- Email: [email protected]
Heparin-loaded zein microsphere film and hemocompatibility
Journal of Controlled Release 105 (2005) 120 – 131 www.elsevier.com/locate/jconrel
Heparin-loaded zein microsphere film and hemocompatibility Hua-Jie Wanga, Zhi-Xin Lina, Xin-Ming Liub, Shi-Yan Shenga, Jin-Ye Wanga,b,T a
School of Life Science and Biotechnology, Shanghai Jiaotong University, 1954 Huashan Road, Shanghai 200030, China b Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China Received 5 June 2004; accepted 25 March 2005 Available online 12 May 2005
Abstract Zein was studied as a drug-eluting coating film composed of zein microspheres for cardiovascular devices (e.g. stent). In vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis showed that both zein film and its degraded product had better biocompatibility compared with Corning culture plate on the growth of human umbilical veins endothelial cells (HUVECs, p b 0.05, n = 6), and the effect of zein degraded product on HUVECs was dose-dependent. The best result was obtained at 0.3 mg/ml of the addition. The encapsulation efficiency of heparin and heparin loading varied with the amount of both zein and heparin, and the highest encapsulation efficiency (heparin 1.33 mg/ml and zein 16 mg/ml) was 22.77 F 1.33% (n = 3). Scanning electron microscope (SEM) observation indicated that the zein film was made of microspheres in diameter from nano- to micrometer, which could be controlled. Sizes of heparin-loaded zein microspheres changed before and after release of heparin because of conglutination among zein microspheres. Release rate of heparin from microsphere film reached to 33.5 F 1.2% within 12 h, and began to get into subsequent bslow releaseQ phase; about 55% of the entrapped heparin was released after 20 days. Both zein film and heparin-loaded zein microsphere film were effective in suppressing platelet adhesion, and the heparin-loaded film showed a better anticoagulation as determined with thrombin time (TT) assay. These results suggest that zein film could be used directly as a new type of coating material for its better biocompatibility with HUVECs. Moreover, the heparin-loaded zein microsphere film can significantly improve the hemocompatibility. D 2005 Elsevier B.V. All rights reserved. Keywords: Film; Hemocompatibility; Microspheres; Platelet adhesion; Zein
Abbreviations: HUVECs, human umbilical veins endothelial cells; ECGS, endothelial cells growth supplement; MTT, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SEM, scanning electron microscope; TT, thrombin time; PBS, phosphate-buffered solution; FCS, fetal calf serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T Corresponding author. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China. Tel.: +86 21 54925330; fax: +86 21 64376697. E-mail address: [email protected] (J.-Y. Wang). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.03.014
1. Introduction The major drawback of angioplasty is restenosis, which occurs in 30–60% of treated vessels, resulting in the need for repeat procedures [1]. As a cardiovascular device, the potential of stenting technique to reduce the morbidity of restenosis and cost of care is enormous; however, the rate of in-
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
stent restenosis still gets to 10–40% due to unsatisfactory biocompatibility of stents [2]. Up to date, there have been four methods used to solve this problem. The first method is to administer anticoagulant to the patient through injection or oral administration for days, which successfully decreases the rate of the thrombus formation, while the rate of massive hemorrhage is increased. And when the anticoagulant is taken orally, it would be denatured in the digestive tract if the suitable carrier were unavailable [3]. The second method is to modify the surface of the stents with polymers, such as the use of phospholipids [4], which could improve the biocompatibility, especially the blood compatibility. The third method is to coat anticoagulant on the surface of the stents [5], but the concentration of the anticoagulant is difficult to be maintained because of the effect of the blood flow. The fourth method is to seed endothelial cells on the surface of the stents with autogenous endothelium, which could avoid contacting the blood with the material, and therefore suppresses the activation of coagulation factors [6]. Among these methods, drug-eluting stents are called brevolutionQ in stenting technique, while coated stents have showed huge promise and great efforts have been made to develop the suitable drug and coating materials of stents, which can maintain the drug concentrations at the site of stents deployment and minimize systemic side effects [7]. These drugs used contain heparin, dexamethasone, sirolimus, everolimus, tacrolimus, carvedilol, paclitaxel, actinomycin-D, flavopiridol, angiopeptin and so on [8,9]. These drugs are often blended with synthetic polymers that act as drug reservoirs to elute the active agent over a period of several weeks or months [10]. Unfortunately, many of the synthetic polymers induce an exaggerated inflammatory response and neointimal hyperplasia in animal models [11,12]. It is noted that most of them are nonbiodegradable, and have to be taken out by surgery, which give patients great discomfort. Therefore, the research focuses on searching a slow eluting material with better biocompatibility and biodegradability. The anticoagulant is encapsulated by or combined with the carrier; the compound of drug and carrier can deliver the drug at a dose low enough to avoid systemic effect but high enough to provide local protection against vascular disease [13].
121
Zein is the major storage protein of corn and accounts for 40–50% of the protein; it is soluble in aqueous alcohol solutions (60–95%). Zein has been used widely, such as adhesive, biodegradable plastics, chewing gum, coating for food products, fiber, cosmetic powder, microencapsulated pesticides and inks. Especially zein has been prepared as microspheres to deliver insulin [14,15]. Our previous work has indicated that zein film, composed of particles in diameter of 100–2500 nm, has good biocompatibilities with both human liver cells and mice fibroblast cells [16]. In present study, zein was considered as a new type of coating material either directly or as a heparin carrier applying for cardiovascular devices such as stent. The biocompatibility of zein film and its degraded product digested with pepsin on human umbilical veins endothelial cells (HUVECs) was studied, the methods of preparing zein microspheres and heparin-loaded microsphere film were investigated, the encapsulation efficiency and release kinetics of heparin-loaded zein microspheres, the morphology of the films, and hemocompatibility (anticoagulation, protein adsorption and platelet adhesion) were tested.
2. Materials and methods 2.1. Materials Zein with biochemical purity was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), endothelial cells growth supplement (ECGS) was purchased from Upstate Biotechnologies Inc. (Lake Placid, NY, USA), heparin and thrombin were from Sigma (St. Louis, MO, USA), and other reagents were reagent grade. 2.2. Preparation of zein film and its degraded product Zein was dissolved in aqueous mixture of ethanol, and the ethanol content was brought to 40% immediately to form zein microspheres suspended solution, then the film was prepared through volatilization at 37 8C. The film was sterilized by UV for at least 1 h and immersed into the RPMI 1640 nutrient fluid before use. Zein degraded product was obtained from zein powder digested with pepsin (zein/pepsin = 10:1 w/w)
122
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
in citric acid–NaH2PO4 (pH 2.2) at 37 8C. The supernatant was freeze-dried to get the degraded powder for use. 2.3. Adhesion and proliferation assays of HUVECs HUVECs were harvested from human umbilical veins as described [17] and cells at passage 3 were digested by incubation with 0.025% trypsin/0.002% EDTA, resuspended with RPMI 1640 medium (20% FBS, 100 units/ml penicillin, 100 Ag/ml streptomycin, 30 Ag/ml ECGS and 90 Ag/ml heparin) and seeded at 2.4 104 cells/ml onto 96-well Corning culture microplate coated with zein film; other groups were 2 mg/ml zein degraded product adding group and Corning culture microplate control group. After 2 h, the culture medium in each well was taken out and the new medium (without or with zein degraded product) was added. The medium was refreshed every 2 days. HUVECs were photographed with inverted optical microscope everyday. At 4 h after being seeded, the non-adhered cells in the medium were counted with a hemocytometer to evaluate the adhesion of HUVECs under different conditions. 3-(4,5-Dimethylthiazol -2yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used to assess the proliferation of HUVECs after 2 h, 1 days, 3 days and 5 days of culture. The attached cells on different substrates were fixed with 2.5% glutaraldehyde in PBS for at least 2 h, washed with Milli-Q water. The samples were mounted on stubs and coated in vacuum with gold. Then cells were examined with a scanning electron microscope (SEM S-450, Hitachi, Japan).
remove unentrapped heparin. The encapsulation efficiency was determined using the method described by Smith [18]. Heparin-loaded microsphere suspended solution was volatilized at room temperature to form heparin-loaded zein microsphere film. SEM was used to observe the surface and internal morphology of the film after vacuum-coated with a gold layer. Heparinloaded zein microsphere film was immersed into 1 ml PBS (pH 7.4) and incubated at 37 8C. In vitro, the releasing medium of each sample was removed and replaced with fresh PBS at a given time interval. The heparin content in the medium was analyzed using the method described by Smith, and the releasing rate of heparin from heparin-loaded zein film was calculated. 2.5. Anticoagulation of zein film and heparin-loaded zein microsphere film [19] This test was divided into four groups: zein film group, Corning culture plate group, physiological saline group and heparin-loaded zein microsphere film group. Samples of the first and the last group were composed of microspheres. Thrombin, anticoagulant plasma from healthy rabbit and calcium chloride solution were prewarmed at 37 8C. Then 0.1 ml plasma, 0.1 ml of 0.85% (w/v) calcium chloride solution and thrombin (100 units per ml) were added into the sample well, and began to record the time until cruor. In order to assess the activity of heparin released from zein microsphere film, we also performed this test at the given time during the releasing process of heparin with thrombin time (TT) assay.
2.4. Heparin-loaded zein microspheres and film
2.6. Plasma protein adsorption and platelet adhesion [5]
The heparin-loaded microspheres were obtained based on the solubility of zein and heparin. Heparin and zein were dissolved in the aqueous mixture of ethanol, respectively; after mixed sufficiently, the ethanol content was brought to 40% immediately to form microspheres suspended solution. Then the suspended solution was rapidly frozen with dry ice and acetone, vacuum-dried overnight and the powder was stored at 4 8C before use. The vacuum-dried microspheres powder was resuspended in Milli-Q water (PALL, USA) and the heparin-loaded zein microspheres were separated by chromatography to
Fresh healthy human blood was provided by Shanghai Blood Center with approval. The blood was centrifuged at 1000 rpm for 10 min at 4 8C to get platelet-rich plasma (PRP), and at 3000 rpm for 10 min at 4 8C to get platelet-poor plasma (PPP). The fresh PRP or PPP samples were used in this study. Zein film and heparin-loaded zein microsphere film were sufficiently rinsed with Milli-Q water, and immersed into platelet-poor plasma, which was placed at 37 8C for 3 h. After being washed three times, proteins adsorbed on the zein film and heparin-loaded zein microsphere film were removed
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
with 1 wt% aqueous solution of sodium dodecyl sulfate (SDS), and freeze-dried. The protein samples were redissolved in Milli-Q water, applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For evaluation of platelet adhesion, zein film and heparin-loaded zein film were placed in contact with 50 Al of human PRP and left at 37 8C for 60–180 min, the number of platelets in the PRP was diluted with PBS to 5 106 cells. The PRP was removed and the film was rinsed three times with PBS [20]. The adhered platelets were fixed by immersing the film into 2.5% solution of glutaraldehyde in PBS for at least 2 h at 4 8C. Samples were freeze-dried, then sputter-coated with gold for SEM observation. The number of platelets adhered was determined with colorimetric method [21]. Briefly, 150 Al of 4-nitropheny-phosphate disodium salt was added in each well and placed at 37 8C for 60 min. The absorption was determined at the wavelength of 405 nm after stopping reaction with 100 Al of 2 N NaOH. 2.7. Statistical analysis All the data were analyzed by one-way factorial ANOVA and multiple comparisons. Significant effects of treatment were defined using Scheffe’s statistic method.
123
In order to evaluate the attachment, spreading, and proliferation changes of HUVECs on the surface treated with zein film and its degraded products, we analyzed the proliferation of cells in 2 h, 1 day, 3 days and 5 days with MTT method (Fig. 1). Mitochondrial redox activity of HUVECs can be evaluated by reduction of MTT to formazan by mitochondrial succinate dehydrogenase in complex II (succinate: ubiquinone oxidoreductase complex), which plays a central role in both oxidative phosphorylation and the tricarboxylic acid cycle. HUVECs attached and adhered onto zein film and the glass plate completely after 1 h culture, the percentage of attached cells was more than 90% in each group and no significant difference was observed. At the third day of culture, an enhancement of mitochondrial activity from cells on zein film was observed compared with cells on Corning culture microplate, but the cells treated with zein degraded product did not show significant difference with the control group according to the result of MTT assay. Significantly higher proliferations were obtained for both zein film and its degraded product adding groups after 5 days of culture. There was no significant difference between zein film group and zein degraded product adding group. We deduced that at earlier state of the cell proliferation, substratum topography on which cells adhered might be, to some extent, important [22]. In summary, the adhesion of cells was not affected by zein degraded product, while
3. Results and discussion 0.35
3.1. Cell adhesion and proliferation assays
0.3
OD490nm
Stent coatings can be divided into two categories, biocompatible materials and drug-eluting coatings. Biocompatible materials currently under investigation are thought to be less thrombogenic and inflammatory, and are thereby potentially able to reduce neointimal hyperplasia. Metallic stents exert a continuous radial pressure on the diseased artery, resulting in compression of atherosclerotic plaques, sealing of dissections, and expansion of the vessel. Improving the surface properties of stents, by applying a biocompatible coating aimed at improving the vascular healing response in an attempt to prevent early thrombosis and late neointimal proliferation, is more logical approach.
**
*
0.25 0.2 0.15 0.1 0.05
0
1
2
3
4
5
6
Time (days) Fig. 1. The proliferation assay of HUVECs on zein film (D), glass control (w), or treated with zein degraded product (5, 2 mg/ml) after being cultured for 5 days. *Represents a statistical significance between zein film group and the control group; **represents statistical significances compared both zein film group and zein degraded product group with the control group ( p b 0.05, n = 6).
124
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
some structures in zein might play an important role on the proliferation of cells at later stage. As shown in Fig. 2, attachment, adhesion and spreading occurred in the first phase of cell/material
interactions, and there were some differences between these groups. Cells cultured in each group maintained their characteristic polygonal morphology; the spreading cells maintained physical contact with each other
Fig. 2. Morphology comparison of HUVECs cultured for 3 days on the glass control (A1, A2), zein film (B1, B2), and treated with zein degraded product (C1, C2, 2 mg/ml) by SEM observation. Bars in left and right columns represent 29 and 9.9 Am, respectively.
OD590nm
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2
*
125
through filopodia or lamellopodia. The morphologies of cells varied greatly on different matrixes, especially on zein film after 3 days of culture. The cytoplasm of HUVECs on zein film was abundant and lacked of thin pinna.
* * *
3.2. Effect of zein degraded product on HUVECs 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Sample Concentration (mg/ml) Fig. 3. Effects of zein degraded product on HUVECs proliferation with MTT analysis after being cultured for 2 days ( p b 0.05, n = 6). *Represents significant difference compared with the control (containing 0 mg/ml of zein degraded product).
After being cultured for 2 days with different concentrations of zein degraded product, MTT showed that the growth of HUVECs cultured with zein degraded product was significantly better than the control ( p b 0.05, n = 6). A lower content presented a higher stimulating effect (Fig. 3) and the effect of zein degraded product on HUVECs was significantly dose-dependent. Fig. 4 shows the mor-
Fig. 4. Effect of zein degraded product on HUVECs. Morphology and contribution of HUVECs on Corning microplate after being cultured with different concentrations of zein degraded product for 2 days were observed by an optical microscope (32 magnification). a: 0 mg/ml (the control group); b: 0.2 mg/ml; c: 0.3 mg/ml; d: 0.5 mg/ml; e: 1 mg/ml; f: 2 mg/ml.
126
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
phology and distribution of HUVECs treated with different concentration of zein degraded product after 2 days of culture with an optical microscope, which also showed more cells in the group treating with lower content of the product and a higher concentration of zein degraded product resulted in diminution of HUVECs to some extent (1 mg/ml), while a round shape was observed when 2 mg/ml of zein degraded product was added. 3.3. Preparation and characterization of heparinloaded protein microspheres Before being processed, feed of zein is the unregulated particle. We utilized the phase separation method based on the solubility of zein to prepare protein microspheres. When the content of zein in 40% of alcohol aqueous solution is 8 mg/ml, we got microspheres in diameter of 40–100 nm. Local drug delivery through the use of drug-eluting stents allows the controlled release of a high concentration of drug at the site of diseased artery without excess systemic effects. In order to develop the function of zein as a heparin carrier, we prepared heparin-loaded zein microspheres through phase separation technique. The encapsulation efficiency of heparin was determined through the method described by Smith. As can be seen in Table 1, encapsulation efficiency and heparin loading depended on the concentration and the ratio of zein to heparin. Both encapsulation efficiency and heparin loading increased with the increase of heparin and zein concentrations at a given ratio of them (except for heparin loading at heparin 5.33 mg/ml and zein 16 mg/ml); both the encapsulation efficiency of heparin and heparin loading increased with the increase of the ratio of heparin to zein (zein 8 mg/ml). Satisfactory result was obtained when the concentration of heparin and zein was 1.33 mg/ml and 16 mg/ml, respectively, in which the encapsulation efficiency of heparin was the highest. The result was in accordance with that we have got previously, but the values were far lower compared with those of a hydrophobic drug, ivermection [23]. According to the SEM result of our previous study, the size of heparin-loaded zein microspheres varied from nano- to micrometer with the increase of zein concentration. So we can say that heparin-loaded per
Table 1 Encapsulation efficiency of heparin and heparin loading in zein microspheres (n = 3) Microspheres preparation (concentration of heparin and zein) Heparin 1.33 mg/ml, zein 4 mg/ml Heparin 1.33 mg/ml, zein 8 mg/ml Heparin 1.33 mg/ml, zein 12 mg/ml Heparin 1.33 mg/ml, zein 16 mg/ml Heparin 2.67 mg/ml, zein 8 mg/ml Heparin 4 mg/ml, zein 8 mg/ml Heparin 5.33 mg/ml, zein 8 mg/ml Heparin 4 mg/ml, zein 12 mg/ml Heparin 5.33 mg/ml, zein 16 mg/ml
Encapsulation efficiency of heparin (w/w %)a
Heparin loading (w/w %)b
1.97 F 0.77
0.34 F 0.17
8.74 F 0.98
0.98 F 0.11
11.94 F 0.85
0.89 F 0.06
22.77 F 1.33
1.28 F 0.07
11.94 F 0.56
1.35 F 0.06
15.38 F 1.11
3.46 F 0.25
16.37 F 0.93
3.67 F 0.21
16.86 F 0.74
2.53 F 0.11
19.56 F 1.30
2.20 F 0.15
a Encapsulation efficiency (w/w %) = Amount of heparin in microspheres/Heparin initially added. b Heparin loading (w/w %) = Amount of heparin in microspheres/ Amount of microspheres.
zein microsphere is different because of the different microsphere sizes. By phase separation technique, we got heparinloaded zein microspheres and the film was prepared from the microspheres. The film thickness was 26.3 Am (Fig. 5a), made from microspheres as shown in Fig. 5b. The size of microspheres was 1–2 Am (Fig. 5c), and some conglutination was observed from the facade. Characteristics of heparin-loaded zein microsphere film before and after release were also observed by SEM randomly. From the facade of the film, sizes of microspheres increased with immersing time of the film in PBS, adjacent microspheres fused with each other (Fig. 5d–f). However, the profile of the film showed that few of the microspheres fused and their size was rather constant (Fig. 5g–i). Fig. 6 shows the biphasic release characteristic of the film. At 12 h, the release rate of heparin reached to 33.5 F 1.2%, which was defined as a dburst effectT (phase 1). Following the phase 1, the release appeared as a sustained release (phase 2). After 20 days, the release rate tended to tail off in the subsequent
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
127
Fig. 5. Characteristics of heparin-loaded zein microsphere film (the profile a: bar = 14 Am; b: bar = 5 Am; the fac¸ade c: bar = 1.9 Am), and morphology of heparin-loaded zein microsphere films sampling randomly before and after release (the fac¸ade c: 0 day, bar = 1.9 Am; d: 1 day, bar = 7.5 Am; e: 3 days, bar = 7.4 Am; f: 6 days, bar = 7.5 Am. The profile g: 1 day, bar = 4.8 Am; h: 3 days, bar = 4.9 Am; i: 6 days, bar = 7.2 Am).
dslowerT phase and about 54.3 F 3.9% of heparin in the film was released at last. Due to the hydrophobic property of zein, it could not be degraded in PBS solution, and our previous study also showed that zein aggregated easily in water solution. So we deduced that the dburst release (phase 1)T attributed to the drug existing at or near the surface of the film. The process of the heparin release formed some micro-channels between zein microspheres, to permit the release of the heparin entrapped into
microspheres in phase 2. The slower releasing rate was probably due to the longer diffusing route of deeper heparin. Existence of repellency between heparin molecules on the surface of zein microspheres may be the reason that zein microspheres could not fuse with each other and still maintain the size as shown in Fig. 5g–i. However, with the disappearance of heparin on the surface, microspheres began to aggregate as observed from the fac¸ade of the film (Fig. 5d–f). The micro-channels also began to
128
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 Table 3 The anticoagulant activity of heparin-loaded zein microsphere film during release of heparin (n = 3)
45
30
15
0
0
5
10
15
20
25
Time (days) Fig. 6. Release kinetics curve of heparin-loaded zein microsphere film in PBS (pH 7.4, 37 8C, n = 3).
disappear. As there was no digestive enzyme existed in the present system, the heparin entrapped into microspheres could not continue to be released by diffusion into the medium after 20 days. So we considered that the release kinetics of heparin–zein microspheres was characterized as no erosionincomplete release. 3.4. Hemocompatibility of zein film and heparinloaded microsphere film The anticoagulation was studied by TT assay. There were no significant differences between the control group, physiological saline group and zein film group as shown in Table 2; the TT time was controlled within 30 s approximately. However, heparin-loaded zein microsphere film group showed the significant anticoagulation and the plasma kept incoagulable all the time. This result indicated that zein film itself did not prevent acute or subacute thrombosis if zein film was coated directly on cardiovascular devices such as stent. However, for heparin-loaded zein microsphere film, which was obtained at room temperature, heparin still kept high Table 2 Anticoagulation of zein film and heparin-loaded microsphere film (n = 3) Groups
TT (s), average F S.D.
Control Physiological saline Zein film Heparin-loaded microsphere film
30.3 F 5.1 29.3 F 5.5 29.3 F 3.2 IncoagulableT
T Significant difference compared with the control ( p b 0.01).
Time of heparin release
Time of formation of fibrin clot
Time of clotting of plasma
Significant difference
Control 2h 6h 12 h 24 h 48 h 96 h 240 h
– – – 7.3 F 1.2 min 2.7 F 0.7 min – – –
20 F 2.5 s Incoagulable Incoagulable 12 F 1 h 37 F 12 min 42 F 11 s 48 F 4.6 s 54 F 13.3 s
– ** ** ** ** * * *
*Statistical significance compared with the control at p b 0.05. **Statistical significance compared with the control at p b 0.005.
activity. We could control the concentration of heparin entrapped into the film by adjusting the preparing process of microspheres or the thickness of the film, to prolong the action of heparin in body. For the given heparin-loaded zein microsphere film in this study, we also designed a simplified test to determine anticoagulating effect of heparin-loaded zein microsphere film during the release process of heparin. Cruor always occurs in several hours after implantation of stent, so the result shown in Table 3 implied that heparin-loaded zein microsphere film could maintain the anticoagulant activity by a slow heparin release, e.g. we could expect to prolong the action of heparin released from heparin-loaded zein
1
*
0.8
OD405nm
Cumulative (%)
60
**
0.6 0.4 0.2 0 Glass
Zein film
Heparin-loaded film
Fig. 7. Platelet adhesion on different material surfaces. Data were expressed as means F standard deviations. *Represents a statistical significance compared with the control group; **represents statistical significances compared with both the control group and zein film group ( p b 0.05, n = 6).
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
microsphere film by increasing the thickness of the film. Platelet adhesion on films from human plasma is an important test for the evaluation of the blood
129
compatibility of materials. Fig. 7 shows that the number of platelets adhered on both the zein film and heparin-loaded zein microsphere film decreased significantly compared with that on glass plate
Fig. 8. SEM observations of blood platelets adhered on different matrixes for 60 min: the glass control (A1, A2); zein film (B1, B2) and heparinloaded zein microsphere film (C1, C2). Bars in left and right columns represent 6 and 2 Am, respectively.
130
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
( p b 0.05), falling about 12% and 22% of it, respectively. Moreover, numbers of adhering platelets on heparin-loaded film was the minimum and significantly lower than zein film. Moreover, the morphology of platelets varied on these surfaces. Platelets adhered and spread, developed characteristic pseudopodia on the glass control (Fig. 8A), but they maintained their original round shape on the zein film. Little platelet adhesion was observed on the zein film (Fig. 8B), especially on the heparin-loaded zein microsphere film (Fig. 8C), although its OD value still got to 0.6 as shown in Fig. 7. We considered that the platelets on heparin-loaded zein film might be eluted during preparation of sample for scanning electron microscope. The platelet adhesion mainly turns on the types and conformation of adsorbed plasma protein; plasma protein adsorption strongly depends on the surface characteristics, e.g. surface hydrophobicity or charge. The higher the surface charge density, the more proteins are adsorbed which may be mainly driven by Coulomb forces [24–26]. Hydrophobic surface has been indicated to be more suitable to protein adsorption than hydrophilic surface [27]. Hydrophobic property of zein might contribute to its high adsorbing ability to proteins from plasma, compared with the glass control. Human serum albumin was the predominant adsorbed protein, as indicated by SDS-PAGE (data not shown). The higher content of albumin adsorbed on the zein film might result in the reduced platelet adhesion [28], while much lower platelet adhesion of heparin-loaded zein film might be due to the electrostatic repulsion between negative charges of platelet and heparin released from the film [29].
4. Conclusion Zein, a native protein, and its degraded product showed a good biocompatibility on the growth of HUVECs, the former also showing the property of suppressing platelet adhesion, which gave us the promise of improving biocompatibility of cardiovascular devices after modification with endothelial cells. In addition, we could control the heparin content at the site of implantation of cardiovascular devices by adjusting the encapsulation efficiency of heparin in microspheres and the thickness of micro-
sphere film, which could avoid massive hemorrhage. The most important was that the released heparin still kept high activity. Superior anticoagulation and inhibition of platelet adhesion of heparin-loaded zein microsphere film was worthwhile to continue the research, which will prevent acute or subacute thrombosis and endow cardiovascular devices, e.g. stent, with biocompatibility. Acknowledgements This study was supported by the Chinese Academy of Sciences (BaiRenJiHua, KGCX2-SW-602-5), the National Hi-Tech Research and Development Plan (863 Project) of China (2002AA327100), and the National Natural Science Foundation of China (30470477). References [1] E.C. Sims, M.T. Rothman, T.D. Warner, M.E.B. Powell, Coronary artery brachytherapy, Clin. Oncol. 14 (2002) 313 – 326. [2] L. Martinez-Elbal, J.M. Ruiz-Nodar, J. Zueco, J.R. LopezMinguez, J. Moreu, I. Calvo, J.A Ramirez, M. Alonso, N. Vazquez, R. Lezaun, C. Rodriguez, Direct coronary stenting versus stenting with balloon pre-dilation: immediate and follow-up results of a multicentre, prospective, randomized study. The DISCO trial. Direct stent of coronary arteries, Eur. Heart J 23 (2002) 633 – 640. [3] A.K. Larsen, Oral heparin results in the appearance of heparin fragments in the plasma of rats, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 2964 – 2968. [4] Y. Iwasaki, A. Yamasaki, K. Ishihara, Platelet compatible blood filtration fabrics using a phosphorylcholine polymer having high surface mobility, Biomaterials 24 (2003) 3599 – 3604. [5] J.F.W. Keuren, S.J.H. Wielders, G.M. Willems, M. Morra, L. Cahaland, P. Cahalan, T. Lindhout, Thrombogenicity of polysaccharide-coated surfaces, Biomaterials 24 (2003) 1917 – 1924. [6] M.J.B. Kutryk, L.M.C. van Dortmont, R.P.G. de Crom, Seeding of intravascular stents by xenotransplantation of genetically modified endothelial cells, Semin. Interv. Cardiol. 3 (1998) 217 – 220. [7] H.T. Moon, Y.K. Lee, J.K. Han, Y. Byun, A novel formulation for controlled release of heparin–DOCA conjugate dispersed as nanoparticles in polyurethane film, Biomaterials 22 (2001) 281 – 289. [8] M.N. Babapulle, M.J. Eisenberg, Coated stents for the prevention of restenosis: Part 1, Circulation 106 (2002) 2734 – 2740.
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 [9] M.N. Babapulle, M.J. Eisenberg, Coated stents for the prevention of restenosis: Part 2, Circulation 106 (2002) 2859 – 2866. [10] Y. Nakayama, J.Y. Kim, S. Nishi, H. Ueno, T. Matsuda, Development of high-performance stent: gelatinous photogelcoated stent that permits drug delivery and gene transfer, J. Biomed. Mater. Res. 57 (2001) 559 – 566. [11] W.J. Van der Giessen, M. Lincoff, R.S. Schwartz, H.M.M. Van Beusekom, P.W. Serruys, D.R. Holmes, S.G. Ellis, E.J. Topol, Marked inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries, Circulation 94 (1996) 1690 – 1697. [12] E. Rechavia, F. Litvack, M.C. Fishbien, M. Nakamura, N. Eigler, Biocompatibility of polyurethane-coated stents: tissue and vascular aspects, Cathet. Cardiovasc. Diagn. 45 (1998) 202 – 207. [13] C. Rogers, E.R. Edelman, Controlled release of heparin reduces neointimal hyperplasia in stented rabbit arteries: Ramifications for local therapy, J. Interv. Cardiol. 5 (1992) 195 – 202. [14] R. Shukla, C. Munir, Zein: the industrial protein from corn, Ind. Crops Prod. 13 (2001) 171 – 192. [15] H. Bernstein, E. Morrel, E. Mathlowitz, K. Schwaller, R. Thomas, Protein microspheres and methods of using them, US Patent No. 5679377, 1997. [16] J. Dong, Q.S. Sun, J.Y. Wang, Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility, Biomaterials 25 (2004) 4691 – 4697. [17] E.A. Jaffe, R.L. Nachman, C.G. Becker, Culture of human endothelial cells derived from umbilical veins, J. Clin. Invest. 52 (1973) 2745 – 2756. [18] P.K. Smith, A.K. Mallia, G.T. Hermanson, Colorimetric method for the assay of heparin content in immobilized heparin preparations, Anal. Biochem. 109 (1980) 466 – 473. [19] W.C. Lin, T.Y. Liu, M.C. Yang, Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate, Biomaterials 25 (2004) 1947 – 1957.
131
[20] J.P. Xu, J. Ji, W.D. Chen, D.Z. Fan, Y.F. Sun, J.C. Shen, Phospholipid based polymer as drug release coating for cardiovascular device, Eur. Polym. J. 20 (2) (2004) 291 – 298. [21] P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F. Manzato, C. Santonastaso, A colorimetric method for the measurement of platelet adhesion in microtiter plates, Anal. Biochem. 216 (1994) 444 – 450. [22] A.M. Rajnicek, C.D. McCaig, Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways, J. Cell. Sci. 110 (1997) 2915 – 2924. [23] X.M. Liu, Q.S. Sun, H.J. Wang, L. Zhang, J.Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system, Biomaterials 26 (2005) 109 – 115. [24] W.B. Tsai, J.M. Grunkemeier, T.A. Horbett, Human plasma fibrinogen adsorption and platelet adhesion to polystyrene, J. Biomed. Mater. Res. 44 (1999) 130 – 139. [25] M. Tanaka, T. Motomura, M. Kawada, T. Anzai, Y. Kasori, T. Shiroya, K. Shimura, M. Onishi, A. Mochizuki, Blood compatible aspects of poly (2-methoxyethylacrylate) (PMEA)—relationship between protein adsorption and platelet adhesion on PMEA surface, Biomaterials 21 (2000) 1471 – 1481. [26] A. Gessner, A. Lieske, B.R. Paulke, R.H. Muller, Influence of surface charge density on protein adsorption on polymeric nanoparticles: analysis by two-dimensional electrophoresis, Eur. J. Pharm. Biopharm. 54 (2) (2002) 165 – 170. [27] V. Tangpasuthadol, N. Ponchaisirikul, V.P. Hoven, Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption, Carbohydr. Res. 338 (9) (2003) 937 – 942. [28] G.A. Abraham, A.A.A. de Queiroz, J.S. Roman, Immobilization of a nonsteroidal anti-inflammatory drug onto commercial segmented polyurethane surface to improve haemocompatibility properties, Biomaterials 23 (2002) 1625 – 1638. [29] I.K. Kang, O.H. Kwon, M.K. Kim, Y.M. Lee, Y.K. Sung, In vitro blood compatibility of group-grafted and heparinimmobilized polyurehanes prepared by plasma glow discharge, Biomaterials 18 (1997) 1099 – 1107.
Heparin-loaded zein microsphere film and hemocompatibility Hua-Jie Wanga, Zhi-Xin Lina, Xin-Ming Liub, Shi-Yan Shenga, Jin-Ye Wanga,b,T a
School of Life Science and Biotechnology, Shanghai Jiaotong University, 1954 Huashan Road, Shanghai 200030, China b Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China Received 5 June 2004; accepted 25 March 2005 Available online 12 May 2005
Abstract Zein was studied as a drug-eluting coating film composed of zein microspheres for cardiovascular devices (e.g. stent). In vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis showed that both zein film and its degraded product had better biocompatibility compared with Corning culture plate on the growth of human umbilical veins endothelial cells (HUVECs, p b 0.05, n = 6), and the effect of zein degraded product on HUVECs was dose-dependent. The best result was obtained at 0.3 mg/ml of the addition. The encapsulation efficiency of heparin and heparin loading varied with the amount of both zein and heparin, and the highest encapsulation efficiency (heparin 1.33 mg/ml and zein 16 mg/ml) was 22.77 F 1.33% (n = 3). Scanning electron microscope (SEM) observation indicated that the zein film was made of microspheres in diameter from nano- to micrometer, which could be controlled. Sizes of heparin-loaded zein microspheres changed before and after release of heparin because of conglutination among zein microspheres. Release rate of heparin from microsphere film reached to 33.5 F 1.2% within 12 h, and began to get into subsequent bslow releaseQ phase; about 55% of the entrapped heparin was released after 20 days. Both zein film and heparin-loaded zein microsphere film were effective in suppressing platelet adhesion, and the heparin-loaded film showed a better anticoagulation as determined with thrombin time (TT) assay. These results suggest that zein film could be used directly as a new type of coating material for its better biocompatibility with HUVECs. Moreover, the heparin-loaded zein microsphere film can significantly improve the hemocompatibility. D 2005 Elsevier B.V. All rights reserved. Keywords: Film; Hemocompatibility; Microspheres; Platelet adhesion; Zein
Abbreviations: HUVECs, human umbilical veins endothelial cells; ECGS, endothelial cells growth supplement; MTT, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SEM, scanning electron microscope; TT, thrombin time; PBS, phosphate-buffered solution; FCS, fetal calf serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T Corresponding author. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China. Tel.: +86 21 54925330; fax: +86 21 64376697. E-mail address: [email protected] (J.-Y. Wang). 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.03.014
1. Introduction The major drawback of angioplasty is restenosis, which occurs in 30–60% of treated vessels, resulting in the need for repeat procedures [1]. As a cardiovascular device, the potential of stenting technique to reduce the morbidity of restenosis and cost of care is enormous; however, the rate of in-
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
stent restenosis still gets to 10–40% due to unsatisfactory biocompatibility of stents [2]. Up to date, there have been four methods used to solve this problem. The first method is to administer anticoagulant to the patient through injection or oral administration for days, which successfully decreases the rate of the thrombus formation, while the rate of massive hemorrhage is increased. And when the anticoagulant is taken orally, it would be denatured in the digestive tract if the suitable carrier were unavailable [3]. The second method is to modify the surface of the stents with polymers, such as the use of phospholipids [4], which could improve the biocompatibility, especially the blood compatibility. The third method is to coat anticoagulant on the surface of the stents [5], but the concentration of the anticoagulant is difficult to be maintained because of the effect of the blood flow. The fourth method is to seed endothelial cells on the surface of the stents with autogenous endothelium, which could avoid contacting the blood with the material, and therefore suppresses the activation of coagulation factors [6]. Among these methods, drug-eluting stents are called brevolutionQ in stenting technique, while coated stents have showed huge promise and great efforts have been made to develop the suitable drug and coating materials of stents, which can maintain the drug concentrations at the site of stents deployment and minimize systemic side effects [7]. These drugs used contain heparin, dexamethasone, sirolimus, everolimus, tacrolimus, carvedilol, paclitaxel, actinomycin-D, flavopiridol, angiopeptin and so on [8,9]. These drugs are often blended with synthetic polymers that act as drug reservoirs to elute the active agent over a period of several weeks or months [10]. Unfortunately, many of the synthetic polymers induce an exaggerated inflammatory response and neointimal hyperplasia in animal models [11,12]. It is noted that most of them are nonbiodegradable, and have to be taken out by surgery, which give patients great discomfort. Therefore, the research focuses on searching a slow eluting material with better biocompatibility and biodegradability. The anticoagulant is encapsulated by or combined with the carrier; the compound of drug and carrier can deliver the drug at a dose low enough to avoid systemic effect but high enough to provide local protection against vascular disease [13].
121
Zein is the major storage protein of corn and accounts for 40–50% of the protein; it is soluble in aqueous alcohol solutions (60–95%). Zein has been used widely, such as adhesive, biodegradable plastics, chewing gum, coating for food products, fiber, cosmetic powder, microencapsulated pesticides and inks. Especially zein has been prepared as microspheres to deliver insulin [14,15]. Our previous work has indicated that zein film, composed of particles in diameter of 100–2500 nm, has good biocompatibilities with both human liver cells and mice fibroblast cells [16]. In present study, zein was considered as a new type of coating material either directly or as a heparin carrier applying for cardiovascular devices such as stent. The biocompatibility of zein film and its degraded product digested with pepsin on human umbilical veins endothelial cells (HUVECs) was studied, the methods of preparing zein microspheres and heparin-loaded microsphere film were investigated, the encapsulation efficiency and release kinetics of heparin-loaded zein microspheres, the morphology of the films, and hemocompatibility (anticoagulation, protein adsorption and platelet adhesion) were tested.
2. Materials and methods 2.1. Materials Zein with biochemical purity was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), endothelial cells growth supplement (ECGS) was purchased from Upstate Biotechnologies Inc. (Lake Placid, NY, USA), heparin and thrombin were from Sigma (St. Louis, MO, USA), and other reagents were reagent grade. 2.2. Preparation of zein film and its degraded product Zein was dissolved in aqueous mixture of ethanol, and the ethanol content was brought to 40% immediately to form zein microspheres suspended solution, then the film was prepared through volatilization at 37 8C. The film was sterilized by UV for at least 1 h and immersed into the RPMI 1640 nutrient fluid before use. Zein degraded product was obtained from zein powder digested with pepsin (zein/pepsin = 10:1 w/w)
122
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
in citric acid–NaH2PO4 (pH 2.2) at 37 8C. The supernatant was freeze-dried to get the degraded powder for use. 2.3. Adhesion and proliferation assays of HUVECs HUVECs were harvested from human umbilical veins as described [17] and cells at passage 3 were digested by incubation with 0.025% trypsin/0.002% EDTA, resuspended with RPMI 1640 medium (20% FBS, 100 units/ml penicillin, 100 Ag/ml streptomycin, 30 Ag/ml ECGS and 90 Ag/ml heparin) and seeded at 2.4 104 cells/ml onto 96-well Corning culture microplate coated with zein film; other groups were 2 mg/ml zein degraded product adding group and Corning culture microplate control group. After 2 h, the culture medium in each well was taken out and the new medium (without or with zein degraded product) was added. The medium was refreshed every 2 days. HUVECs were photographed with inverted optical microscope everyday. At 4 h after being seeded, the non-adhered cells in the medium were counted with a hemocytometer to evaluate the adhesion of HUVECs under different conditions. 3-(4,5-Dimethylthiazol -2yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used to assess the proliferation of HUVECs after 2 h, 1 days, 3 days and 5 days of culture. The attached cells on different substrates were fixed with 2.5% glutaraldehyde in PBS for at least 2 h, washed with Milli-Q water. The samples were mounted on stubs and coated in vacuum with gold. Then cells were examined with a scanning electron microscope (SEM S-450, Hitachi, Japan).
remove unentrapped heparin. The encapsulation efficiency was determined using the method described by Smith [18]. Heparin-loaded microsphere suspended solution was volatilized at room temperature to form heparin-loaded zein microsphere film. SEM was used to observe the surface and internal morphology of the film after vacuum-coated with a gold layer. Heparinloaded zein microsphere film was immersed into 1 ml PBS (pH 7.4) and incubated at 37 8C. In vitro, the releasing medium of each sample was removed and replaced with fresh PBS at a given time interval. The heparin content in the medium was analyzed using the method described by Smith, and the releasing rate of heparin from heparin-loaded zein film was calculated. 2.5. Anticoagulation of zein film and heparin-loaded zein microsphere film [19] This test was divided into four groups: zein film group, Corning culture plate group, physiological saline group and heparin-loaded zein microsphere film group. Samples of the first and the last group were composed of microspheres. Thrombin, anticoagulant plasma from healthy rabbit and calcium chloride solution were prewarmed at 37 8C. Then 0.1 ml plasma, 0.1 ml of 0.85% (w/v) calcium chloride solution and thrombin (100 units per ml) were added into the sample well, and began to record the time until cruor. In order to assess the activity of heparin released from zein microsphere film, we also performed this test at the given time during the releasing process of heparin with thrombin time (TT) assay.
2.4. Heparin-loaded zein microspheres and film
2.6. Plasma protein adsorption and platelet adhesion [5]
The heparin-loaded microspheres were obtained based on the solubility of zein and heparin. Heparin and zein were dissolved in the aqueous mixture of ethanol, respectively; after mixed sufficiently, the ethanol content was brought to 40% immediately to form microspheres suspended solution. Then the suspended solution was rapidly frozen with dry ice and acetone, vacuum-dried overnight and the powder was stored at 4 8C before use. The vacuum-dried microspheres powder was resuspended in Milli-Q water (PALL, USA) and the heparin-loaded zein microspheres were separated by chromatography to
Fresh healthy human blood was provided by Shanghai Blood Center with approval. The blood was centrifuged at 1000 rpm for 10 min at 4 8C to get platelet-rich plasma (PRP), and at 3000 rpm for 10 min at 4 8C to get platelet-poor plasma (PPP). The fresh PRP or PPP samples were used in this study. Zein film and heparin-loaded zein microsphere film were sufficiently rinsed with Milli-Q water, and immersed into platelet-poor plasma, which was placed at 37 8C for 3 h. After being washed three times, proteins adsorbed on the zein film and heparin-loaded zein microsphere film were removed
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
with 1 wt% aqueous solution of sodium dodecyl sulfate (SDS), and freeze-dried. The protein samples were redissolved in Milli-Q water, applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For evaluation of platelet adhesion, zein film and heparin-loaded zein film were placed in contact with 50 Al of human PRP and left at 37 8C for 60–180 min, the number of platelets in the PRP was diluted with PBS to 5 106 cells. The PRP was removed and the film was rinsed three times with PBS [20]. The adhered platelets were fixed by immersing the film into 2.5% solution of glutaraldehyde in PBS for at least 2 h at 4 8C. Samples were freeze-dried, then sputter-coated with gold for SEM observation. The number of platelets adhered was determined with colorimetric method [21]. Briefly, 150 Al of 4-nitropheny-phosphate disodium salt was added in each well and placed at 37 8C for 60 min. The absorption was determined at the wavelength of 405 nm after stopping reaction with 100 Al of 2 N NaOH. 2.7. Statistical analysis All the data were analyzed by one-way factorial ANOVA and multiple comparisons. Significant effects of treatment were defined using Scheffe’s statistic method.
123
In order to evaluate the attachment, spreading, and proliferation changes of HUVECs on the surface treated with zein film and its degraded products, we analyzed the proliferation of cells in 2 h, 1 day, 3 days and 5 days with MTT method (Fig. 1). Mitochondrial redox activity of HUVECs can be evaluated by reduction of MTT to formazan by mitochondrial succinate dehydrogenase in complex II (succinate: ubiquinone oxidoreductase complex), which plays a central role in both oxidative phosphorylation and the tricarboxylic acid cycle. HUVECs attached and adhered onto zein film and the glass plate completely after 1 h culture, the percentage of attached cells was more than 90% in each group and no significant difference was observed. At the third day of culture, an enhancement of mitochondrial activity from cells on zein film was observed compared with cells on Corning culture microplate, but the cells treated with zein degraded product did not show significant difference with the control group according to the result of MTT assay. Significantly higher proliferations were obtained for both zein film and its degraded product adding groups after 5 days of culture. There was no significant difference between zein film group and zein degraded product adding group. We deduced that at earlier state of the cell proliferation, substratum topography on which cells adhered might be, to some extent, important [22]. In summary, the adhesion of cells was not affected by zein degraded product, while
3. Results and discussion 0.35
3.1. Cell adhesion and proliferation assays
0.3
OD490nm
Stent coatings can be divided into two categories, biocompatible materials and drug-eluting coatings. Biocompatible materials currently under investigation are thought to be less thrombogenic and inflammatory, and are thereby potentially able to reduce neointimal hyperplasia. Metallic stents exert a continuous radial pressure on the diseased artery, resulting in compression of atherosclerotic plaques, sealing of dissections, and expansion of the vessel. Improving the surface properties of stents, by applying a biocompatible coating aimed at improving the vascular healing response in an attempt to prevent early thrombosis and late neointimal proliferation, is more logical approach.
**
*
0.25 0.2 0.15 0.1 0.05
0
1
2
3
4
5
6
Time (days) Fig. 1. The proliferation assay of HUVECs on zein film (D), glass control (w), or treated with zein degraded product (5, 2 mg/ml) after being cultured for 5 days. *Represents a statistical significance between zein film group and the control group; **represents statistical significances compared both zein film group and zein degraded product group with the control group ( p b 0.05, n = 6).
124
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
some structures in zein might play an important role on the proliferation of cells at later stage. As shown in Fig. 2, attachment, adhesion and spreading occurred in the first phase of cell/material
interactions, and there were some differences between these groups. Cells cultured in each group maintained their characteristic polygonal morphology; the spreading cells maintained physical contact with each other
Fig. 2. Morphology comparison of HUVECs cultured for 3 days on the glass control (A1, A2), zein film (B1, B2), and treated with zein degraded product (C1, C2, 2 mg/ml) by SEM observation. Bars in left and right columns represent 29 and 9.9 Am, respectively.
OD590nm
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2
*
125
through filopodia or lamellopodia. The morphologies of cells varied greatly on different matrixes, especially on zein film after 3 days of culture. The cytoplasm of HUVECs on zein film was abundant and lacked of thin pinna.
* * *
3.2. Effect of zein degraded product on HUVECs 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Sample Concentration (mg/ml) Fig. 3. Effects of zein degraded product on HUVECs proliferation with MTT analysis after being cultured for 2 days ( p b 0.05, n = 6). *Represents significant difference compared with the control (containing 0 mg/ml of zein degraded product).
After being cultured for 2 days with different concentrations of zein degraded product, MTT showed that the growth of HUVECs cultured with zein degraded product was significantly better than the control ( p b 0.05, n = 6). A lower content presented a higher stimulating effect (Fig. 3) and the effect of zein degraded product on HUVECs was significantly dose-dependent. Fig. 4 shows the mor-
Fig. 4. Effect of zein degraded product on HUVECs. Morphology and contribution of HUVECs on Corning microplate after being cultured with different concentrations of zein degraded product for 2 days were observed by an optical microscope (32 magnification). a: 0 mg/ml (the control group); b: 0.2 mg/ml; c: 0.3 mg/ml; d: 0.5 mg/ml; e: 1 mg/ml; f: 2 mg/ml.
126
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
phology and distribution of HUVECs treated with different concentration of zein degraded product after 2 days of culture with an optical microscope, which also showed more cells in the group treating with lower content of the product and a higher concentration of zein degraded product resulted in diminution of HUVECs to some extent (1 mg/ml), while a round shape was observed when 2 mg/ml of zein degraded product was added. 3.3. Preparation and characterization of heparinloaded protein microspheres Before being processed, feed of zein is the unregulated particle. We utilized the phase separation method based on the solubility of zein to prepare protein microspheres. When the content of zein in 40% of alcohol aqueous solution is 8 mg/ml, we got microspheres in diameter of 40–100 nm. Local drug delivery through the use of drug-eluting stents allows the controlled release of a high concentration of drug at the site of diseased artery without excess systemic effects. In order to develop the function of zein as a heparin carrier, we prepared heparin-loaded zein microspheres through phase separation technique. The encapsulation efficiency of heparin was determined through the method described by Smith. As can be seen in Table 1, encapsulation efficiency and heparin loading depended on the concentration and the ratio of zein to heparin. Both encapsulation efficiency and heparin loading increased with the increase of heparin and zein concentrations at a given ratio of them (except for heparin loading at heparin 5.33 mg/ml and zein 16 mg/ml); both the encapsulation efficiency of heparin and heparin loading increased with the increase of the ratio of heparin to zein (zein 8 mg/ml). Satisfactory result was obtained when the concentration of heparin and zein was 1.33 mg/ml and 16 mg/ml, respectively, in which the encapsulation efficiency of heparin was the highest. The result was in accordance with that we have got previously, but the values were far lower compared with those of a hydrophobic drug, ivermection [23]. According to the SEM result of our previous study, the size of heparin-loaded zein microspheres varied from nano- to micrometer with the increase of zein concentration. So we can say that heparin-loaded per
Table 1 Encapsulation efficiency of heparin and heparin loading in zein microspheres (n = 3) Microspheres preparation (concentration of heparin and zein) Heparin 1.33 mg/ml, zein 4 mg/ml Heparin 1.33 mg/ml, zein 8 mg/ml Heparin 1.33 mg/ml, zein 12 mg/ml Heparin 1.33 mg/ml, zein 16 mg/ml Heparin 2.67 mg/ml, zein 8 mg/ml Heparin 4 mg/ml, zein 8 mg/ml Heparin 5.33 mg/ml, zein 8 mg/ml Heparin 4 mg/ml, zein 12 mg/ml Heparin 5.33 mg/ml, zein 16 mg/ml
Encapsulation efficiency of heparin (w/w %)a
Heparin loading (w/w %)b
1.97 F 0.77
0.34 F 0.17
8.74 F 0.98
0.98 F 0.11
11.94 F 0.85
0.89 F 0.06
22.77 F 1.33
1.28 F 0.07
11.94 F 0.56
1.35 F 0.06
15.38 F 1.11
3.46 F 0.25
16.37 F 0.93
3.67 F 0.21
16.86 F 0.74
2.53 F 0.11
19.56 F 1.30
2.20 F 0.15
a Encapsulation efficiency (w/w %) = Amount of heparin in microspheres/Heparin initially added. b Heparin loading (w/w %) = Amount of heparin in microspheres/ Amount of microspheres.
zein microsphere is different because of the different microsphere sizes. By phase separation technique, we got heparinloaded zein microspheres and the film was prepared from the microspheres. The film thickness was 26.3 Am (Fig. 5a), made from microspheres as shown in Fig. 5b. The size of microspheres was 1–2 Am (Fig. 5c), and some conglutination was observed from the facade. Characteristics of heparin-loaded zein microsphere film before and after release were also observed by SEM randomly. From the facade of the film, sizes of microspheres increased with immersing time of the film in PBS, adjacent microspheres fused with each other (Fig. 5d–f). However, the profile of the film showed that few of the microspheres fused and their size was rather constant (Fig. 5g–i). Fig. 6 shows the biphasic release characteristic of the film. At 12 h, the release rate of heparin reached to 33.5 F 1.2%, which was defined as a dburst effectT (phase 1). Following the phase 1, the release appeared as a sustained release (phase 2). After 20 days, the release rate tended to tail off in the subsequent
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
127
Fig. 5. Characteristics of heparin-loaded zein microsphere film (the profile a: bar = 14 Am; b: bar = 5 Am; the fac¸ade c: bar = 1.9 Am), and morphology of heparin-loaded zein microsphere films sampling randomly before and after release (the fac¸ade c: 0 day, bar = 1.9 Am; d: 1 day, bar = 7.5 Am; e: 3 days, bar = 7.4 Am; f: 6 days, bar = 7.5 Am. The profile g: 1 day, bar = 4.8 Am; h: 3 days, bar = 4.9 Am; i: 6 days, bar = 7.2 Am).
dslowerT phase and about 54.3 F 3.9% of heparin in the film was released at last. Due to the hydrophobic property of zein, it could not be degraded in PBS solution, and our previous study also showed that zein aggregated easily in water solution. So we deduced that the dburst release (phase 1)T attributed to the drug existing at or near the surface of the film. The process of the heparin release formed some micro-channels between zein microspheres, to permit the release of the heparin entrapped into
microspheres in phase 2. The slower releasing rate was probably due to the longer diffusing route of deeper heparin. Existence of repellency between heparin molecules on the surface of zein microspheres may be the reason that zein microspheres could not fuse with each other and still maintain the size as shown in Fig. 5g–i. However, with the disappearance of heparin on the surface, microspheres began to aggregate as observed from the fac¸ade of the film (Fig. 5d–f). The micro-channels also began to
128
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 Table 3 The anticoagulant activity of heparin-loaded zein microsphere film during release of heparin (n = 3)
45
30
15
0
0
5
10
15
20
25
Time (days) Fig. 6. Release kinetics curve of heparin-loaded zein microsphere film in PBS (pH 7.4, 37 8C, n = 3).
disappear. As there was no digestive enzyme existed in the present system, the heparin entrapped into microspheres could not continue to be released by diffusion into the medium after 20 days. So we considered that the release kinetics of heparin–zein microspheres was characterized as no erosionincomplete release. 3.4. Hemocompatibility of zein film and heparinloaded microsphere film The anticoagulation was studied by TT assay. There were no significant differences between the control group, physiological saline group and zein film group as shown in Table 2; the TT time was controlled within 30 s approximately. However, heparin-loaded zein microsphere film group showed the significant anticoagulation and the plasma kept incoagulable all the time. This result indicated that zein film itself did not prevent acute or subacute thrombosis if zein film was coated directly on cardiovascular devices such as stent. However, for heparin-loaded zein microsphere film, which was obtained at room temperature, heparin still kept high Table 2 Anticoagulation of zein film and heparin-loaded microsphere film (n = 3) Groups
TT (s), average F S.D.
Control Physiological saline Zein film Heparin-loaded microsphere film
30.3 F 5.1 29.3 F 5.5 29.3 F 3.2 IncoagulableT
T Significant difference compared with the control ( p b 0.01).
Time of heparin release
Time of formation of fibrin clot
Time of clotting of plasma
Significant difference
Control 2h 6h 12 h 24 h 48 h 96 h 240 h
– – – 7.3 F 1.2 min 2.7 F 0.7 min – – –
20 F 2.5 s Incoagulable Incoagulable 12 F 1 h 37 F 12 min 42 F 11 s 48 F 4.6 s 54 F 13.3 s
– ** ** ** ** * * *
*Statistical significance compared with the control at p b 0.05. **Statistical significance compared with the control at p b 0.005.
activity. We could control the concentration of heparin entrapped into the film by adjusting the preparing process of microspheres or the thickness of the film, to prolong the action of heparin in body. For the given heparin-loaded zein microsphere film in this study, we also designed a simplified test to determine anticoagulating effect of heparin-loaded zein microsphere film during the release process of heparin. Cruor always occurs in several hours after implantation of stent, so the result shown in Table 3 implied that heparin-loaded zein microsphere film could maintain the anticoagulant activity by a slow heparin release, e.g. we could expect to prolong the action of heparin released from heparin-loaded zein
1
*
0.8
OD405nm
Cumulative (%)
60
**
0.6 0.4 0.2 0 Glass
Zein film
Heparin-loaded film
Fig. 7. Platelet adhesion on different material surfaces. Data were expressed as means F standard deviations. *Represents a statistical significance compared with the control group; **represents statistical significances compared with both the control group and zein film group ( p b 0.05, n = 6).
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
microsphere film by increasing the thickness of the film. Platelet adhesion on films from human plasma is an important test for the evaluation of the blood
129
compatibility of materials. Fig. 7 shows that the number of platelets adhered on both the zein film and heparin-loaded zein microsphere film decreased significantly compared with that on glass plate
Fig. 8. SEM observations of blood platelets adhered on different matrixes for 60 min: the glass control (A1, A2); zein film (B1, B2) and heparinloaded zein microsphere film (C1, C2). Bars in left and right columns represent 6 and 2 Am, respectively.
130
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131
( p b 0.05), falling about 12% and 22% of it, respectively. Moreover, numbers of adhering platelets on heparin-loaded film was the minimum and significantly lower than zein film. Moreover, the morphology of platelets varied on these surfaces. Platelets adhered and spread, developed characteristic pseudopodia on the glass control (Fig. 8A), but they maintained their original round shape on the zein film. Little platelet adhesion was observed on the zein film (Fig. 8B), especially on the heparin-loaded zein microsphere film (Fig. 8C), although its OD value still got to 0.6 as shown in Fig. 7. We considered that the platelets on heparin-loaded zein film might be eluted during preparation of sample for scanning electron microscope. The platelet adhesion mainly turns on the types and conformation of adsorbed plasma protein; plasma protein adsorption strongly depends on the surface characteristics, e.g. surface hydrophobicity or charge. The higher the surface charge density, the more proteins are adsorbed which may be mainly driven by Coulomb forces [24–26]. Hydrophobic surface has been indicated to be more suitable to protein adsorption than hydrophilic surface [27]. Hydrophobic property of zein might contribute to its high adsorbing ability to proteins from plasma, compared with the glass control. Human serum albumin was the predominant adsorbed protein, as indicated by SDS-PAGE (data not shown). The higher content of albumin adsorbed on the zein film might result in the reduced platelet adhesion [28], while much lower platelet adhesion of heparin-loaded zein film might be due to the electrostatic repulsion between negative charges of platelet and heparin released from the film [29].
4. Conclusion Zein, a native protein, and its degraded product showed a good biocompatibility on the growth of HUVECs, the former also showing the property of suppressing platelet adhesion, which gave us the promise of improving biocompatibility of cardiovascular devices after modification with endothelial cells. In addition, we could control the heparin content at the site of implantation of cardiovascular devices by adjusting the encapsulation efficiency of heparin in microspheres and the thickness of micro-
sphere film, which could avoid massive hemorrhage. The most important was that the released heparin still kept high activity. Superior anticoagulation and inhibition of platelet adhesion of heparin-loaded zein microsphere film was worthwhile to continue the research, which will prevent acute or subacute thrombosis and endow cardiovascular devices, e.g. stent, with biocompatibility. Acknowledgements This study was supported by the Chinese Academy of Sciences (BaiRenJiHua, KGCX2-SW-602-5), the National Hi-Tech Research and Development Plan (863 Project) of China (2002AA327100), and the National Natural Science Foundation of China (30470477). References [1] E.C. Sims, M.T. Rothman, T.D. Warner, M.E.B. Powell, Coronary artery brachytherapy, Clin. Oncol. 14 (2002) 313 – 326. [2] L. Martinez-Elbal, J.M. Ruiz-Nodar, J. Zueco, J.R. LopezMinguez, J. Moreu, I. Calvo, J.A Ramirez, M. Alonso, N. Vazquez, R. Lezaun, C. Rodriguez, Direct coronary stenting versus stenting with balloon pre-dilation: immediate and follow-up results of a multicentre, prospective, randomized study. The DISCO trial. Direct stent of coronary arteries, Eur. Heart J 23 (2002) 633 – 640. [3] A.K. Larsen, Oral heparin results in the appearance of heparin fragments in the plasma of rats, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 2964 – 2968. [4] Y. Iwasaki, A. Yamasaki, K. Ishihara, Platelet compatible blood filtration fabrics using a phosphorylcholine polymer having high surface mobility, Biomaterials 24 (2003) 3599 – 3604. [5] J.F.W. Keuren, S.J.H. Wielders, G.M. Willems, M. Morra, L. Cahaland, P. Cahalan, T. Lindhout, Thrombogenicity of polysaccharide-coated surfaces, Biomaterials 24 (2003) 1917 – 1924. [6] M.J.B. Kutryk, L.M.C. van Dortmont, R.P.G. de Crom, Seeding of intravascular stents by xenotransplantation of genetically modified endothelial cells, Semin. Interv. Cardiol. 3 (1998) 217 – 220. [7] H.T. Moon, Y.K. Lee, J.K. Han, Y. Byun, A novel formulation for controlled release of heparin–DOCA conjugate dispersed as nanoparticles in polyurethane film, Biomaterials 22 (2001) 281 – 289. [8] M.N. Babapulle, M.J. Eisenberg, Coated stents for the prevention of restenosis: Part 1, Circulation 106 (2002) 2734 – 2740.
H.-J. Wang et al. / Journal of Controlled Release 105 (2005) 120–131 [9] M.N. Babapulle, M.J. Eisenberg, Coated stents for the prevention of restenosis: Part 2, Circulation 106 (2002) 2859 – 2866. [10] Y. Nakayama, J.Y. Kim, S. Nishi, H. Ueno, T. Matsuda, Development of high-performance stent: gelatinous photogelcoated stent that permits drug delivery and gene transfer, J. Biomed. Mater. Res. 57 (2001) 559 – 566. [11] W.J. Van der Giessen, M. Lincoff, R.S. Schwartz, H.M.M. Van Beusekom, P.W. Serruys, D.R. Holmes, S.G. Ellis, E.J. Topol, Marked inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries, Circulation 94 (1996) 1690 – 1697. [12] E. Rechavia, F. Litvack, M.C. Fishbien, M. Nakamura, N. Eigler, Biocompatibility of polyurethane-coated stents: tissue and vascular aspects, Cathet. Cardiovasc. Diagn. 45 (1998) 202 – 207. [13] C. Rogers, E.R. Edelman, Controlled release of heparin reduces neointimal hyperplasia in stented rabbit arteries: Ramifications for local therapy, J. Interv. Cardiol. 5 (1992) 195 – 202. [14] R. Shukla, C. Munir, Zein: the industrial protein from corn, Ind. Crops Prod. 13 (2001) 171 – 192. [15] H. Bernstein, E. Morrel, E. Mathlowitz, K. Schwaller, R. Thomas, Protein microspheres and methods of using them, US Patent No. 5679377, 1997. [16] J. Dong, Q.S. Sun, J.Y. Wang, Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility, Biomaterials 25 (2004) 4691 – 4697. [17] E.A. Jaffe, R.L. Nachman, C.G. Becker, Culture of human endothelial cells derived from umbilical veins, J. Clin. Invest. 52 (1973) 2745 – 2756. [18] P.K. Smith, A.K. Mallia, G.T. Hermanson, Colorimetric method for the assay of heparin content in immobilized heparin preparations, Anal. Biochem. 109 (1980) 466 – 473. [19] W.C. Lin, T.Y. Liu, M.C. Yang, Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate, Biomaterials 25 (2004) 1947 – 1957.
131
[20] J.P. Xu, J. Ji, W.D. Chen, D.Z. Fan, Y.F. Sun, J.C. Shen, Phospholipid based polymer as drug release coating for cardiovascular device, Eur. Polym. J. 20 (2) (2004) 291 – 298. [21] P. Bellavite, G. Andrioli, P. Guzzo, P. Arigliano, S. Chirumbolo, F. Manzato, C. Santonastaso, A colorimetric method for the measurement of platelet adhesion in microtiter plates, Anal. Biochem. 216 (1994) 444 – 450. [22] A.M. Rajnicek, C.D. McCaig, Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways, J. Cell. Sci. 110 (1997) 2915 – 2924. [23] X.M. Liu, Q.S. Sun, H.J. Wang, L. Zhang, J.Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system, Biomaterials 26 (2005) 109 – 115. [24] W.B. Tsai, J.M. Grunkemeier, T.A. Horbett, Human plasma fibrinogen adsorption and platelet adhesion to polystyrene, J. Biomed. Mater. Res. 44 (1999) 130 – 139. [25] M. Tanaka, T. Motomura, M. Kawada, T. Anzai, Y. Kasori, T. Shiroya, K. Shimura, M. Onishi, A. Mochizuki, Blood compatible aspects of poly (2-methoxyethylacrylate) (PMEA)—relationship between protein adsorption and platelet adhesion on PMEA surface, Biomaterials 21 (2000) 1471 – 1481. [26] A. Gessner, A. Lieske, B.R. Paulke, R.H. Muller, Influence of surface charge density on protein adsorption on polymeric nanoparticles: analysis by two-dimensional electrophoresis, Eur. J. Pharm. Biopharm. 54 (2) (2002) 165 – 170. [27] V. Tangpasuthadol, N. Ponchaisirikul, V.P. Hoven, Surface modification of chitosan films. Effects of hydrophobicity on protein adsorption, Carbohydr. Res. 338 (9) (2003) 937 – 942. [28] G.A. Abraham, A.A.A. de Queiroz, J.S. Roman, Immobilization of a nonsteroidal anti-inflammatory drug onto commercial segmented polyurethane surface to improve haemocompatibility properties, Biomaterials 23 (2002) 1625 – 1638. [29] I.K. Kang, O.H. Kwon, M.K. Kim, Y.M. Lee, Y.K. Sung, In vitro blood compatibility of group-grafted and heparinimmobilized polyurehanes prepared by plasma glow discharge, Biomaterials 18 (1997) 1099 – 1107.