- Email: [email protected]
Nasal administration of heparin-loaded microspheres based on poly(lactic acid)
Il Farmaco 60 (2005) 919–924 http://france.elsevier.com/direct/FARMAC/
Original article
Nasal administration of heparin-loaded microspheres based on poly(lactic acid) Ayca Yıldız a, Alper Okyar b, Gül Baktır b, Ahmet Araman a, Yıldız Özsoy a,* a
Department of Pharmaceutical Technology, Faculty of Pharmacy, Istanbul University, Beyazt, 34116-Universite, Istanbul, Turkey b Department of Pharmacology, Faculty of Pharmacy, Istanbul University, 34116-Universite, Istanbul, Turkey Received 13 January 2005; received in revised form 1 August 2005; accepted 4 August 2005
Abstract In this study, heparin-loaded microspheres having smooth surface and small particle size were designed in order to provide the absorption of heparin through nasal mucosa. For this purpose, microspheres at different polymer/drug ratios (1:10, 1:2.5 and 1:1) and at different concentrations of polyvinyl alcohol, emulsifying agent (1.5% and 2.5% w/v) were prepared by solvent evaporation method with poly(lactic acid). The microspheres were for evaluated shape and surface properties, particle size, production yield, encapsulation efficiency and in vitro drug release. Based on the in vitro data, selected microspheres were applied by nasal route to Wistar albino rats. According to in vivo studies, heparin-loaded microspheres may be used by nasal route as an alternative to parenteral route. © 2005 Elsevier SAS. All rights reserved. Keywords: Heparin; Poly(lactic acid); Microspheres; Nasal route; aPTT
1. Introduction Heparin is one of the most potent anticoagulant widely used for the treatment and prevention of deep vein thrombosis [1]. The mean half-life observed in practically human cases amounts to approximately 1.5 h [2]. Heparin is degraded to ineffective fragments after oral ingestion [3] and while subcutaneous administration is efficacious, it is uncomfortable and associated with poor patient compliance [4]. In recent years, the nasal cavity has been widely investigated as a potential site for non-invasive drug delivery. As a site for drug delivery, nasal cavity possesses many advantages such as a large surface area for absorption with a highly vascularized subepithelial layer. In addition, blood is drained directly from the nose into the systemic circulation, thereby avoiding first-pass metabolism by the liver [5]. Microspheres of different materials have been evaluated in vivo as nasal drug delivery system [6]. Among the biodegradable polymers, poly(lactic acid) (PLA), a polyester, is the most widely used material for preparing microspheres because of its biodegradability and low toxicity [7]. * Corresponding author. Tel.: +90 212 440 0000/13498; fax: +90 212 440 0252. E-mail address: [email protected] (Y. Özsoy). 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.08.004
In the literature, heparin implants have been prepared with PLA [8], ethylene-vinyl acetate [9] and poly(D,L-lactic-coglycolic acid) (PLGA) [10–12] for local treatment; heparinloaded microparticles prepared using biodegradable (poly-ecaprolactone, PLA, PLGA) and nonbiodegradable (Eudragit RS and RL) polymers have been evaluated after oral administration in rabbits [13–15]; conjugates of heparin with deoxycholic acid have been synthesized in order to enhance heparin absorption from the gastrointestinal tract [16]. Yang et al. [17] has reported that low-molecular-weight heparin with cyclodextrins can be used nasal delivery. There were no data on heparin microspheres applied via nasal route in the literature. In this study, heparin-loaded microspheres were prepared with PLA using solvent evaporation method. The surface morphology of microspheres was evaluated. The influence of drug-polymer ratio and emulsifying agent concentration on the formation of microspheres, drug loading capacity and particle size were also investigated. The formulations were characterized by in vitro release study and the formulations providing suitable highest drug release were selected for in vivo study via nasal route. After nasal administration of these formulations to rats, the time course of the anticoagulant activ-
920
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
ity was investigated by means of aPTT and compared with heparin solution.
2. Materials and methods 2.1. Materials PLA was purchased from Boehringer Ingelheim (Resomer L104). Polyvinyl alcohol (PVA) (Mw: 13,000–23,000, 88% hydrolyzed) was obtained from E. Merck. Heparin (195.3 IU/mg) was supplied by Mustafa Nevzat Pharmaceutical Company, Istanbul, Turkey. Sodium azide, methylene chloride, sodium hydroxide, hydrochloric acid, sodium chloride were from E. Merck. Azure A was from Eastman and methylcellulose was from Sigma.
ant primary emulsion was added to 75 ml of an aqueous PVA solution (1.5% or 2.5%, w/v) at 13500 rpm for 5 min. This water–oil–water (w/o/w) emulsion was stirred with a magnetic stirrer overnight at room temperature to allow the solvent to evaporate. The particles were separated by centrifugation, washed three times with distilled water, lyophilized coded (D1–D6) and kept under room temperature until analysis. All formulations were prepared as triplicate. 2.3. Morphology of microspheres Prepared microspheres were coated with gold–palladium under an argon atmosphere at room temperature and then the surface morphology of the microspheres was determined by scanning electron microscopy using a JEOL 840A JXA. 2.4. Differential scanning calorimetry
2.2. Preparation of heparin microspheres Heparin-loaded microspheres (12 formulations) were prepared with single and double emulsions using a solvent evaporation technique [18]. The codes of the prepared microspheres can be seen in (Table 1). Apart from these formulations, blank microspheres without heparin were also prepared using same methods for comparison.
Thermograms of the blank microspheres and heparinloaded microspheres were produced using a computerinterfaced Perkin–Elmer System 4 differential scanning calorimeter. Samples (4–8 mg) were sealed into aluminum pans and measurements were run from 25 to 300 °C against an empty pan. 2.5. Particle size distribution
2.2.1. Single emulsion (o/w) Two hundred fifty milligrams of PLA were dissolved in dichloromethane and different amounts of the drug (25, 100 and 250 mg) were dispersed in this solution. It was then dropped into 75 ml of the aqueous solution of PVA (1.5%, 2.5% w/v) to form oil–water (o/w) emulsion and stirred using a high speed homogenizer (CAT-X620) at a constant rate of 13500 rpm for 5 min. The resultant emulsion was left overnight at room temperature by continuously stirring with a magnetic stirrer to evaporate organic phase. The microspheres formed were separated by centrifugation at 3000 rpm. The prepared microspheres were washed three times with distilled water. Subsequently, the microspheres were lyophilized, coded (S1–S6) and kept under room temperature until analysis. All formulations were prepared as triplicate. 2.2.2. Double emulsion (w/o/w) The primary emulsion (w/o) containing required amounts of heparin (25, 100 and 250 mg) with PVA (12.5% w/v) and methylcellulose (1.25%, w/v) was formed using a high speed homogenizer (CAT-X620) at 13500 rpm for 2 min. The result-
The lyophilized microspheres were dispersed in filtered (0.22 µm membrane filter) 0.9% (w/w) sodium chloride. Volume size distributions were obtained using a Malvern MasterSizer (Malvern Instruments, Malvern, UK). The size of microspheres is expressed as d10, d50 and d90 representing the size below which 10%, 50%, and 90% by weight of sample falls, respectively. The values (d50) were expressed for all formulations as mean size range. 2.6. Determination of actual drug loading and encapsulation effıciency The amount of heparin entrapped in microspheres was determined with Azure A colorimetric method [19] based on metachromasy by measuring the amount of non-entrapped drug in the external aqueous solution recovered after centrifugation and washing of the microspheres. Azure A is reacted with a heparin relies on the metachromatic shift of the absorbance of the heparin–Azure A complex from 620 to 510 nm. Typically, aliquots (1 ml) of aqueous samples were reacted
Table 1 The code of heparin-loaded microspheres with single (S1–S6) and double (D1–D6) emulsion Formulation code S1, D1 S2, D2 S3, D3 S4, D4 S5, D5 S6, D6
Heparin amount (mg) 25 100 250 25 100 250
Polymer amount (mg) 250 250 250 250 250 250
Polymer/drug ratio 1:10 1:2.5 1:1 1:10 1:2.5 1:1
PVA amount (%, w/v) 1.5 1.5 1.5 2.5 2.5 2.5
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
with 1.43 ml of Azure A solution (0.09 mg/ml, w/v) and 1.43 ml barbital buffer (pH 8.61 ± 0.02). The absorbance of solutions was measured at 510 nm within 30 min using Shimadzu UV–Vis-1601 spectrophotometer. Assay studies were performed in triplicate. Polymer solution did not interfere with assay method. The actual drug loading and percentage of encapsulation efficiency were calculated as follows: Actual load (mg) = A – B A = Theoretical heparin content (mg) B = Calculated amount of the heparin in the external aqueous solution (mg) Encapsulation efficiency 共 % 兲 = Actual load 共 % w/w 兲 Theorical heparin content 共 % w/w 兲
× 100
2.7. In vitro release experiments For in vitro release study, 25 mg of heparin-loaded microspheres were suspended in 10 ml of barbital buffer (pH 8.61 ± 0.02) containing 0.01% (w/v) sodium azide, an antibacterial agent. The microsphere suspension was gently stirred (100 rpm) using horizontal shaking (Minishaker, B. Braun, Model Kühner) at room temperature. Two milliliters of samples were withdrawn at appropriate intervals (1–8 h) from the dissolution medium and replaced by 2 ml of fresh medium. The samples were centrifuged for 5 min. The heparin amount of the samples was assayed according to the colorimetric method described above. All release studies were performed in triplicate. 2.8. In vivo study Normal female Wistar albino rats (about 160–210 g body weight) were used for in vivo experiments. They were fasted overnight before dosing and divided into three groups of three animals each. Microsphere formulations (S1 and D6) selected according to in vitro release experiments were suspended in 0.9% (w/v) sodium chloride solution and were administered
921
as a 20 µl aliquot to the nostrils via micropipet to rats placed in supine position. The drops were administered slowly to avoid any leakage. The heparin amount applied was 31.25 mg/kg for formulation S1 and 42.5 mg/kg for formulation D6. Heparin solution (10 mg/kg) was administered by nasal route for comparison. Blood samples (1.8 ml) were withdrawn before and 0.5, 1, 2, 3, 4, 5, 6, 24 and 48 h after administration of each dosage form from ear veins into vials containing 50 µl of an aqueous 3.8% (w/v) sodium citrate solution. The blood samples were immediately centrifuged at 2500 rpm for 5 min. The clotting time of heparin in the plasma were measured by aPTT assay. 2.8.1. Activated partial thromboplastin time (aPTT) assay The anticoagulant activity of heparin in plasma sample was determined by aPTT assay according to the literature [20]. Briefly, 0.1 ml of plasma samples was incubated with 0.1 ml of aPTT reagent for 2 min at 37 °C. After incubation, 0.1 ml of 0.02 M calcium chloride solution was added, and the time for formation of fibrin clot was recorded. Same procedure was performed for heparin-loaded microspheres before dosing for heparin solution administration, and for blank plasma samples taken from the rats as a control. 2.9. Statistical analysis In vitro release data obtained from each experiment were subjected to statistical analysis using a computer programme called GraphPad-Prism 3.0 software, for a one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls multiple comparisons test. The significance of the differences was evaluated by Student’s t-test. The level of significance chosen was P < 0.05.
3. Results and discussion The scanning electron microphotograph results exhibited good spherical shape and smooth surface for all microsphere formulations (S1–S6 and D1–D6). Fig. 1 shows typical scan-
Fig. 1. Scanning electron micrographs of heparin-loaded microspheres (a) single (b) double emulsion methods (× 6000) [Bars are 2 µm].
922
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
Fig. 3. Size distribution of heparin-loaded microspheres.
Fig. 2. DSC spectra of heparin-loaded microspheres (A) and blank microspheres (B).
ning electron microphotographs of heparin-loaded microspheres prepared using single and double emulsion technique. Thermal analyses of blank and heparin-loaded microspheres were performed by differential scanning calorimetry (DSC). The values of the glass transition (Tg), crystallization (Tc) and melting (Mp) of the polymer and heparin-loaded microspheres were observed at 57.81 and 59.02, 83.62 °C and 83.73, 141.63 and 141.12 °C, respectively (Fig. 2). These data were in agreement with previous studies [10,21,22]. These results indicated no specific interactions between heparin and PLA. The average particle sizes of heparin-loaded microspheres ranged as between 1.4 and 2.9 µm for all microsphere formulations (S1–S6 and D1–D6) (Table 2). The average particle sizes of microsphere formulations prepared at different polymer/drug ratios (1:10, 1:2.5, 1:1) did not show significant differences from each other (P > 0.05). Increasing the PVA concentration from 1.5% to 2.5% (w/v) resulted in no
significant change with this respect (P > 0.05). These data are in agreement with literature [23]. Because of similarity of size distribution pattern of all formulations, a representative example of microsphere formulations prepared is shown in (Fig. 3). As can be seen in this figure, the particle size range was narrow. The d50 values of all the prepared microsphere formulations except formulations S1 and D1 are smaller than 2 µm (Table 2). Such small particles have been considered to be suitable for nasal administration [6]. As can be seen in Table 2, the drug loading capacity increased with the increasing amount of theoretical drug. The encapsulation efficiency of all microsphere formulations was found higher than 20% except for formulations S4 and D4. Similar findings have been obtained in some studies on solvent evaporation technique [23]. Formulation D5 had the highest encapsulation efficiency (37.01 ± 0.83%). When the microsphere yields were compared with each other, the results showed that microsphere yield decreased with increasing amount of theoretical heparin (Table 2). This may be due to high solubility of heparin in water [2]. In addition, when single and double emulsion methods were compared, encapsulation efficiencies of formulations prepared with double emulsion (D1–D6) was found higher than those obtained with single emulsion. It has been reported that double emulsion is a suitable method for especially hydrophilic drugs [24]. In vitro release study results indicated that microspheres prepared with single (S1–S6) and double (D1–D6) emulsion
Table 2 The theoretical drug amount, encapsulation efficiency and mean particle size of all heparin-loaded microspheres Code S1 S2 S3 S4 S5 S6 D1 D2 D3 D4 D5 D6
Theoretical load (mg) 25 100 250 25 100 250 25 100 250 25 100 250
Actual load (mg) 6.26 ± 0.54 30.67 ± 1.09 51.38 ± 0.22 3.53 ± 1.2 32.03 ± 0.95 80.85 ± 0.32 6.84 ± 0.18 35.11 ± 1.61 89.45 ± 0.85 4.71 ± 1.73 37.01 ± 0.83 85.13 ± 1.22
Data were expressed as the mean ± S.D. (N = 3).
Encapsulation efficiency (%) 25.03 ± 2.24 30.67 ± 1.09 20.55 ± 0.31 14.12 ± 5.79 32.03 ± 0.95 32.26 ± 0.05 27.35 ± 0.8 35.11 ± 1.61 35.78 ± 0.63 18.85 ± 8.31 37.01 ± 0.83 34.05 ± 1.90
Yield efficiency (%) 65.37 ± 0.08 51.35 ± 0.73 30.75 ± 0.21 63.07 ± 2.23 47.02 ± 0.74 29.03 ± 3.09 62.31 ± 0.04 47.53 ± 0.92 29.15 ± 0.78 60.91 ± 1.42 45.54 ± 1.56 29.25 ± 2.42
Mean particle size (µm) 1.394 ± 0.016 1.688 ± 0.076 1.597 ± 0.046 1.532 ± 0.014 1.426 ± 0.040 1.517 ± 0.047 1.692 ± 0.026 1.688 ± 0.018 1.625 ± 0.099 1.514 ± 0.016 1.534 ± 0.036 1.425 ± 0.122
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
methods provide a sustained–release effect up to 8 h (Figs. 4 and 5). At the end of the eighth, heparin amount released from S2 to S6 and D1–D5 was found to be < 0.12 and < 0.13 mg, whereas S1 and D6 formulations were reached 0.16 and 0.60 mg, respectively. When the release data were evaluated statistically (GraphPad-Prism 3.0 Software), it was found that released heparin amounts from S1 and D6 were significantly higher (P < 0.05) than all other formulations (S2–S6 and D1–D5). For this reason, the two formulations (S1 and D6) providing highest heparin release were chosen for in vivo evaluation in rats via nasal route. After nasal administration of heparin-loaded microspheres (S1 and D6) and heparin solution to rats, the mean clotting time determined by the aPTT (which reflects both the anti-IIa and anti-Xa activity) was evaluated in blood plasma as a function of time (Fig. 6). The normal clotting time in rats was found to be approximately 23.0 ± 3.1 s whereas maximum aPTT level was obtained with
923
heparin solution at 3 h (100.2 ± 9.4 s), heparin-loaded microsphere formulations (S1 and S6) led to maximum anticoagulant activity at 5 h, the maximal aPTT levels being 52.6 ± 3.9 and 94.6 ± 4.2 s for S1 and D6, respectively. The anticoagulant effects of microsphere formulations S1 and D6 were significant at 3 h, reached its maximum at 5 h and was maintained for 48 h at significant level (differences at 24 and 48 h were significant (P < 0.05) for both formulations when compared to heparin solution) (Fig. 6). The delay in onset of effect (aPTT level) observed with both microsphere formulations can be explained by the slower release of heparin. Based on the area under the curve (AUC) of the clotting time as a function of time between 0 and 48 h, a tentative pharmacological bioavailability was calculated (Table 3). Relative pharmacological bioavailability data calculated according to AUC and applicated dosage was found to be 61.31% and 143.63% for S1 and D6, respectively. This result indicated that especially the D6 formulation provides sustained–release effect during 48 h. In conclusion, in this study heparin-loaded microspheres having smooth surface and small particle size were obtained by using a PLA. In vitro release studies showed that heparin
Fig. 4. In vitro release profiles of heparin-loaded microspheres prepared with single emulsion: S1: (1:10) drug/polymer ratio with %1.5 PVA (♦), S2: (1:1) drug/polymer ratio, %1.5 PVA (n) , S3: (1:2.5) drug/polymer ratio, %1.5 PVA (m), S4: (1:10) drug/polymer ratio with %2.5 PVA (·), S5: (1:1) drug/polymer ratio, %2.5 PVA (M) , S6: (1:2.5) drug/polymer ratio, %2.5PVA (*). Experiments were performed in barbital buffer (pH 8.61 ± 0.02). Data are mean ± S.D. (N = 3).
Fig. 6. Clotting time profiles of heparin solution and heparin-loaded microspheres after nasal administration to rats. Heparin solution (10 mg/kg) (n); formulation S1 (31.25 mg/kg) (*); formulation D6 (42.50 mg/kg) (m). The clotting time was measured by aPTT assay. The data are plotted as mean ± S.D., (N = 3). Table 3 Main pharmacokinetic parameters obtained for heparin solution and heparinloaded microspheres after nasal administration in rats
Fig. 5. In vitro release profiles of heparin-loaded microspheres prepared with double emulsion: D1: (1:10) drug/polymer ratio with %1.5 PVA (♦), D2: (1:1) drug/polymer ratio, %1.5 PVA (n) , D3: (1:2.5) drug/polymer ratio,%1.5 PVA (m), D4: (1:10) drug/polymer ratio with %2.5 PVA (·), D5: (1:1) drug/polymer ratio, %2.5 PVA (M) , D6: (1:2.5) drug/polymer ratio, %2.5 PVA (*). Experiments were performed in barbital buffer (pH 8.61 ± 0.02). Data are mean ± S.D. (N = 3).
Parameters Applied dose (mg/kg) Maximal aPTTa level (s) tmax (h) AUC0-48 (s.h/kg) RBb (%)
Formulations Heparin solution 10.00 100.2 ± 9.4 3 210.29 –
S1 31.25 52.6 ± 3.9 5 402.92 61.31c
AUC indicates area under the curve. a The data are shown as mean ± S.D. (N = 3). b Relative bioavailability. c Statistically different from heparin solution (P < 0.05).
D6 42.50 94.6 ± 4.2 5 1285.63 143.63c
924
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
microspheres provide sustained–release effect and in vivo studies indicated that heparin-loaded microspheres could be applied via nasal route.
[9]
[10]
Acknowledgements [11]
This work was supported by the Research Fund of Istanbul University. Project number: T-953/06112000. Authors wish to thank Mustafa Nevzat Pharmaceutical Company for kindly supplying heparin. In vitro part of this study was presented at the Fourth World Meeting on Pharmaceutics, Biopharmaceutics, Pharmaceutical Technology, April 8–11, 2002, Florence, Italy. In vivo part of this study was resented at the Fifth International Conference and Workshop on Cell Culture and In vitro Models of Drug Absorption and Delivery, February 25–March 5, 2004, Saarbrücken, Germany.
[12]
[13]
[14]
[15]
References [1] [2]
[3]
[4]
[5]
[6] [7]
[8]
Martindale, in: The Extra Pharmacopeia, The Pharmaceutical Press, London, 2002, pp. 902–905 (33rd ed.). F. Nachtmann, G. Atzl, W.D. Roth, Heparin sodium, in: K. Florey (Ed.), Analytical Profiles, Academic Press, New York, 1983, pp. 215– 276. P.W. Majerus, G.J. Broze, J.P. Miletich, D.M. Tollefsen, Anticoagulant, Thrombolytic, and Antiplatelet Drugs, in: J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, A.G. Gilman (Eds.), Goodman & Gilman’s The Pharmacological Basis of Therapeutics, ninth ed, The Mc Graw-Hill Companies Inc., New York, 1996, pp. 1341–1359. J. Dawes, C.V. Prowse, D.S. Pepper, Absorption of heparin, LMW heparin and SP-54 after subcutaneous injection, assessed by competitive binding assay, Thromb. Res. 44 (1986) 683–693. C.R. Behl, H.K. Pimplaskar, A.P. Sileno, J. deMeireles, V.D. Romeo, Effects of physicochemical properties and other factors on sytemic nasal drug delivery, Adv. Drug Deliv. 29 (1998) 89–116. L. Pereswetoff-Morath, Microspheres as nasal drug delivery systems, Adv. Drug Deliv. 29 (1998) 185–194. S. Cohen, T. Yoshioka, M. Lucarelli, L.H. Hwang, R. Larger, Controlled delivery systems for proteins based on poly (lactic/glycolic acid) microspheres, Pharm. Res. 8 (1991) 713–720. D. Teomim, I. Fishbien, G. Golomb, L. Orloff, M. Mayberg, A.J. Domb, Perivascular delivery of heparin for the reduction of smooth muscle cell proliferation after endothelial injury, J. Control. Release 60 (1999) 129–142.
[16]
[17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
E.R. Edelman, D.A. Adams, M.J. Karnovsky, Effect of controlled adventitial heparin delivery on smooth muscle all proliferation following endothelial injury, Proc. Natl. Acad. Sci. USA 87 (1990) 3773–3777. M.R. Kreitz, J.A. Domm, E. Mathiowitz, Controlled delivery of therapeutics from microporous membranes, II. In vitro degradation and release of heparin-loaded poly(D,L-lactide-co-glycolide), Biomaterials 18 (1997) 1645–1651. Z. Yang, P. Birkenhauer, F. Julmy, D. Chickering, J.P. Ranieri, H.P. Merkle, T.F. Lüscher, B. Gander, Sustained release of heparin from polymeric particles for inhibition of human vascular smooth muscle cell proliferation, J. Control. Release 60 (1999) 269–277. E.R. Edelman, A. Nathan, M. Katada, J. Gates, M.J. Karnovsky, Perivascular graft heparin delivery using biodegradable polymer wraps, Biomaterials 21 (2000) 2279–2286. Y. Jiao, N. Ubrich, M.M. Arvier, C. Vigneron, M. Hoffman, T. Lecompte, P. Maincent, In vitro and in vivo evaluation of oral heparin-loaded polymeric nanoparticles in rabbits, Circulations 105 (2002) 230–235. Y. Jiao, N. Ubrich, V. Hoffart, M.M. Arvier, C. Vigneron, M. Hoffman, P. Maincent, Anticoagulant activity of heparin following oral administration of heparin-loaded microparticles in rabbits, J. Pharm. Sci. 91 (2002) 760–768. L.P. Tan, S.S. Venkatraman, P.F. Sung, X.T. Wang, Effect of plasticization on heparin release from biodegradable matrices, Int. J. Pharm. 283 (2004) 89–96. Y.K. Lee, J.H. Nam, H.C. Shin, Y. Byun, Conjugation of lowmolecular-weight heparin and deoxycholic acid for the development of a new oral anticoagulant agent, Circulation 104 (2001) 3116–3120. T. Yang, A. Hussain, J. Paulson, T.J. Abbruscato, F. Ahsan, Cyclodextrins in nasal delivery of low-molecular-weight heparins: in vivo and in vitro studies, Pharm. Res. 21 (2004) 1127–1136. S. Benita, J.P. Benoit, F. Puisieux, C. Thies, Characterisation of drug-loaded poly(D,L-lactide) microspheres, J. Pharm. Sci. 73 (1984) 1721–1724. S.R. Gundry, M.D. Klein, R.A. Drongowsky, M.M. Kirsh, Clinical evaluation of a new rapid heparin assay using the dye Azure A, Am. J. Surg. 148 (1984) 191–194. C.D. Marci, D. Prager, A review of the clinical indications for the plasma heparin assay, Clin. Pathol. 99 (1993) 546–550. M. Cavalier, J.P. Benoit, C. Thies, The formation and characterization of hydrocortisone-loaded poly((±)-lactide) microspheres, J. Pharm. Pharmacol. 38 (1986) 249–253. B.R. Conway, H.O. Alpar, Double emulsion microencapsulation of proteins as model antigens using polylactide polymers: effect of emulsifier on the microsphere characteristics and release kinetics, Eur. J. Pharm. Biopharm. 42 (1996) 42–48. J.P. McGee, M. Singh, X.M. Li, H. Qiu, D.T. O’Hagan, The encapsulation of model protein in poly (D,L lactide-co-glycolide) microparticles of various sizes: an evaluation of process reproducibility, J. Microencapsul. 14 (1997) 197–210. T. Uchida, K. Yoshida, Y. Nakada, N. Nagareya, Y. Konishi, A. Nakai, M. Nishikata, K. Matsuyama, Preparation and characterization of polylactic acid microspheres containing water-soluble anesthetics with mall molecular weight, Chem. Pharm. Bull. (Tokyo) 45 (1997) 513–517.
Original article
Nasal administration of heparin-loaded microspheres based on poly(lactic acid) Ayca Yıldız a, Alper Okyar b, Gül Baktır b, Ahmet Araman a, Yıldız Özsoy a,* a
Department of Pharmaceutical Technology, Faculty of Pharmacy, Istanbul University, Beyazt, 34116-Universite, Istanbul, Turkey b Department of Pharmacology, Faculty of Pharmacy, Istanbul University, 34116-Universite, Istanbul, Turkey Received 13 January 2005; received in revised form 1 August 2005; accepted 4 August 2005
Abstract In this study, heparin-loaded microspheres having smooth surface and small particle size were designed in order to provide the absorption of heparin through nasal mucosa. For this purpose, microspheres at different polymer/drug ratios (1:10, 1:2.5 and 1:1) and at different concentrations of polyvinyl alcohol, emulsifying agent (1.5% and 2.5% w/v) were prepared by solvent evaporation method with poly(lactic acid). The microspheres were for evaluated shape and surface properties, particle size, production yield, encapsulation efficiency and in vitro drug release. Based on the in vitro data, selected microspheres were applied by nasal route to Wistar albino rats. According to in vivo studies, heparin-loaded microspheres may be used by nasal route as an alternative to parenteral route. © 2005 Elsevier SAS. All rights reserved. Keywords: Heparin; Poly(lactic acid); Microspheres; Nasal route; aPTT
1. Introduction Heparin is one of the most potent anticoagulant widely used for the treatment and prevention of deep vein thrombosis [1]. The mean half-life observed in practically human cases amounts to approximately 1.5 h [2]. Heparin is degraded to ineffective fragments after oral ingestion [3] and while subcutaneous administration is efficacious, it is uncomfortable and associated with poor patient compliance [4]. In recent years, the nasal cavity has been widely investigated as a potential site for non-invasive drug delivery. As a site for drug delivery, nasal cavity possesses many advantages such as a large surface area for absorption with a highly vascularized subepithelial layer. In addition, blood is drained directly from the nose into the systemic circulation, thereby avoiding first-pass metabolism by the liver [5]. Microspheres of different materials have been evaluated in vivo as nasal drug delivery system [6]. Among the biodegradable polymers, poly(lactic acid) (PLA), a polyester, is the most widely used material for preparing microspheres because of its biodegradability and low toxicity [7]. * Corresponding author. Tel.: +90 212 440 0000/13498; fax: +90 212 440 0252. E-mail address: [email protected] (Y. Özsoy). 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.08.004
In the literature, heparin implants have been prepared with PLA [8], ethylene-vinyl acetate [9] and poly(D,L-lactic-coglycolic acid) (PLGA) [10–12] for local treatment; heparinloaded microparticles prepared using biodegradable (poly-ecaprolactone, PLA, PLGA) and nonbiodegradable (Eudragit RS and RL) polymers have been evaluated after oral administration in rabbits [13–15]; conjugates of heparin with deoxycholic acid have been synthesized in order to enhance heparin absorption from the gastrointestinal tract [16]. Yang et al. [17] has reported that low-molecular-weight heparin with cyclodextrins can be used nasal delivery. There were no data on heparin microspheres applied via nasal route in the literature. In this study, heparin-loaded microspheres were prepared with PLA using solvent evaporation method. The surface morphology of microspheres was evaluated. The influence of drug-polymer ratio and emulsifying agent concentration on the formation of microspheres, drug loading capacity and particle size were also investigated. The formulations were characterized by in vitro release study and the formulations providing suitable highest drug release were selected for in vivo study via nasal route. After nasal administration of these formulations to rats, the time course of the anticoagulant activ-
920
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
ity was investigated by means of aPTT and compared with heparin solution.
2. Materials and methods 2.1. Materials PLA was purchased from Boehringer Ingelheim (Resomer L104). Polyvinyl alcohol (PVA) (Mw: 13,000–23,000, 88% hydrolyzed) was obtained from E. Merck. Heparin (195.3 IU/mg) was supplied by Mustafa Nevzat Pharmaceutical Company, Istanbul, Turkey. Sodium azide, methylene chloride, sodium hydroxide, hydrochloric acid, sodium chloride were from E. Merck. Azure A was from Eastman and methylcellulose was from Sigma.
ant primary emulsion was added to 75 ml of an aqueous PVA solution (1.5% or 2.5%, w/v) at 13500 rpm for 5 min. This water–oil–water (w/o/w) emulsion was stirred with a magnetic stirrer overnight at room temperature to allow the solvent to evaporate. The particles were separated by centrifugation, washed three times with distilled water, lyophilized coded (D1–D6) and kept under room temperature until analysis. All formulations were prepared as triplicate. 2.3. Morphology of microspheres Prepared microspheres were coated with gold–palladium under an argon atmosphere at room temperature and then the surface morphology of the microspheres was determined by scanning electron microscopy using a JEOL 840A JXA. 2.4. Differential scanning calorimetry
2.2. Preparation of heparin microspheres Heparin-loaded microspheres (12 formulations) were prepared with single and double emulsions using a solvent evaporation technique [18]. The codes of the prepared microspheres can be seen in (Table 1). Apart from these formulations, blank microspheres without heparin were also prepared using same methods for comparison.
Thermograms of the blank microspheres and heparinloaded microspheres were produced using a computerinterfaced Perkin–Elmer System 4 differential scanning calorimeter. Samples (4–8 mg) were sealed into aluminum pans and measurements were run from 25 to 300 °C against an empty pan. 2.5. Particle size distribution
2.2.1. Single emulsion (o/w) Two hundred fifty milligrams of PLA were dissolved in dichloromethane and different amounts of the drug (25, 100 and 250 mg) were dispersed in this solution. It was then dropped into 75 ml of the aqueous solution of PVA (1.5%, 2.5% w/v) to form oil–water (o/w) emulsion and stirred using a high speed homogenizer (CAT-X620) at a constant rate of 13500 rpm for 5 min. The resultant emulsion was left overnight at room temperature by continuously stirring with a magnetic stirrer to evaporate organic phase. The microspheres formed were separated by centrifugation at 3000 rpm. The prepared microspheres were washed three times with distilled water. Subsequently, the microspheres were lyophilized, coded (S1–S6) and kept under room temperature until analysis. All formulations were prepared as triplicate. 2.2.2. Double emulsion (w/o/w) The primary emulsion (w/o) containing required amounts of heparin (25, 100 and 250 mg) with PVA (12.5% w/v) and methylcellulose (1.25%, w/v) was formed using a high speed homogenizer (CAT-X620) at 13500 rpm for 2 min. The result-
The lyophilized microspheres were dispersed in filtered (0.22 µm membrane filter) 0.9% (w/w) sodium chloride. Volume size distributions were obtained using a Malvern MasterSizer (Malvern Instruments, Malvern, UK). The size of microspheres is expressed as d10, d50 and d90 representing the size below which 10%, 50%, and 90% by weight of sample falls, respectively. The values (d50) were expressed for all formulations as mean size range. 2.6. Determination of actual drug loading and encapsulation effıciency The amount of heparin entrapped in microspheres was determined with Azure A colorimetric method [19] based on metachromasy by measuring the amount of non-entrapped drug in the external aqueous solution recovered after centrifugation and washing of the microspheres. Azure A is reacted with a heparin relies on the metachromatic shift of the absorbance of the heparin–Azure A complex from 620 to 510 nm. Typically, aliquots (1 ml) of aqueous samples were reacted
Table 1 The code of heparin-loaded microspheres with single (S1–S6) and double (D1–D6) emulsion Formulation code S1, D1 S2, D2 S3, D3 S4, D4 S5, D5 S6, D6
Heparin amount (mg) 25 100 250 25 100 250
Polymer amount (mg) 250 250 250 250 250 250
Polymer/drug ratio 1:10 1:2.5 1:1 1:10 1:2.5 1:1
PVA amount (%, w/v) 1.5 1.5 1.5 2.5 2.5 2.5
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
with 1.43 ml of Azure A solution (0.09 mg/ml, w/v) and 1.43 ml barbital buffer (pH 8.61 ± 0.02). The absorbance of solutions was measured at 510 nm within 30 min using Shimadzu UV–Vis-1601 spectrophotometer. Assay studies were performed in triplicate. Polymer solution did not interfere with assay method. The actual drug loading and percentage of encapsulation efficiency were calculated as follows: Actual load (mg) = A – B A = Theoretical heparin content (mg) B = Calculated amount of the heparin in the external aqueous solution (mg) Encapsulation efficiency 共 % 兲 = Actual load 共 % w/w 兲 Theorical heparin content 共 % w/w 兲
× 100
2.7. In vitro release experiments For in vitro release study, 25 mg of heparin-loaded microspheres were suspended in 10 ml of barbital buffer (pH 8.61 ± 0.02) containing 0.01% (w/v) sodium azide, an antibacterial agent. The microsphere suspension was gently stirred (100 rpm) using horizontal shaking (Minishaker, B. Braun, Model Kühner) at room temperature. Two milliliters of samples were withdrawn at appropriate intervals (1–8 h) from the dissolution medium and replaced by 2 ml of fresh medium. The samples were centrifuged for 5 min. The heparin amount of the samples was assayed according to the colorimetric method described above. All release studies were performed in triplicate. 2.8. In vivo study Normal female Wistar albino rats (about 160–210 g body weight) were used for in vivo experiments. They were fasted overnight before dosing and divided into three groups of three animals each. Microsphere formulations (S1 and D6) selected according to in vitro release experiments were suspended in 0.9% (w/v) sodium chloride solution and were administered
921
as a 20 µl aliquot to the nostrils via micropipet to rats placed in supine position. The drops were administered slowly to avoid any leakage. The heparin amount applied was 31.25 mg/kg for formulation S1 and 42.5 mg/kg for formulation D6. Heparin solution (10 mg/kg) was administered by nasal route for comparison. Blood samples (1.8 ml) were withdrawn before and 0.5, 1, 2, 3, 4, 5, 6, 24 and 48 h after administration of each dosage form from ear veins into vials containing 50 µl of an aqueous 3.8% (w/v) sodium citrate solution. The blood samples were immediately centrifuged at 2500 rpm for 5 min. The clotting time of heparin in the plasma were measured by aPTT assay. 2.8.1. Activated partial thromboplastin time (aPTT) assay The anticoagulant activity of heparin in plasma sample was determined by aPTT assay according to the literature [20]. Briefly, 0.1 ml of plasma samples was incubated with 0.1 ml of aPTT reagent for 2 min at 37 °C. After incubation, 0.1 ml of 0.02 M calcium chloride solution was added, and the time for formation of fibrin clot was recorded. Same procedure was performed for heparin-loaded microspheres before dosing for heparin solution administration, and for blank plasma samples taken from the rats as a control. 2.9. Statistical analysis In vitro release data obtained from each experiment were subjected to statistical analysis using a computer programme called GraphPad-Prism 3.0 software, for a one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls multiple comparisons test. The significance of the differences was evaluated by Student’s t-test. The level of significance chosen was P < 0.05.
3. Results and discussion The scanning electron microphotograph results exhibited good spherical shape and smooth surface for all microsphere formulations (S1–S6 and D1–D6). Fig. 1 shows typical scan-
Fig. 1. Scanning electron micrographs of heparin-loaded microspheres (a) single (b) double emulsion methods (× 6000) [Bars are 2 µm].
922
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
Fig. 3. Size distribution of heparin-loaded microspheres.
Fig. 2. DSC spectra of heparin-loaded microspheres (A) and blank microspheres (B).
ning electron microphotographs of heparin-loaded microspheres prepared using single and double emulsion technique. Thermal analyses of blank and heparin-loaded microspheres were performed by differential scanning calorimetry (DSC). The values of the glass transition (Tg), crystallization (Tc) and melting (Mp) of the polymer and heparin-loaded microspheres were observed at 57.81 and 59.02, 83.62 °C and 83.73, 141.63 and 141.12 °C, respectively (Fig. 2). These data were in agreement with previous studies [10,21,22]. These results indicated no specific interactions between heparin and PLA. The average particle sizes of heparin-loaded microspheres ranged as between 1.4 and 2.9 µm for all microsphere formulations (S1–S6 and D1–D6) (Table 2). The average particle sizes of microsphere formulations prepared at different polymer/drug ratios (1:10, 1:2.5, 1:1) did not show significant differences from each other (P > 0.05). Increasing the PVA concentration from 1.5% to 2.5% (w/v) resulted in no
significant change with this respect (P > 0.05). These data are in agreement with literature [23]. Because of similarity of size distribution pattern of all formulations, a representative example of microsphere formulations prepared is shown in (Fig. 3). As can be seen in this figure, the particle size range was narrow. The d50 values of all the prepared microsphere formulations except formulations S1 and D1 are smaller than 2 µm (Table 2). Such small particles have been considered to be suitable for nasal administration [6]. As can be seen in Table 2, the drug loading capacity increased with the increasing amount of theoretical drug. The encapsulation efficiency of all microsphere formulations was found higher than 20% except for formulations S4 and D4. Similar findings have been obtained in some studies on solvent evaporation technique [23]. Formulation D5 had the highest encapsulation efficiency (37.01 ± 0.83%). When the microsphere yields were compared with each other, the results showed that microsphere yield decreased with increasing amount of theoretical heparin (Table 2). This may be due to high solubility of heparin in water [2]. In addition, when single and double emulsion methods were compared, encapsulation efficiencies of formulations prepared with double emulsion (D1–D6) was found higher than those obtained with single emulsion. It has been reported that double emulsion is a suitable method for especially hydrophilic drugs [24]. In vitro release study results indicated that microspheres prepared with single (S1–S6) and double (D1–D6) emulsion
Table 2 The theoretical drug amount, encapsulation efficiency and mean particle size of all heparin-loaded microspheres Code S1 S2 S3 S4 S5 S6 D1 D2 D3 D4 D5 D6
Theoretical load (mg) 25 100 250 25 100 250 25 100 250 25 100 250
Actual load (mg) 6.26 ± 0.54 30.67 ± 1.09 51.38 ± 0.22 3.53 ± 1.2 32.03 ± 0.95 80.85 ± 0.32 6.84 ± 0.18 35.11 ± 1.61 89.45 ± 0.85 4.71 ± 1.73 37.01 ± 0.83 85.13 ± 1.22
Data were expressed as the mean ± S.D. (N = 3).
Encapsulation efficiency (%) 25.03 ± 2.24 30.67 ± 1.09 20.55 ± 0.31 14.12 ± 5.79 32.03 ± 0.95 32.26 ± 0.05 27.35 ± 0.8 35.11 ± 1.61 35.78 ± 0.63 18.85 ± 8.31 37.01 ± 0.83 34.05 ± 1.90
Yield efficiency (%) 65.37 ± 0.08 51.35 ± 0.73 30.75 ± 0.21 63.07 ± 2.23 47.02 ± 0.74 29.03 ± 3.09 62.31 ± 0.04 47.53 ± 0.92 29.15 ± 0.78 60.91 ± 1.42 45.54 ± 1.56 29.25 ± 2.42
Mean particle size (µm) 1.394 ± 0.016 1.688 ± 0.076 1.597 ± 0.046 1.532 ± 0.014 1.426 ± 0.040 1.517 ± 0.047 1.692 ± 0.026 1.688 ± 0.018 1.625 ± 0.099 1.514 ± 0.016 1.534 ± 0.036 1.425 ± 0.122
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
methods provide a sustained–release effect up to 8 h (Figs. 4 and 5). At the end of the eighth, heparin amount released from S2 to S6 and D1–D5 was found to be < 0.12 and < 0.13 mg, whereas S1 and D6 formulations were reached 0.16 and 0.60 mg, respectively. When the release data were evaluated statistically (GraphPad-Prism 3.0 Software), it was found that released heparin amounts from S1 and D6 were significantly higher (P < 0.05) than all other formulations (S2–S6 and D1–D5). For this reason, the two formulations (S1 and D6) providing highest heparin release were chosen for in vivo evaluation in rats via nasal route. After nasal administration of heparin-loaded microspheres (S1 and D6) and heparin solution to rats, the mean clotting time determined by the aPTT (which reflects both the anti-IIa and anti-Xa activity) was evaluated in blood plasma as a function of time (Fig. 6). The normal clotting time in rats was found to be approximately 23.0 ± 3.1 s whereas maximum aPTT level was obtained with
923
heparin solution at 3 h (100.2 ± 9.4 s), heparin-loaded microsphere formulations (S1 and S6) led to maximum anticoagulant activity at 5 h, the maximal aPTT levels being 52.6 ± 3.9 and 94.6 ± 4.2 s for S1 and D6, respectively. The anticoagulant effects of microsphere formulations S1 and D6 were significant at 3 h, reached its maximum at 5 h and was maintained for 48 h at significant level (differences at 24 and 48 h were significant (P < 0.05) for both formulations when compared to heparin solution) (Fig. 6). The delay in onset of effect (aPTT level) observed with both microsphere formulations can be explained by the slower release of heparin. Based on the area under the curve (AUC) of the clotting time as a function of time between 0 and 48 h, a tentative pharmacological bioavailability was calculated (Table 3). Relative pharmacological bioavailability data calculated according to AUC and applicated dosage was found to be 61.31% and 143.63% for S1 and D6, respectively. This result indicated that especially the D6 formulation provides sustained–release effect during 48 h. In conclusion, in this study heparin-loaded microspheres having smooth surface and small particle size were obtained by using a PLA. In vitro release studies showed that heparin
Fig. 4. In vitro release profiles of heparin-loaded microspheres prepared with single emulsion: S1: (1:10) drug/polymer ratio with %1.5 PVA (♦), S2: (1:1) drug/polymer ratio, %1.5 PVA (n) , S3: (1:2.5) drug/polymer ratio, %1.5 PVA (m), S4: (1:10) drug/polymer ratio with %2.5 PVA (·), S5: (1:1) drug/polymer ratio, %2.5 PVA (M) , S6: (1:2.5) drug/polymer ratio, %2.5PVA (*). Experiments were performed in barbital buffer (pH 8.61 ± 0.02). Data are mean ± S.D. (N = 3).
Fig. 6. Clotting time profiles of heparin solution and heparin-loaded microspheres after nasal administration to rats. Heparin solution (10 mg/kg) (n); formulation S1 (31.25 mg/kg) (*); formulation D6 (42.50 mg/kg) (m). The clotting time was measured by aPTT assay. The data are plotted as mean ± S.D., (N = 3). Table 3 Main pharmacokinetic parameters obtained for heparin solution and heparinloaded microspheres after nasal administration in rats
Fig. 5. In vitro release profiles of heparin-loaded microspheres prepared with double emulsion: D1: (1:10) drug/polymer ratio with %1.5 PVA (♦), D2: (1:1) drug/polymer ratio, %1.5 PVA (n) , D3: (1:2.5) drug/polymer ratio,%1.5 PVA (m), D4: (1:10) drug/polymer ratio with %2.5 PVA (·), D5: (1:1) drug/polymer ratio, %2.5 PVA (M) , D6: (1:2.5) drug/polymer ratio, %2.5 PVA (*). Experiments were performed in barbital buffer (pH 8.61 ± 0.02). Data are mean ± S.D. (N = 3).
Parameters Applied dose (mg/kg) Maximal aPTTa level (s) tmax (h) AUC0-48 (s.h/kg) RBb (%)
Formulations Heparin solution 10.00 100.2 ± 9.4 3 210.29 –
S1 31.25 52.6 ± 3.9 5 402.92 61.31c
AUC indicates area under the curve. a The data are shown as mean ± S.D. (N = 3). b Relative bioavailability. c Statistically different from heparin solution (P < 0.05).
D6 42.50 94.6 ± 4.2 5 1285.63 143.63c
924
A. Yıldız et al. / Il Farmaco 60 (2005) 919–924
microspheres provide sustained–release effect and in vivo studies indicated that heparin-loaded microspheres could be applied via nasal route.
[9]
[10]
Acknowledgements [11]
This work was supported by the Research Fund of Istanbul University. Project number: T-953/06112000. Authors wish to thank Mustafa Nevzat Pharmaceutical Company for kindly supplying heparin. In vitro part of this study was presented at the Fourth World Meeting on Pharmaceutics, Biopharmaceutics, Pharmaceutical Technology, April 8–11, 2002, Florence, Italy. In vivo part of this study was resented at the Fifth International Conference and Workshop on Cell Culture and In vitro Models of Drug Absorption and Delivery, February 25–March 5, 2004, Saarbrücken, Germany.
[12]
[13]
[14]
[15]
References [1] [2]
[3]
[4]
[5]
[6] [7]
[8]
Martindale, in: The Extra Pharmacopeia, The Pharmaceutical Press, London, 2002, pp. 902–905 (33rd ed.). F. Nachtmann, G. Atzl, W.D. Roth, Heparin sodium, in: K. Florey (Ed.), Analytical Profiles, Academic Press, New York, 1983, pp. 215– 276. P.W. Majerus, G.J. Broze, J.P. Miletich, D.M. Tollefsen, Anticoagulant, Thrombolytic, and Antiplatelet Drugs, in: J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, A.G. Gilman (Eds.), Goodman & Gilman’s The Pharmacological Basis of Therapeutics, ninth ed, The Mc Graw-Hill Companies Inc., New York, 1996, pp. 1341–1359. J. Dawes, C.V. Prowse, D.S. Pepper, Absorption of heparin, LMW heparin and SP-54 after subcutaneous injection, assessed by competitive binding assay, Thromb. Res. 44 (1986) 683–693. C.R. Behl, H.K. Pimplaskar, A.P. Sileno, J. deMeireles, V.D. Romeo, Effects of physicochemical properties and other factors on sytemic nasal drug delivery, Adv. Drug Deliv. 29 (1998) 89–116. L. Pereswetoff-Morath, Microspheres as nasal drug delivery systems, Adv. Drug Deliv. 29 (1998) 185–194. S. Cohen, T. Yoshioka, M. Lucarelli, L.H. Hwang, R. Larger, Controlled delivery systems for proteins based on poly (lactic/glycolic acid) microspheres, Pharm. Res. 8 (1991) 713–720. D. Teomim, I. Fishbien, G. Golomb, L. Orloff, M. Mayberg, A.J. Domb, Perivascular delivery of heparin for the reduction of smooth muscle cell proliferation after endothelial injury, J. Control. Release 60 (1999) 129–142.
[16]
[17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
E.R. Edelman, D.A. Adams, M.J. Karnovsky, Effect of controlled adventitial heparin delivery on smooth muscle all proliferation following endothelial injury, Proc. Natl. Acad. Sci. USA 87 (1990) 3773–3777. M.R. Kreitz, J.A. Domm, E. Mathiowitz, Controlled delivery of therapeutics from microporous membranes, II. In vitro degradation and release of heparin-loaded poly(D,L-lactide-co-glycolide), Biomaterials 18 (1997) 1645–1651. Z. Yang, P. Birkenhauer, F. Julmy, D. Chickering, J.P. Ranieri, H.P. Merkle, T.F. Lüscher, B. Gander, Sustained release of heparin from polymeric particles for inhibition of human vascular smooth muscle cell proliferation, J. Control. Release 60 (1999) 269–277. E.R. Edelman, A. Nathan, M. Katada, J. Gates, M.J. Karnovsky, Perivascular graft heparin delivery using biodegradable polymer wraps, Biomaterials 21 (2000) 2279–2286. Y. Jiao, N. Ubrich, M.M. Arvier, C. Vigneron, M. Hoffman, T. Lecompte, P. Maincent, In vitro and in vivo evaluation of oral heparin-loaded polymeric nanoparticles in rabbits, Circulations 105 (2002) 230–235. Y. Jiao, N. Ubrich, V. Hoffart, M.M. Arvier, C. Vigneron, M. Hoffman, P. Maincent, Anticoagulant activity of heparin following oral administration of heparin-loaded microparticles in rabbits, J. Pharm. Sci. 91 (2002) 760–768. L.P. Tan, S.S. Venkatraman, P.F. Sung, X.T. Wang, Effect of plasticization on heparin release from biodegradable matrices, Int. J. Pharm. 283 (2004) 89–96. Y.K. Lee, J.H. Nam, H.C. Shin, Y. Byun, Conjugation of lowmolecular-weight heparin and deoxycholic acid for the development of a new oral anticoagulant agent, Circulation 104 (2001) 3116–3120. T. Yang, A. Hussain, J. Paulson, T.J. Abbruscato, F. Ahsan, Cyclodextrins in nasal delivery of low-molecular-weight heparins: in vivo and in vitro studies, Pharm. Res. 21 (2004) 1127–1136. S. Benita, J.P. Benoit, F. Puisieux, C. Thies, Characterisation of drug-loaded poly(D,L-lactide) microspheres, J. Pharm. Sci. 73 (1984) 1721–1724. S.R. Gundry, M.D. Klein, R.A. Drongowsky, M.M. Kirsh, Clinical evaluation of a new rapid heparin assay using the dye Azure A, Am. J. Surg. 148 (1984) 191–194. C.D. Marci, D. Prager, A review of the clinical indications for the plasma heparin assay, Clin. Pathol. 99 (1993) 546–550. M. Cavalier, J.P. Benoit, C. Thies, The formation and characterization of hydrocortisone-loaded poly((±)-lactide) microspheres, J. Pharm. Pharmacol. 38 (1986) 249–253. B.R. Conway, H.O. Alpar, Double emulsion microencapsulation of proteins as model antigens using polylactide polymers: effect of emulsifier on the microsphere characteristics and release kinetics, Eur. J. Pharm. Biopharm. 42 (1996) 42–48. J.P. McGee, M. Singh, X.M. Li, H. Qiu, D.T. O’Hagan, The encapsulation of model protein in poly (D,L lactide-co-glycolide) microparticles of various sizes: an evaluation of process reproducibility, J. Microencapsul. 14 (1997) 197–210. T. Uchida, K. Yoshida, Y. Nakada, N. Nagareya, Y. Konishi, A. Nakai, M. Nishikata, K. Matsuyama, Preparation and characterization of polylactic acid microspheres containing water-soluble anesthetics with mall molecular weight, Chem. Pharm. Bull. (Tokyo) 45 (1997) 513–517.