiffer and rougher surfaces compared to Ca-beads. The latter exhibited the lowest roughness and the highest surface homogeneity of all microspheres. Similar values of average surface roughness were obtained for SA/Ca/ PLL, demonstrating that subsequent reaction of Caalginate beads with PLL does not influence the surface physically. All capsules revealed significantly higher surface roughness. Although smoothing was possible by additional coating, the surface remained rougher than that of beads. Despite similar surface roughness, the Young’s modulus values clearly revealed differences in the local elastic properties with higher values if the Ba-beads were (31) Martinsen, A.; Skjåk-Bræk, G.; Smidsrød, O. Biotechnol. Bioeng. 1989, 33, 79-89. (32) Gaumann, A.; Laudes, M.; Jacob, B.; Pommersheim, R.; Laue, C.; Vogt, W.; Schrezenmeir, J. Biomaterials 2000, 21, 1911-1917.

Hydrogel Microspheres

Figure 14. Young’s modulus (2) and compression work (9) as a function of the microsphere types.

prepared from alginate of higher molar mass. While coating made the capsule surfaces smoother, higher capsule surface stiffness resulted from this treatment. A comparative analysis of surface and bulk mechanical properties considering the influence of the microsphere compositions and preparation conditions allows further conclusions, which are supported by the plots in Figure 14. Both the Young’s modulus of the microsphere surface and the compression work result from the characteristics of the highly swollen polyelectrolyte network as well as its structure and homogeneity. As can be seen from Figure 14, similar tendencies of the Young’s modulus and Wb are observed for the relatively homogeneous Ba-beads. A stronger influence of the alginate concentration on Wb is obvious compared to the effect that this modification generates on the local surface elastic modulus. SA molar mass is influencing both bulk and surface compressibility. The Ca-beads with a more heterogeneous gel network

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possess higher resistance to compression while having less stiff surfaces. A particular behavior of the Young’s modulus and the compression work was observed for the capsules. For C1, both characteristics are much higher compared to the beads. Coating with SA and SCS produces different effects on capsule surface and bulk mechanical properties, which can be explained by the modification of the capsule membrane composition and dimensions. The results from analyzing surface morphology, local elastic properties, and bulk mechanical properties of hydrogel microspheres are significant for the goal-directed design of such materials and are expected to have an impact on further improvement of their biocompatibility. In particular for the bicomponent alginate hydrogels, which are under investigation for various applications such as tissue engineering and immunoisolation, the modification of the surface properties without changing the chemical composition offers the potential for biocompatibility.27 For the capsules, which find application when higher mechanical stability and lower molecular cutoff are required, coating could improve this parameter. Acknowledgment. This work was supported by the Swiss National Science Foundation, Grants 200063876.00/1 and 200020-100524/1. The authors extend their gratitude to B. Strand, Norwegian University, Trondheim, for her support with NMR analyses as well as to Kuraray Medical, Inc., Japan, for supplying alginate samples. LA048389H