Enhanced antithrombotic effect of hirudin by bovine serum albumin nanoparticles

Abstract

The aim of this study was to design hirudin-loaded bovine serum albumin (BSA) nanoparticles to control release and improve antithrombotic effect of hirudin. BSA nanoparticles were designed as carriers for delivery of hirudin. Hirudin–BSA nanoparticles were prepared by a desolvation procedure and cross linked on the wall material of BSA. The hirudin–BSA nanoparticles were characterised by particle size distribution, zeta potential, entrapment efficiency, differential scanning calorimetry (DSC), and powder X-ray diffractometry (PXRD). The in vitro release characteristics and pharmacological availability were investigated. The morphology of hirudin–BSA nanoparticles was approximately spherical. The mean particle size was 164.1 ± 5.40 nm and the zeta potential was −20.41 ± 0.64 mV. The mean entrapment efficiency and drug loading were 85.14% ± 4.79% and 66.38% ± 3.54%, respectively. Results from DSC and PXRD revealed that hirudin in BSA existed in an amorphous state. The release behaviours of hirudin from BSA nanoparticles in phosphate buffer solution were fitted to the bioexponential model. The in vivo result obtained after intravenous injection of hirudin–BSA nanoparticles in normal rats demonstrated that BSA nanoparticles could prolong the antithrombotic effect of hirudin in comparison with hirudin solution. These results suggest that hirudin–BSA nanoparticles may be a promising drug delivery system for thrombosis and disseminated intravascular coagulation therapy.

KEYWORDS:

• hirudin
• nanoparticles
• desolvation–chemical cross-link method; bovine serum albumin

1. Introduction

Hirudin is a potent antithrombotic peptide extracted from leeches (Hirudo medicinalis). Hirudin has become the most specific tight-binding thrombin inhibitor since it was isolated in the late 1950s,[1] and it has been applied in the clinic to treat acute coronary artery disease,[2] deep vein thrombosis [3] and disseminated intravascular coagulation [4] due to its lower side effects of haemorrhage, non-allergic reaction, and non-toxicity compared with heparin.[5] Although hirudin is a promising anticoagulant, it also has some drawbacks. Clinical pharmacokinetic studies of hirudin performed in human volunteers showed that hirudin was rapidly distributed into the extracellular space after intravenous injection and eliminated with a half-life of 60–100 min.[5] Hence, repeated injections are required due to the rapid clearance of hirudin from the circulation. Considering the high price of hirudin, the therapy becomes more expensive. Moreover, repeated injections may be potentially life-threatening and unsafe for patients. These disadvantages have significantly limited the clinical application of hirudin.[6–8] Consequently, recent efforts have been directed to the development of drug delivery systems to solve the problems referred to.

Attempts at other administration routes have been made, such as thermosensitive hydrogel for the controlled release of hirudin by subcutaneous administration,[9] chitosan/polyethylene glycol–alginate microcapsules for oral delivery of hirudin,[10] and nasal spray with chitosan enhancer.[11] Although the residence time of hirudin in blood was prolonged for these administration routes, the absolute bioavailability was still lower. Hence, there has been an urgent request to develop an efficient and adequate vehicle that can carry and deliver hirudin to the intended site without provoking any adverse reaction.

Albumin is the most abundant protein in plasma (35–50 g/L, human serum) with a molecular weight of 66.5 kDa, which is stable in the pH range of 4–9, soluble in 40% ethanol, and can be heated at 60 °C for up to 10 h without deleterious effects. These properties, ready availability, biodegradability, and lack of toxicity and immunogenicity, make serum albumin an ideal candidate for drug delivery. Recent applications of serum albumin (human and bovine) have demonstrated some advantages as a natural and, therefore, biocompatible and biodegradable carrier for drugs.[12–14] Nanoparticles of human-serum-albumin-bound paclitaxel (Abraxane, an example of nabTM (nanometre-albumin-bound) technology), have been approved by the Food and Drug Administration and a Phase III trial of a variety of cancer.[15,16] Clinical studies have shown that nab-paclitaxel is significantly more effective than paclitaxel formulated as Cremophor EL® (CrEL, Taxol®, CrEL-paclitaxel), with almost double the response rate and increased time to disease progression.[17] Besides, the albumin-based drug delivery system could control drug release and prolong the half-life of drug. Therefore, the bovine serum albumin (BSA) nanoparticles are a promising carrier to increase the drug loading (DL) efficiency and prolong the half-life of hirudin.

In the present study, hirudin-loaded BSA nanoparticles were prepared by the desolvation–chemical cross-link method. Characterisation of the nanoparticles, including size distribution and zeta potential, was carried out. Drug entrapment efficiency (EE), DL efficiency, and release properties in vitro were also tested. In vivo pharmacological efficacy of hirudin–BSA nanoparticles comparing hirudin and hirudin–BSA nanoparticles’ physical mixture were investigated. The results of in vitro and in vivo studies showed that hirudin–BSA nanoparticles could control the release and prolong the antithrombotic effect of hirudin.

2. Material and methods

2.1. Materials

Hirudin (5006 ATU/g) was provided by Zhengzhou Jiaruo Biotech. Co., Ltd. (Zhengzhou, China). BSA was purchased from Roche Co., Ltd. (New York, NY, USA). Kits for determining thrombin time (TT) were purchased from Sun Biotech Co., Ltd. (Shanghai, China). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially.

2.2. Methods

2.2.1. Preparation of hirudin–BSA

Hirudin–BSA was prepared by the desolvation–chemical cross-link method. For the preparation of nanoparticles, 40 mg of BSA and 60 mg of hirudin were first dissolved in deionised water. Ethanol was slowly (1 mL/min) injected by a micro-syringe pump (Kd Scientific, Fabriqe'auxetats-unis, MA, USA) into aqueous solution, under mechanical agitate (DC-40, Hangzhou Electrical Engineering Instruments, China) with 800 rpm in water bath at 25 °C. Thereafter, 0.25% glutaraldehyde solution was added to cross link the amino groups in the nanoparticles. The cross-linking process was performed during stirring (600 rpm) of the suspension for 10 h. Ethanol was removed by evaporation using a rotary evaporator (RE52-98, Shanghai Yarong Biochemistry Instrument Factory, Shangai, China) and the hirudin–BSA nanoparticles were obtained. The suspension produced was freeze-dried for 48 h to obtain a fine powder of nanoparticles.

2.2.2. Formulation optimisation

The parameters such as weight ratio of drug to total weight of drug and BSA (D/T ratios), the volume of ethanol (E), the amount of glutaraldehyde (G), the stirring rate (R), and the emulsifying temperature (°C) were optimised each at three or four levels taking the EE as index. When one factor was under investigated, the others were fixed. The fixed parameters (D/T ratios, E, and G) mainly affect the EE. Six pieces of formulations were designed according to uniform design, in order to optimise the formulation and technology of preparation. The formulations and manufacturing parameters were optimised concerning drug entrapment efficiency and particle size.

2.2.3. Determination of entrapment efficiency and drug loading

The EE of hirudin–BSA nanoparticles was determined using the ultracentrifugation method.[18] Briefly, 10 mL of hirudin–BSA nanoparticles were centrifuged at 200g for 20 min (TGL-16G-A, ShangHai Anting Scientific Instrument Factory, Shanghai, China). The free hirudin content in the supernatant was measured by thrombin titration method. The drug content in the original hirudin–BSA nanoparticles was detected by the same thrombin titration method. The drug EE and DL were, respectively, calculated as follows: (1) (2) where W_free is the analysed weight of free drug in the supernatant; W_total is the analysed weight of drug in the original hirudin–BSA nanoparticles; and W_{nanoparticles} is the total weight of hirudin–BSA nanoparticles.

Being a specific thrombin inhibitor, it is logical to use the thrombin titration method to determine the potency of hirudin. The method described is simple, rapid, and highly reproducible (coefficient of variation of this method was 2.1%). The assay is based on the 1:1 stoichiometric reaction between hirudin and thrombin. About 100 µL of simple and 200 µL of 0.5% cattle fibrinogen tris (hydroxymethyl) aminomethane hydrochloride buffer were added into the test tube. After shaking well, 5 µL of thrombin solution (1 mL = 2 units) was added into the test tube every 5 min in water bath at 40 ± 0.5 °C. When the solution was solidified, the volume of consumption of thrombin solution should be noted down. The drug contents were calculated as follows: (3) where U is the 1 g containing thrombin activity units (U/g); C₁ is the concentration of thrombin solution (u/mL); C₂ is the concentration of sample (g/mL); V₁ is the volume of consumption of thrombin solution (µL); V₂ is the volume of sample solution (µL); and W is the weight of the sample.

2.2.4. Characterisation of hirudin–BSA

The morphology of hirudin–BSA nanoparticles was examined by transmission electron microscopy (JEM-1200EX, Japan). Samples were prepared by placing a drop of hirudin–BSA nanoparticle suspensions onto a copper grid and air-dried, following negative staining with a drop of 2% aqueous solution of sodium phosphotungstate for contrast enhancement.

The particle size of hirudin–BSA nanoparticles was measured by photon correlation spectroscopy using a particle sizer (Zetasizer 3000SH, Malvern Instruments Ltd., UK) at a fixed angle of 90° at 25 °C. The particle size analysis data were evaluated using the volume distribution. Zeta potential was measured by the laser Doppler anemometry on ZetaPlus Zeta Potential Analyzer (Brookheaven Instruments Corporation, USA). All measurements were performed at 25 ºC. Calculation of the size and polydispersity indices was achieved using the software provided by the manufacturer. The diameter mean value was calculated from the measurements performed at least in triplicates.

2.2.5. Differential scanning calorimetry (DSC)

DSC analysis was performed to characterise the physical state of the albumin core in nanoparticles and incorporated hirudin. Hirudin–BSA nanoparticles were lyophilised without cryoprotectant to prevent the interruption of melting transition peak of cryoprotectant. The thermal characteristics of BSA nanoparticles and hirudin were determined by a differential scanning calorimeter (DSC CDR-4P, TA Instrument, China). Aliquots weighing 10 mg were levelled in an aluminium pan and crimped with an aluminium lid. DSC was used to analyse the samples from 0 to 400 ºC with a heating rate of 10 ºC/min under a nitrogen flow of 20 mL/min. Aluminium oxide was used as standard for calibrating the temperature.

2.2.6. Powder X-ray diffractometry (PXRD)

Powder X-ray diffraction patterns were determined for hirudin and BSA nanoparticles. BSA nanoparticles were lyophilised without cryoprotectant to prevent the interruption of crystalline peak of cryoprotectant. The powder X-ray diffraction pattern was obtained using a powder X-ray diffractometer (D/max r-B, Rigaku, Japan). The operation was performed with a voltage of 40 kV and a current of 100 mA in the region of 2.5° ≤ 2θ ≤ 50° in step scan mode of 0.02° per second.

2.2.7. The in vitro release studies

The in vitro release kinetics of hirudin–BSA nanoparticles in the phosphate buffer solution (PBS, pH 7.4) were studied by dynamic dialysis system. The dialysis bags (molecular weight cut-off 3500 Da, Sigma) were soaked in deionised water for 24 h before use. Typically, 20 mL of hirudin solution (4 mg/mL, diluted with PBS) and 20 mL of hirudin–BSA nanoparticles (4 mg/mL, suspended in PBS) were placed in dialysis bags with the two ends fixed by thread, respectively. The dialysis bag was put into a basket which was immersed in flasks containing 80 mL dissolution medium (PBS, pH 7.4), and agitated with paddle rotation at 100 rpm and 37 ± 0.5 ºC water bath. Aliquots of the dissolution medium (2 mL) were withdrawn at regular time intervals (0, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, 48 h) and the same volume of fresh dissolution medium was added to the flask to maintain the constant volume. Drug concentrations in the dissolution medium were finally analysed using the thrombin titration method. The release rate was calculated as follows: (4) where Q_n is the accumulative drug release mass; C_n is the drug concentration in the release medium of each time interval; V₀ is the total volume of the release medium; V_i is the volume of the withdrawn medium; C_i is the drug concentration in the release medium at time; and W is the total drug content of the release sample.

2.2.8. In vivo pharmacodynamic experiments

2.2.8.1. Animals

For each optimised hirudin formulation studied, male Wistar rats (Laboratory Animal Center of Qingdao Institute For Drug Control, Qingdao, China), weighing 230–250 g, were acclimated for one week under standardised environment with free access to standard food and water. All studies were conducted in accordance with the approval of the Committee of Experimental Animal Administration of the Qingdao University, and were strictly in accordance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (document no. 55, 2001)

2.2.8.2. Pharmacodynamic experiments

Three groups of Wistar rats (six rats per group) were used to determine the pharmacological efficacy of hirudin formulations as follows: (1(1) ) hirudin in 5% glucose, (2(2) ) hirudin–BSA nanoparticles (diluted in 5% glucose), and (3(3) ) hirudin–BSA nanoparticles physical mixture (diluted in 5% glucose). All hirudin formulations (containing 0.2 mg/mL hirudin) were given intravenously to rats by tail vein, at the doses of 0.5 mg/kg. Blood samples (about 0.25 mL) for the evaluation of clotting times were collected under anaesthesia from the orbital sinus before and at 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after administration. Blood samples were placed in tubes containing 3.8% sodium citrate and centrifuged (9:1, v/v) at 4000 rpm for 10 min. The plasma obtained was frozen at −20 °C until assay. The TT for each sample was determined by commercially available kits following corresponding instructions. Anticoagulant effect of hirudin for different groups was expressed as prolongation in TT compared with normal values before injection for each rat.

2.2.9. Data analysis

Results were presented as mean ± SD. Statistical comparisons were made by t-test or ANOVA analysis. The accepted level of significance was P < 0.05.

3. Results and discussion

3.1. Preparation of hirudin–BSA

The parameters such as drug to total weight of drug and BSA (D/T ratios), the volume of ethanol (V₁), and the amount of 0.25% glutaral (V₂) were optimised each at three levels taking the EE as index (Table 1). When one factor was under investigated, the other two were fixed.

Table 1. The levels of experimental factors and hirudin–BSA entrapment efficiencies (n = 3).

	X1 (w/w)	EE (%)	X2 (mL)	EE (%)	X3 (mL)	EE (%)
1	0.4	64.21	20	59.89	20	59.18
2	0.3	65.29	30	65.30	40	66.26
3	0.2	67.64	40	60.14

Note: X₁ is the weight ratio of drug to total weight of drug and BSA (D/T ratios); X₂ is the volume of ethanol (V₁); and X₃ is the amount of 0.25% glutaral (V₂).

The effects of the three influential factors, D/T ratios (X₁), V₁ (X₂), and V₂ (X₃), on encapsulation efficiency (EE) of hirudin–BSA nanoparticles were investigated. The results showed that the maximal value of EE could be reached when D/T ratio was set at 0.2. The DL could be enhanced with the increase of D/L ratio, while a higher BSA improved EE in this study. Moreover, the EE value increased with the increase of the amount of 0.25% glutaral. Ultimately, each factor was set as two or three levels for uniform experiment. The D/L ratio was set at 0.4, 0.3, and 0.2 for gaining the higher drug loading. The levels of the other two factors were set like Table 1. Results of uniform experiment of hirudin–BSA were shown in Table 2.

Table 2. Uniform design and experimental results (n = 3).

	Factor
Experiment	D/T	E (mL)	G (μL)	EE (%)
1	0.4	30	20	0.692
2	0.4	20	40	0.725
3	0.3	40	20	0.612
4	0.3	30	40	0.670
5	0.2	20	20	0.484
6	0.2	40	40	0.617

The optimised formulation was as follows: the weight ratio of drug to total weight of drug and BSA was 0.4, the volume of ethanol was 30 mL, and the volume of 0.25% glutaral was 40 μL. The average EE and the average DL of hirudin–BSA were 85.14% ± 4.79% and 66.38% ± 3.54%, respectively.

3.2. Characterisation of hirudin–BSA

The hirudin–BSA nanoparticles were spherical or ellipsoidal in shape (Figure 1). The mean particle size of hirudin–BSA nanoparticles was 164.1 ± 5.40 nm (polydispersivity index 0.197 ± 0.009, Figure 2). The zeta potential was −20.41 ± 0.64 mV and the nanoparticles were stable.

Figure 1. Transmission electron photomicrograms of hirudin–BSA: (A) ×5800; (B) ×10,000).

Figure 2. The size distribution of hirudin–BSA.

3.3. Differential scanning calorimetry (DSC)

DSC was used to investigate the existing form of hirudin in binary BSA nanoparticles. As shown in Figure 3(A), the melting endothermic peak of hirudin was observed at 167.92 °C, while the thermograms of the lyophilised hirudin-incorporated BSA nanoparticles did not show the endothermic peak for hirudin(Figure 3(D)). This suggests that hirudin was not in crystalline state but in amorphous state. Melting point depression of BSA was also observed in hirudin–BSA nanoparticles. Endothermic peak of BSA used as carrier was observed at 310.76 (Figure 3(B)). The endothermic peak of BSA was reduced as hirudin incorporated. These results from DSC revealed that hirudin in BSA exists in an amorphous state.

Figure 3. Differential scanning calorimetry of samples: (A) hirudin; (B) BSA; (C) physical mixtures; (D) hirudin–BSA nanoparticles.

3.4. Powder X-ray diffractometry (PXRD)

PXRD is one of the most common techniques used to identify the crystalline structure of bulk materials.[19] Thus, PXRD was used to determine the crystalline or non-crystalline nature of hirudin in the lyophilised nanoparticles powder. BSA nanoparticles were lyophilised without cryoprotectant to prevent the interruption of crystalline peak of cryoprotectant. As shown in Figure 4(A), BSA powder showed strong typical peaks of crystalline hirudin at 2θ scattered angles 22.040º and 22.700º. As shown in Figure 4(B), hirudin powder showed strong typical peaks of crystalline hirudin at 2θ scattered angles 14.580º, 18.740º and 23.380º. The presence of sharp peaks indicates the crystalline nature of hirudin. There were no characteristic peaks for hirudin in lyophilised hirudin–BSA nanoparticles (Figure 4(D)), which indicates that hirudin was at an amorphous state in the hirudin–BSA nanoparticles.

Figure 4. X-ray diffraction patterns of samples: (A) BSA; (B) hirudin; (C) physical mixtures; (D) hirudin–BSA nanoparticles

3.5. The in vitro release studies

The release experiment was conducted under sink conditions and the dynamic dialysis was employed for separation of free drug from hirudin–BSA nanoparticles. The release behaviours of hirudin from hirudin–BSA nanoparticles in PBS (pH 7.4) were fitted to the bioexponential equation and could be expressed by the following equation: 100 − Q = 80.23e^−0.2503t + 69.08e^−0.0592t, R_a = 0.6532, R_b = 0.9872. The initial fast release of the drug from the hirudin–BSA nanoparticles was observed in the first 0.5 h, which could be probably due to the portion of the drug that leaked out of nanoparticles and the unloaded drug.[20] Subsequently, the release of drug from nanoparticles was slower and a release plateaux was obtained from 0.5 to 48 h (Figure 5). It was obvious that hirudin released much slower from hirudin–BSA than from hirudin solution. At about 48 h, 70% hirudin of hirudin–BSA nanoparticles was released. In contrast, the release of hirudin from hirudin solution was fast and approximately 95% of the drug was released after incubated for 48 h.

Figure 5. Release profiles of hirudin from hirudin–BSA in PBS (pH 7.4) at 37 °C.

The cross-linking process with glutaraldehyde plays a major role in the stability and drug release from the BSA nanoparticles.[21] The difference between the release properties of hirudin from solution and nanoparticles is evidently attributed to the prolonged release function of BSA nanoparticles. Hirudin was held by the albumin core of the BSA and the drug released mainly through erosion and degradation. These results indicated that hirudin could be released slowly from BSA nanoparticles and be kept at constant concentration for relative long period, thus the frequency of administration might be reduced.

3.6. Pharmacodynamic experiments

TT was used to evaluate the pharmacological efficacy of hirudin. The percentage of changes (vs. normal value) in TT after intravenous administration of hirudin formulations at 0.5 mg/kg was shown in Figure 6. After intravenous injection of hirudin solution, the clotting times dropped rapidly due to fast elimination of hirudin, consistent with previous reports.[22] The TT returned to normal values after 1.5 h. There were no changes of the two parameters for solution group and hirudin–BSA nanoparticles physical mixture group. These results showed that BSA itself had not any contribution to the pharmacological efficacy. But they were significantly prolonged by the hirudin–BSA nanoparticle group (p < 0.01). The TT remained above normal value for up to 6 h. All these data demonstrate that the hirudin–BSA nanoparticles are beneficial to improving the pharmacological availability of hirudin.

Figure 6. Profiles of TT prolongation versus time after IV injection of hirudin solution, hirudin–BSA nanoparticles physical mixture, and hirudin–BSA nanoparticle in rats.

Compared with the hirudin solution, the hirudin–BSA nanoparticle improved the pharmacological availability of hirudin. The possible explanation may be the controlled release property of the BSA nanoparticles. Because the elimination of hirudin from the body is very fast with the half time of about 60–100 min,[5] the hirudin was released more slowly from the BSA nanoparticles into the circulation in comparison with simple solution. The less the hirudin eliminated in a given period of time, the higher the pharmacological availability of hirudin observed.

4. Conclusion

In this study, hirudin–BSA nanoparticles have been successfully synthesised and characterised by a desolvation technique. The results showed that it is simple to prepare hirudin–BSA nanoparticles with good encapsulation and certain sustained-release capacity. Those results of DSC and PXRD revealed that hirudin in BSA nanoparticles exists in an amorphous state. The antithrombotic effect of hirudin was significantly prolonged after intravenous injection of hirudin–BSA nanoparticles in normal rats. This is good for the clinical application.

Our data demonstrated the potential of BSA nanoparticles to optimise the therapeutic efficacy of hirudin and consequently improve the clinical outcomes. The patent of this research was applied for its potential clinical application. Further studies are needed to focus on the pharmacokinetic of hirudin–BSA nanoparticles, and the safety and efficiency of hirudin–BSA nanoparticles to evaluate the potential clinical application value.

Acknowledgements

The project was supported by project for Qingdao-applied basic research (09-1-3-77-jch).

Disclosure statement

No potential conflict of interest was reported by the authors.