Platelet adhesion and activation on an amphoteric chitosan derivative bearing sulfonate groups

Colloids and Surfaces B: Biointerfaces 10 (1998) 263–271

Platelet adhesion and activation on an amphoteric chitosan derivative bearing sulfonate groups Mansoor M. Amiji * Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA Received 22 September 1997; accepted 9 December 1997

Abstract To improve blood compatibility of chitosan, a linear cationic polymer of -glucosamine, we have synthesized an amphoteric derivative containing sulfonate functional groups. Unlike chitosan which is soluble only in acidic pH (<5.0), the sulfonated derivative was soluble over a wide pH range. Elemental analysis of N-sulfofurfuryl chitosan showed 5.20% sulfur content and the degree of substitution analysis confirmed 23.4% sulfofurfuryl substitution. Widescan electron spectroscope for chemical analysis (ESCA) showed the presence of sulfur (3.30%) and sodium (6.00%) on N-sulfofurfuryl chitosan surface. High resolution S2p spectra was entirely due to the -S-0- functional moiety of the sulfonate residues. As compared with an average of more than 73.0 platelets per 25 000 mm2 on unmodified chitosan, only 4.50 platelets were present on the sulfonated chitosan derivative. The extent of platelet activation was also significantly reduced on the N-sulfofurfuryl chitosan surface. N-sulfofurfuryl chitosan, an amphoteric watersoluble derivative, does appear to possess non-thrombogenic properties and may be suitable for some blood-contacting applications. © 1998 Elsevier Science B.V. Keywords: Amphoteric derivative; Chitosan; Platelet adhesion and activation; Sulfonate groups

1. Introduction Chitosan is a linear polymer of -glucosamine obtained by alkaline -deacetylation of chitin. Chitin, the second-most abundant natural polymer, is harvested mainly from the exoskeleton of marine crustaceans such as crabs, krill, lobsters and shrimp [1]. Chitosan does possess the physical, chemical and mechanical properties for use in various medical and pharmaceutical applications [2,3]. For instance, inter- and intramolecular hydrogen bonding, similar to cellulose, imparts * Corresponding author. Tel: 617 373-3137; Fax: 617 373-8886; e-mail: [email protected] 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 00 5 - 8

excellent film and fiber-forming properties in chitosan for the development of hemodialysis membranes, artificial skin, wound dressings, physical barriers to prevent post-surgical adhesions and suture materials [4–7]. The polycationic structure of chitosan allows the formation of pH-sensitive drug delivery systems, polylelectrolyte complexes and microcapsules, bile and fatty acid binders and self-assembling nucleic acid delivery systems [8– 15]. Although chitosan does possess the necessary properties for biomedical product development, for those applications that involve blood contact, chitosan promotes thrombosis and embolization [6,16 ]. Surface-induced thrombosis on polymeric biomaterials is initiated by the adsorption of

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plasma proteins, followed by adhesion and activation of platelets [17]. Upon adhesion, platelets undergo surface-induced spreading and degranulation. Release of platelet granular content such as adenosine triphosphate, serotonin and thrombin further activate other resting platelets leading to the formation of platelet aggregates on the surface [18,19]. In addition, the platelet release reaction also triggers the activation of the coagulation reaction. The overall outcome of platelet activation on a biomaterial surface is the formation of mural thrombi consisting of platelets and other blood elements entrapped in a fibrin network [20]. The loose thrombus can easily dislodge from the surface and enter the blood stream as an emboli. Attempts have been made to improve blood compatibility of chitosan with physical blends [21,22], surface modification [6,23,24] and synthesis of blood-compatible derivatives [5,25]. We have developed a unique complexation interpenetration method for chitosan surface modification with negatively-charged modifiers such as heparin, dextran sulfate and poly(ethylene glycol ) sulfonate [6,24]. Hirano et al. [25,26 ] have prepared fatty acid and sulfated derivatives of chitosan with improved blood compatibility properties. In addition, Muzzarelli [26,27] has synthesized N-carboxymethyl, N-carboxybutyl and methyl pyrrolidinone chitosan derivatives that were biocompatible, enzymatically-degradable and soluble in aqueous medium over a wide pH range. Lee et al. [28] have prepared N-acylated derivatives of chitosan containing 20–50% acyl content. The N-hexanoyl derivative was found to be highly blood compatible and degradable with lysozyme. Recent studies have shown that ionomers containing sulfonic acid have favorable blood-contacting responses including anticoagulant [29] nonthrombogenic [30–33], reduced complement activity [34] and anti-calcification properties [35]. In the present study, we have synthesized an amphoteric derivative of chitosan by reacting the polymer with the sodium salt of 5-formyl2-furansulfonic acid to obtain the sulfonated chitosan derivative. In vitro blood compatibility of the sulfonated chitosan was evaluated by measuring the number of adherent platelets and the extent of platelet activation.

2. Materials and methods 2.1. Materials Chitosan with a degree of deacetylation of 86.7% and an average molecular weight of 750 000 daltons was purchased from Fluka Chemika/ Biochemika (Ronkonkoma, NY ). The sulfonation reagent 5-formyl-2-furansulfonic acid, sodium salt ( FFSA) and sodium borohydride were purchased from Aldrich Chemicals Company (Milwaukee, WI ). Deionized distilled water (DDW, NANOpure II, Barnsted/Thermolyne, Dubuque, IO) was used exclusively to prepare all aqueous solutions. All other reagents and chemicals were of analytical grade or better. 2.2. Synthesis of N-sulfofurfuryl chitosan As shown in Fig. 1, N-sulfofurfuryl chitosan was synthesized according to a modified method of Muzzarelli [36 ]. Chitosan was dissolved in 0.10 M acetic acid to prepare a 2.0% (w/v) solution. Onehundred ml of the chitosan solution was slowly mixed with 100 ml of dehydrated methanol containing 1.0% (w/v) triethanolamine. Upon complete mixing, chitosan precipitated due to the increase in the pH of the final solution. The chitosan slurry was stirred for at least 5.0 h at room temperature to ensure complete swelling of the polymer in the solvent system. FFSA (1.5 g) was slowly added to the chitosan slurry and the reaction between the aldehyde group of FFSA and the primary amine group of the -glucosamine residue was allowed to proceed overnight at room temperature. As the reaction continued, chitosan slowly dissolved to form a viscous solution. The Schiffs base thus formed was reduced by slow addition of 0.50 g of sodium borohydride. Nsulfofurfuryl chitosan was precipitated in methanol and washed extensively with methanol and acetone to remove unreacted FFSA. The polymer was dried in a vacuum desiccator and milled to produce fine particles. Typical yield was greater than 90%. 2.3. Bulk characterization Elemental analysis of N-sulfofurfuryl chitosan was performed at the Shwarzkopf Microanalytical

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Fig. 1. Reaction scheme for the synthesis of N-sulfofurfuryl chitosan.

Laboratory ( Woodside, NY ). The degree of substitution of the primary amine groups in chitosan was determined by using 2,4,6,trinitrobenzenesulfonic acid ( TNBS ) assay as described by Snyder and Sobocinski [37]. Briefly, 5.0 ml of 0.25% (w/v) solution of unmodified chitosan or N-sulfofurfuryl chitosan, prepared in 0.05 M acetic acid-sodium acetate buffer (pH 5.0) was mixed with 250 ml of 0.05 M TNBS reagent. The reaction was allowed to proceed for 1.0 h at room temperature. The absorbance of the conjugate at 420 nm was mea-

sured against a blank prepared with 5.0 ml of buffer and TNBS solution. The degree of substitution of N-sulfofurfuryl chitosan was determined from the absorbance values and compared with those of unmodified chitosan. 2.4. Surface characterization Electron spectroscopy for chemical analysis ( ESCA) is a surface analytical technique that measures the elemental composition and identifies

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the chemical functional groups on the surface at ˚ thick layer. Clean glass slide, unmodiup to 100 A fied chitosan-coated glass and N-sulfofurfuryl chitosan-coated glass were analyzed by ESCA to measure the surface elemental composition and the identity of the chemical functional groups. The ESCA experiments were carried out at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) at the University of Washington, (Seattle, WA). Analysis was performed using an XProbe ESCA instrument (Surface Science Instruments, Mountain View, CA) equipped with an aluminum Ka monochro1,2 matized X-ray source. An electron flood gun set at 5.0 eV was used to minimize surface charging. Surface elemental composition was determined using the standard Scofield photoemission crosssections [38]. The identities of chemical functional groups were obtained from the high resolution peak analysis of carbon-1s (C1s), oxygen-1s (O1s), nitrogen-1s (N1s) and sulfur-2p (S2p) envelopes.

tion for 1.50 h. Stained platelets were observed using a Nikon LabophotA II (Melville, NY ) light microscope at 40X magnification. The images of adherent platelets were transferred to a Sony TrinitronA video display monitor using a Hamamatsu CCDA camera (Hamamatsu City, Japan). The Hamamatsu Argus-10A image processor was used to calculate the number of platelets per 25 000 mm2 surface area in every field of observation. The extent of platelet activation was determined qualitatively from the spreading behavior of adherent platelets. The morphology of surface-adherent platelets was observed with a scanning electron microscope (SEM ). SEM analysis was performed as previously described [39] with an AMR-1000 (Amray Instruments, Bedford, MA) scanning electron microscope at an accelerating voltage of 10 kV.

3. Results and discussion 2.5. Platelet adhesion and activation studies 3.1. Synthesis of an amphoteric chitosan derivative The number of adherent platelets and the extent of activation on control and N-sulfofurfuryl chitosan surfaces was determined as previously described [6,24]. A platelet observation chamber was assembled consisting of clean glass slide, unmodified chitosan-coated slide, or chitosan sulfonate-coated slide, two polyethylene spacers, and a glass coverslip. Human blood, obtained from healthy volunteers after informed consent, was collected in heparin-containing evacuated containers ( VacutainersA, Becton–Dickinson, Rutherford, NJ ). Heparinized blood was centrifuged at 100 g for 10 min to obtain platelet-rich plasma (PRP). PRP (200 ml ) was instilled into the platelet observation chamber. Platelets in PRP were allowed to adhere and activate on the control and N-sulfofurfuryl chitosan surfaces for 1.0 h at room temperature. Non-adherent platelets and plasma proteins were removed by washing the chamber with phosphate-buffered saline (PBS, pH 7.4). Adherent platelets were fixed with 2.0% (w/v) glutaraldehyde in PBS for 1.0 h. After washing with PBS, the platelets were stained with 0.10% (w/v) Coomassie Briallian Blue (Bio-Rad, Hercules, CA) dye solu-

N-sulfofurfuryl chitosan was synthesized by a mild reaction via a Schiff ’s base intermediate between chitosan and FSSA, as shown in Fig. 1, to improve blood compatibility properties of chitosan. Table 1 shows the data from elemental analysis of the parent polymer and the amphoteric derivative. Analysis of the unmodified chitosan showed 40.3% carbon (C ), 44.3% oxygen (O), 7.1% hydrogen (H ) and 7.5% nitrogen (N ). The elemental composition was almost identical to the theoretical values predicted for -glucosamine of 40.2% C, 44.7% 0, 7.3% H and 7.8% N. The slight difference in the H and N composition could be attributed to the random distribution of the acetyl-glucosamine residues in the chitosan chain. Elemental analysis of chitosan sulfonate showed a distinctive sulfur (S) signal of 5.2%. The degree of substitution, as measured by the TNBS assay, showed that about 23.4% of the -glucosamine residues of chitosan were converted into the sulfofurfuryl derivative. Unlike chitosan which is soluble only in acidic medium (pH<5.0), N-sulfofurfuryl chitosan was found to be soluble in aque-

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M.M. Amiji / Colloids Surfaces B: Biointerfaces 10 (1998) 263–271 Table 1 Characteristics of chitosan and sulfonated chitosan Chitosan

Sulfonated chitosan

Elemental analysis (%)a Carbon Oxygen Hydrogen Nitrogen Sulfur

40.3 44.3 7.1 7.5 —

36.5 47.9 5.6 4.8 5.2

Degree of substitution (%)b Acetylation Free amine N-sulfofurfuryl

13.3 86.7 —

13.3 63.3 23.4

aElemental analysis of chitosan (Mw 750 000 Da) and sulfonated chitosan was performed at the Schwarzkopf Microanalytical Laboratory ( Woodside, NY ). bThe degree of substitution in sulfonated chitosan was determined using 2,4,6-trinitrobenzenesulfonic acid ( TNBS) assay.

ous medium over a range of pH values from 2.0 to 12.0. 3.2. Surface analysis Since the interactions between blood and polymer surfaces are dictated by the solid–liquid interfacial properties, ESCA was used as an important analytical technique to determine the surface composition and chemistry of the amphoteric chitosan derivative. The surface elemental composition of clean glass showed a characteristic C, O, sodium (Na) and silicone (Si) peaks as shown in Table 2. The atomic composition of 16.2% C, 50.4% 0, 5.90% Na and 23.5% Si on clean glass are consistent with the values that have been reported earlier (6,24,40). The chitosan coated glass slide had

66.0% C, 27.3% O and 3.6% N. There were no Na or Si peaks on the chitosan-coated which suggests that the coating was evenly distributed across the entire glass surface. In addition to a decrease in the composition of C (52.4%) and N (4.60%) on N-sulfofurfuryl chitosan as compared with unmodified chitosan, there was a characteristic S (3.30%) and Na (6.00%) signals due to the exposure of the sulfonate residues on the polymer surface. ESCA data are also rich with information on the surface chemistry of the control and modified chitosan. Table 3 shows the high resolution C1s, O1s, N1s and S2p peaks which were used to determine the chemical identity of the functional groups that were associated with these elements. The peak at the binding energy of 285.0 eV, for instance, is associated with the -C-H- (or hydrocarbon) component of the C1s envelope on the surface. On clean glass, 75.6% of the carbon was associated with -C-H-, 7.40% with -C-O- (or ether) and 17.0% with -CNO- (or carboxyl ) functional groups. The presence of a -CNO- peak of carbon and the -ONC- peak of oxygen are typically due to the unavoidable environmental contamination as a result of adsorption of carbon dioxide from air [6,24].A high resolution scan of the carbon envelope on chitosan-coated glass resolved the hydrocarbon peak (47.9%), ether (39.5%) and the carboxyl (10.5%) peaks. The presence of the carboxyl peak could be due to preferential orientation of the acetyl--glucosamine residues on the surface in the dry state. The O1s envelope on chitosancoated glass was associated with two peaks corresponding to -O-C- and -ONC–species at 85.7% and 14.3%, respectively. The N1s envelope was fitted to only one peak at 399.6 eV binding energy

Table 2 Surface elemental composition on control and sulfonated chitosana Surface type

Clean glass Chitosan-coated glass Sulfonated chitosan-coated glass

Percent atomic composition C

O

N

S

Na

Si

16.2 66.0 52.4

50.4 27.3 33.6

— 6.70 4.60

— — 3.30

5.90 — 6.00

23.5 — —

aSurface elemental composition was obtained from electron spectroscopy for chemical analysis ( ESCA) survey scans. ESCA was performed at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO), University of Washington (Seattle, WA).

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Table 3 High-resolution peaks of ESCA on control and sulfonated chitosoana Surface type

Relative peak intensity

Clean glass Chitosan-coated glass Sulfonated chitosan-coated glass

-C-H (285.0 eV )

-C-O(286.5 eV )

-CNO(288.0 eV )

-O-C(533.0 eV )

-ONC(531.5 eV )

-N-C(399.6 eV )

-S-O(168.3 eV )

75.6 47.9 29.7

7.40 39.5 56.7

17.0 10.5 13.7

79.2 85.7 62.7

20.8 14.3 30.8

– 100 100

– – 100

aHigh-resolution peak analysis was peformed at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) at the University of Washington (Seattle, WA). Table 4 Evaluation of platelet activation from surface-induced spreading Platelet activation stagea

Approximate spread area mm2

Remarks

1 2 3 4

10–15 15–25 25–35 35–45

5

>45

Contact-adherent stage. Platelets adhere but retain their round or discoid shape Partially-activated stage. Initiation of pseudopod formation and extension Partially-activated stage. Extension of pseudopods and initiation of the release reaction Partially-activated stage. Significant extension of pseudopods. Complete release of the granular contents Fully-activated stage. Retraction of pseudopods leading to flat or ‘‘pancake’’ shape

aThe different stages of platelet activation based on surface-induced spreading behavior as reported by Lin, et al [30].

which corresponds with the -N-C- functionality. On N-sulfofurfuryl chitosan surface, there was a marked reduction in the -C-H- peak intensity (29.7%) and a corresponding increase in the -C-O(56.7%) and -CNO- (13.7%) signals as compared with unmodified chitosan. The increase in ether and carboxyl peaks was most likely due to the furan ring structure in the sulfofurfuryl residues on chitosan sulfonate. The S2p envelop on sulfonated chitosan surface was entirely due to the -SO-functionality. The results of ESCA on chitosan sulfonate show a distintictive sulfur signal corresponding to the sulfonate groups on the polymer surface. The accessibility of sulfonate groups to the liquid–polymer interface during blood contact is essential for non-thrombogenicity of the sulfonated chitosan derivative. 3.3. Platelet adhesion and activation The extent of platelet adhesion and surfaceinduced activation is considered to be an early indicator of the thrombogenic potential of blood-

contacting biomaterials [41,42]. The extent of platelet adhesion was determined by counting the number of platelets per 25 000 mm2 surface area. Surface-induced platelet activation was measured qualitatively from the spreading behaviour of adherent platelets as shown in Table 4. When in contact with polymeric surfaces, platelets initially retain their discoid shape present in the resting state and the spread area is typically between 10–15 mm2. Upon activation, platelets extend their pseudopods and initiate the release of granular contents. During the partial activation stage, the area of the spread platelet can increase to about 35 mm. When the platelets are fully-activated they retract the pseudopods to form circular or ‘‘pancake’’ shape and the spread area increases to 45 or 50 mm2 [39,42]. The spreading profiles of activated platelets were used to create five activation stages as described by Lin et al. [30] Clean glass, as shown in previous studies [6,24,40], does promote platelet adhesion and activation. As shown in Table 5, an average of almost 148 adherent platelets were present per 25 000 mm2 on the glass surface. The degree of

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M.M. Amiji / Colloids Surfaces B: Biointerfaces 10 (1998) 263–271 Table 5 Number of adherent platelets and the extent of platelet activation on control and sulfonated chitosana Surface type

Number of platelets/25 000 mm2

Extent of activation

Clean glass Chitosan-coated glass Sulfonated chitosan-coated glass

147.8±35.2b 73.3±9.70 4.50±1.31

4.50±0.22 3.20±0.46 1.20±0.19

aPlatelets in platelet-rich plasma were allowed to adhere and activate on the control and sulfonated chitosan surfaces for 1 h at room temperature. bMean±S.D. (n=24).

activation of 4.50 was also very high to indicate that the platelets were fully-activated. Chitosan also is highly thrombogenic and allowed on the average 73 platelets to adhere per 25 000 mm2. The degree of platelet activation on chitosan-coated glass of 3.20 was significantly less than that on control glass. Due to the hydrophilic nature of the surface, chitosan does not promote complete platelet activation. On N-sulfofurfuryl-modified chitosan, there were only 4.50 adherent platelets per 25 000 mm2. Furthermore, all of the adherent platelets had retained their discoid shape that is present in the resting platelets. Adherent platelet morphology is also evident from the SEM micrographs as shown in Fig. 2. On unmodified chitosan, several platelets had adhered and residues in N-sulfofurfuryl chitosan, partially activated. The presence of sulfonate on the other hand, prevented platelet adhesion and activation completely. Okkema et al. [43] suggested that reduced platelet adhesion and activation on the sulfonated polymer could be either due to enhanced platelet consumption or tight binding of fibrinogen in a conformation that does not favor interactions with platelet membrane glycoprotein receptors. Santerre et al. [44] observed that fibrinogen adsorbed on sulfonated polyurethanes could not be readily displaced by other plasma proteins.

4. Conclusions As a material obtained from the exoskeleton of crustaceans, chitin and chitosan present an enormous opportunity for commercialization in the development of medical and pharmaceutical pro-

(a)

(b) Fig. 2. Scanning electron micrographs of platelets adhered and activated on unmodified chitosan (a) and N-sulfofurfuryl chitosan (b). Platelets in platelet-rich plasma were allowed to adhere and activate for 1.0 h at room temperature. The scale bar is equal to 10 mm.

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ducts. For those application that involve blood contact, however, chitosan promotes thrombosis and embolization. In this study, we have synthesized N-sulfofurfuryl chitosan, an amphoteric derivative, containing sulfonate groups to impart non-thrombogenic properties. N-sulfofurfuryl chitosan was soluble in aqueous medium over the pH range of 2.0–12.0. Elemental analysis and degree of substitution studies confirmed the presence of sulfofurfuryl residues in the derivative. Since the interactions with blood are dictated by the surface of the polymer, we used ESCA to determine the surface elemental composition and chemical functionality. ESCA’s survery scan showed a characteristic S (3.30%) and Na (6.00%) signal in the modified chitosan to indicate that the sulfonate groups were excessible to the surface. High resolution C1s analysis showed an increase in the ether (-C-O-) signal probably due to the furan ring structure. The number of adherent platelets and the extent of platelet activation was significantly reduced on N-sulfofurfuryl chitosan as compared with unmodified chitosan. In contrast to an average of 73 fully-activated platelets on unmodified chitosan, only 4.50 contact-adherent platelets were present on N-sulfofurfuryl chitosan surface per 25 000 mm2. The results of this study show that N-sulfofurfuryl chitosan, an amphoteric derivative with sulfonic acid residues, can be synthesized to impart aqueous solubility in the polymer over a wide pH range and significantly lower the extent of platelet adhesion and activation.

Acknowledgment This study was supported in part by the Research and Scholarship Development Grant administered by Northeastern University. The author is extremely grateful for the assistance provided by Ms Roula Qaqish and Ms Fiona Duncan.

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