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The role of daidzein-loaded sterically stabilized solid lipid nanoparticles in therapy for cardio-cerebrovascular diseases
Biomaterials 29 (2008) 4129–4136
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The role of daidzein-loaded sterically stabilized solid lipid nanoparticles in therapy for cardio-cerebrovascular diseases Yu Gao 1, Wangwen Gu 1, Lingli Chen, Zhenghong Xu, Yaping Li* Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2008 Accepted 3 July 2008 Available online 29 July 2008
Daidzein is a very good candidate for treating cardio-cerebrovascular diseases, but its poor oral absorption and bioavailability limit its curative efficacy. In this work, daidzein-loaded solid lipid nanoparticles (SLNs) with PEGylated phospholipid as stabilizer were successfully prepared by hot homogenization method. SLNs showed the mean particle size 126 14 nm with entrapment efficiency 82.5 3.7%. In vitro release of SLNs demonstrated a sustained release manner with cumulative release over 90% within 120 h in bovine serum albumin solution (4%, w/v). The pharmacokinetic behavior showed that SLNs loading daidzein could significantly increase circulation time compared with orally administrated daidzein suspension or intravenously delivered daidzein solution. SLNs showed the better effect on cardiovascular system of the anesthetic dogs by reducing the myocardial oxygen consumption (MOC) and the coronary resistance (CR) in heart compared with oral suspension or intravenous solution. The SLNs demonstrated the best effect on cerebrovascular system by increasing cerebral blood flow (CeBF) and reducing cerebrovascular resistance (CeR) in anesthetized dogs, and the protective effect on rats with ischemia-reperfusion injury model among three formulations. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Daidzein Cardiovascular disease Cerebrovascular diseases Nanoparticles
1. Introduction It is well known that cardiovascular and cerebrovascular diseases including the coronary artery disease, heart failure, acute myocardial infarction, arrhythmias et al., are the common causes of morbidity and mortality in the world. Although there are many drugs for therapy of cardiovascular and cerebrovascular disease on the market, most of them show side effect and contraindication [1], and some drugs may even increase the risk of cardio-cerebrovascular accident [2]. Daidzein is one of the major soy isoflavones found in Leguminosae and certain Traditional Chinese Medicinal herbs such as Kudzu. Several studies have shown that daidzein has various biological activity such as a weak estrogenic or antiestrogenic effect by binding to the nuclear estrogen receptor [3], inhibition of the growth of cancer cells [4,5], prevention of diabetes onset [6], promotion of the proliferation of osteoblast cells [7], and alleviation of menopausal symptom [8]. Apart from these beneficial effects on human health, daidzein is also a good candidate for the treatment of cardiovascular disease, which is its most important
* Corresponding author. Tel./fax: þ86 21 5080 6820. E-mail address: [email protected] (Y. Li). 1 These authors contributed equally. 0142-9612/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2008.07.008
pharmacological activity and the primary reason being used as a drug entity in China [9,10]. Daidzein could reduce the low-density lipoprotein (LDL) cholesterol, inhibit pro-inflammatory cytokines, cell adhesion proteins and platelet aggregation, induce nitric oxide production, and improve vascular reactivity. Daidzein could be a good candidate in treating cardiovascular diseases in virtue of the advantages of low cost and abundance as well as its clinical safe, and the Food and Drugs Administration has approved a health claim for soy based clinical trials [10]. However, its poor oral absorption and bioavailability limit its curative efficacy, which could result from its poor solubility in water, low partition coefficient of oil/water, in particular, the strong metabolism that occurs in the intestine and liver [11]. In addition, these properties bring about difficulty for preparing its injection, which could be used for first aid in clinic. Therefore, it is of great importance to investigate a new safe delivery system for daidzein with high effect on cardio-cerebrovascular diseases. We are interested in designing and developing an injectable delivery system for daidzein. In the present work, a sterically stabilized solid lipid nanoparticle (SLN) as daidzein carrier was investigated because of its good handling properties, very low intrinsic toxicity toward normal tissues, manageable burst effect issue that are commonly associated with many other colloidal formulations [12], in particular, good effect on brain targeting [13]. The aim of this work was to prepare the SLNs loading daidzein,
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determine their physicochemical characteristics and evaluate their in vivo behavior and pharmacological effect including the myocardial oxygen consumption (MOC), myocardial oxygen uptake (MOU), coronary blood flow (CBF) and the coronary resistance (CR), cerebrovascular blood flow (CeBF), and cerebrovascular resistance (CeR) in anesthetized dogs and the protective effect on the ischemia-reperfusion injury model in rats. 2. Materials and methods 2.1. Materials Daidzein (purity >99%) was obtained from Huike Plants Exploiture Co. (Shanxi, China). CompritolÒ888 ATO and Carboxymethyl cellulose sodium (CMC-Na) were purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Egg phosphatidylcholine (ePC) and poly(ethylene glycol) phosphatidylethanolamine (PEG2000-PE) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). TR-FIA kit for daidzein was purchased from Labmaster Co. (Truku, Finland). b-glucuronidase was obtained from Roche Pharma, Ltd. (Switzerland). Sulfatase was purchased from Sigma (St. Louis, MO, USA). All other reagents and solvents were analytical grade. 2.2. Animals Sprague–Dawley rats (Grade II, 215 15 g) and male Beagle dogs (8–10 kg) were supplied by Shanghai Experimental Animal Center, CAS. The animals were acclimatized at 25 1 C and a relative humidity of 75 5%. All animal procedures were performed following the protocol approved by the Institutional Animal Care and Use Committee at the Second Military Medical University. 2.3. Preparation and characteristics of SLNs loading daidzein SLNs loading daidzein were prepared by the hot homogenization method as described previously [14]. Briefly, 20 mg daidzein was added into 500 mg melted lipid materials (CompritolÒ888 ATO:ePC:mPEG2000-PE ¼ 18:6:1, w/w) in 70 C water bath, and sonicated for 30 min to dissolve daidzein with a probe sonicator (250W, JY88-II, China). Then, the solution was quickly dispersed in 50 ml prewarmed distilled water and vigorously stirred to form hot, milky emulsion in 65 C water bath (RET, IKA, Germany). The resultant emulsion was cooled to obtain SLNs. Finally, SLNs were purified by gel filtration on Sephadex G-25 eluted with water and lyophilized. The particle size, size distribution and z potential of SLNs loading daidzein were measured by laser light scattering using a Nicomp 380/ZLS zeta potential analyzer (Particle Sizing System, USA). The morphology of SLNs was examined using a transmission electron microscope (TEM, CM12, Philips, Netherlands) after negative staining with sodium phosphotungstate solution (0.2%, w/v). In order to know drug entrapment efficiency, the amount of daidzein incorporated in SLNs was determined using HPLC. Briefly, 10 mg of SLNs was added to 20 ml of methanol. After lipids were dissolved when heated to 65 C, the solution was cooled to room temperature and centrifuged at 20,000 rpm for 10 min to remove CompritolÒ888 ATO. The supernatant (10 ml) was directly injected into HPLC with C18 column, the mobile phase: acetonitrile-water (65:35), detection wavelength: 249 nm, the flow rate: 1 ml/min. The drug entrapment efficiency was defined as the percentage of daidzein recovered from SLNs compared with the initial drug amount. The drug loading was calculated as the amount entrapped daidzein compared with the total amount of SLNs. In vitro release of daidzein from SLNs was measured using the dialysis method at 37 C. The SLNs were dispersed in 5 ml physiological saline solution containing bovine serum albumin (BSA, 4%, w/v), then surged vertically for 3 min and filled into dialysis bag (molecular cutoff of 5000 Da). The dialysis bag was put into a container with 100 ml solution with BSA (4%, w/v). The solution was stirred at 300 rpm at 37 C. Subsequently, 0.5 ml of the solution was withdrawn from the system at predetermined time point and analyzed by HPLC. As control, the release profile of daidzein in physiological saline solution without BSA was also performed. 2.4. Pharmacokinetics and biodistribution of SLNs The health adult Sprague–Dawley rats were randomly divided into three groups (n ¼ 20), one group received free daidzein suspended in CMC-Na solution (0.5%, w/ v) by oral administration at a dose of 20 mg/kg. Another two groups were administrated with daidzein in DMSO solution (20 mg/kg) or SLNs dispersed in saline solution at a dose of 20 mg/kg by intravenous route, respectively. At predetermined point, blood sample (0.2 ml) was collected via caudal vein with heparinized tubes. Then, the samples were centrifuged at 2500 g for 10 min. Plasma was transferred into microcentrifuge tubes and stored at 20 C until analysis. The daidzein in plasma was determined using the TR-FIA kit. After samples were treated with hydrolysis and extraction, the values of fluorescence were measured by a timeresolved fluorometry Victor3 1420 multilabel counter (PerkinElmer, USA) according
to the protocol recommended by the manufacturer. Pharmacokinetic parameters were obtained using the Practical Pharmacokinetic Program Version 97. For study on biodistribution of daidzein, the animals were sacrificed at predetermine time point, and then tissues including heart, liver, spleen, lung, kidney and brain were harvested and stored at 50 C until analysis. The daidzein in tissue was determined according to Janning’s report [15]. Briefly, tissue was cut into small pieces and triturated into powder under liquid nitrogen. To 100 mg tissue aliquots, 500 ml lysis buffer (NaCl 75 mM; EDTA 25 mM with 1% SDS, w/v) was added, and samples were allowed to lyse for 1.5–2 h at 4 C. The internal standard (1 mg/ml, 10 ml genistein) was then added. After addition of hydrolysis buffer (1 M sodium acetate containing 1% (w/v) ascorbic acid and 0.1% (w/v) EDTA), 8 ml b-glucuronidase and 4 ml sulfatase, the samples were kept at 37 C for at least 15 h. Then, methanol as extraction solvent was added and extracted three times. After centrifugation, the organic phase was combined and dried under a stream of nitrogen at 37 C. The concentration of daidzein after dissolved in 100 ml methanol was determined by HPLC analysis. 2.5. Measurement of MOC, MOU, CBF and CR Twelve male Beagle dogs were randomly divided into three groups (n ¼ 4): group 1: control group, treated with free daidzein (4 mg/kg) suspended in CMC-Na solution (0.5%, w/v) by oral administration; group 2: treated with free drug (4 mg/ kg) dissolved in DMSO solution, and group 3: treated with SLNs (4 mg/kg) dispersed in saline solution by intravenous route. The dogs were anesthetized with 3% sodium pentobarbital intravenously (30 mg/kg). The trachea was connected to a respirator (SC-M5, Shanghai) through a midline cervical incision with respiratory rate of 16– 18 times/min. The esophagus was separated and a trachea was inserted into gastric canal for oral administration of the daidzein suspension. The arterial blood pressure (ABP) was measured through femoral artery catheterization by pressure transducer (MPU20.5A, Nihon Khonden, Japan). Then, thoracotomy was performed in the fourth left intercostal space and the heart was suspended in a pericardial cradle. Subsequently, the left circumflex branch of coronary artery and the aortic arch were isolated, and then the probes of electromagnetic flowmeter (MFV-1100/1200, Nihon Kohden, Japan) were placed to determine CBF and myocardial blood flow (MBF). Through the cervical incision, the right common carotid artery and internal jugular vein were isolated, and two catheters were inserted separately. The blood samples were extracted synchronously to determine PO2 and pH (DH-1830, Blood Gas Analyzer, China). Finally, the blood oxygen content in coronary venous sinus (VO2) and in artery (AO2) was calculated. After the operation was completed and the observation parameters displayed stable, the free drug (4 mg/kg) dissolved in DMSO solution and SLNs dispersed in saline solution were infused through femoral vein with an electro-driven constant flow pump (SH-88AB, Quanzhou, China) for 30 min. Free daidzein suspension (4 mg/ kg) was delivered though the gastric canal. The above parameters were recorded at preadministration (0) and 10, 30 and 60 min post administration. After the record finished, the heart was taken out, washed with normal saline solution and weighed. The MOC, MOU and CR were calculated from (Eqs. (1)–(3)). MOCðml=min=100gÞ ¼ MBFðml=minÞ ðAO2 ðml=LÞ VO2 ðml=LÞÞ=100g
(1)
Rate of MOUð%Þ ¼ ðAO2 ðml=LÞ VO2 ðml=LÞÞ=AO2 ðml=LÞ
(2)
CRðKpa=ml=minÞ ¼ ABPðKpaÞ=CBFðml=minÞ
(3)
2.6. Effect on rats with middle cerebral artery (MCA) occlusion and reperfusion model The protection against cerebral ischemia of SLNs was investigated by a MCA occlusion/reperfusion model in rats according to Longa’s method [16]. Briefly, eighty male Sprague–Dawley rats were divided into five groups (n ¼ 16): group 1: sham control group; group 2: control group, treated with saline solution by intravenous administration; group 3: oral group, treated with free daidzein suspension (12 mg/kg) by oral administration; group 4: treated with free daidzein solution (12 mg/kg) and group 5: treated with SLNs (12 mg/kg) by intravenous route. Rats were anesthetized with 10% chloral hydrate (3 ml/kg). The right carotid artery bifurcation was exposed, and the right common carotid artery (CCA) and internal carotid artery (ICA) were isolated and ligated transiently. The external carotid artery (ECA) was isolated and an 18 mm length of 4-0 nylon suture was introduced into the ECA lumen and advanced into the ICA to block the origin of MCA. Saline solution, free daidzein solution or SLNs were intravenously administered just 1 h after occlusion. Free daidzein suspension was administrated by direct gastric gavage at 1 h after occlusion. The suture was left in place for another 1 h, and then restoration of MCA blood flow was achieved by withdrawing the suture to the ECA. The sham control rats received the same surgery procedures but had not the suture inserted. Twenty-three hours after the reperfusion, the neurological symptom score was determined according to Longa’s five score regulation [16], which consists of
Y. Gao et al. / Biomaterials 29 (2008) 4129–4136 0 grade: no neurological defect; 1 grade: failure to extend right forepaw fully; 2 grade: circling to the right; 3 grade: circling to the reverse of the right-the left; 4 grade: rats did not walk spontaneously and had a depressed level of consciousness. After the evaluation of neurological symptoms, rats were sacrificed to excise their brains. In each group, one half of the brain (n ¼ 8) was used to detect the brain water content, and the other half was used to measure the infarct volume. The water content was assessed by comparing the wet weight with the dry weight, which were determined immediately after excision and after staying in an oven at 105 C for 24 h, respectively. The water content were calculated from (Eq. (4)): Water content ð%Þ ¼ ðwet weight dry weightÞ 100=wet weight:
(4)
The infarct volume measurement was performed by slicing the brain into seven sections (2-mm thick) and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 C for 30 min with shaking, then immersed in 10% formaldehyde neutral buffer solution for preservation. Photograph of each section was taken with a digital camera (Fujifilm, Tokyo, Japan) and the infarct area was measured with a software (Medbrain 2.0). The percentage of infarction (infarct ratio) was calculated from the data of infarct volume and total coronal section according to (Eq. (5)): Ratioð%Þ ¼ infarct volume mm3 100=total coronal section mm3
(5)
2.7. Measurement of CeBF and CeR in anesthetic dogs Twelve male Beagle dogs were divided into three groups (n ¼ 4): group 1: control group, treated with free daidzein suspension (4 mg/kg) by oral administration; group 2: treated with free drug solution (4 mg/kg); and group 3: treated with SLNs (4 mg/kg) dispersed in saline solution. The dogs were anesthetized with 3% sodium pentobarbital 30 mg/kg intravenously. After a midline cervical incision, the esophagus was separated and a trachea was inserted into the gastric canal for oral administration of the daidzein suspension. Then, CCA and ECA were isolated, and ECA were ligated transiently. A probe of electromagnetic flowmeter with diameter of 2.0 w 2.5 mm was placed into CCA to determine the blood flow, which was denoted as ICA blood flow. Then, the femoral artery was isolated, and ABP was measured through femoral artery catheterization by pressure transducer. The catheter was also implanted into the femoral vein for infusion of daidzein solution and SLNs. After the surgical, the animals were placed to stabilize for 30 min, and the above parameters were recorded at preadministration (0 min). After the free drug solution or SLNs infused through femoral vein with an electro-driven constant flow pump for 30 min, and the free daidzein suspension was administrated though the gastric canal, the parameters were recorded at 5, 15, 30 and 60 min. When the record finished, the brain was taken out, washed with normal saline solution and weighed. The CeBF and CeR were calculated from Eqs. (6) and (7). CeBFðml=min=100gÞ ¼ 2 CCA blood flow ðml=min=100gÞ
(6)
CeR ¼ ABPðkPaÞ=½CBFðml=minÞ,100g brain
(7)
2.8. Statistical analysis Data were presented as the mean SD of each group. Statistical analysis of the data from the physicochemical studies was performed by applying the Student’s t-test. A1-way ANOVA followed by the Bonferroni correction was used to assess
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statistical differences for the physiological variables. Analysis of variance (ANVOA) followed by Duncan’s multiple-range test was used for the scores obtained in the neurological symptom. Values of p < 0.05 were indicative of significant differences, and p < 0.01 indicative of a very significant difference.
3. Results 3.1. Physicochemical characteristics of SLNs In order to improve the pharmacological activity of daidzein on cardio-cerebrovascular diseases, SLNs loading daidzein were successfully prepared by the hot homogenization method with CompritolÒ888 ATO and ePC as solid core, and PEG2000-PE as stabilizer to increase the circulation time and drug accumulation in brain [13]. SLNs were round and uniform with the mean particle size of 126 14 nm (Fig. 1A), polydispersity indexes of 0.226 0.023 and zeta potentials of 34.5 3.4 mV. It has been reported that the zeta potential is a very important factor to evaluate the stability of colloidal dispersion because particles could be dispersed stably when absolute value of zeta potential is over 30 mV due to the electric repulsion between particles [17]. In addition, SLNs showed the drug entrapment efficiency of 82.5 3.7% with the drug loading 3.5 0.4%. In order to know the release behavior of daidzein from SLNs, we performed the release experiment in physiological saline solution with or without BSA (4%, w/v). The drug release profiles from SLNs were shown in Fig. 1B. The SLNs showed a burst drug release (45%) within the initial 12 h in BSA solution. Subsequently, the release rate gradually slowed, and the cumulative release of drug was over 90% within 120 h. As control, the release of daidzein from SLNs in physiological saline solution without BSA showed a sustained release manner without obvious burst drug release, and the cumulative release of daidzein was about 40% within 120 h. 3.2. Pharmacokinetics and biodistribution The pharmacokinetic behavior of daidzein encapsulated in SLNs was investigated in healthy Sprague–Dawley rats after intravenous administration. The drug concentration-time curves of daidzein were shown in Fig. 2A. After a single dose intravenous administration, SLNs showed a prolonged circulation time of daidzein in blood, which could still be measured in plasma at 48 h. However, the free drug in DMSO solution was quickly removed from the circulation system. As control, daidzein by oral route showed very low drug concentration and bioavailability, which was accordant with Qiu’s report [18]. The pharmacokinetic profiles for daidzein in rats were best described by a non-compartmental model. The main pharmacokinetic parameters of SLNs were t1/2 (12.62 1.36 h),
Fig. 1. Transmission electron microscopy of SLNs (A) (Bar ¼ 200 nm); release profiles of daidzein from SLNs in saline solution (
) or BSA solution (
) at 37 C (B).
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Daidzein concentration (ng/mL)
1000000
suspension, SLNs exhibited a bias to brain with more drug accumulation in brain. At 12 h, the SLNs showed an increasing accumulation in brain, which could result from the brain targeting of SLNs [13]. In addition, SLNs still kept higher drug level in plasma after 12 h than that of other two formulations, which should attribute to the PEG chains on the surface of SLNs that increased the circulatory half-life time and decreased the drug uptake in reticuloendothelial system (RES) sites.
A
10000
3.3. In vivo pharmacological efficacy
100
1
Daidzein amount (µg/g tissue)
Daidzein amount µg/g tissue) (µ
25
0
12
24 Time (h)
36
48
B
20 15 10 5 0 25 20
*
*
blood heart liver spleen lung kidney brain
C
15 10 5 0
*
*
blood heart liver spleen lung kidney brain
Fig. 2. Pharmacokinetics and biodistribution of SLNs loading daidzein. (A) Blood clearance curves of daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ), free daidzein dissolved in DMSO solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ) at a dose of 20 mg/kg. Data were given as mean SD of six to eight rats per group. (B) Tissue distribution of daidzein in rat at 6 and 12 h (C). Free daidzein suspension by oral administration ( ), free daidzein solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ) at a dose of 20 mg/kg. Data were given as mean SD of four to five rats per group. *p < 0.01 compared with oral suspension.
MRT (5.59 0.76 h), CL (0.24 0.13 L/(h kg)) and AUC0 w N (83.62 1.89 mg h/ml), which showed significant difference with free drug in DMSO solution with t1/2 (5.62 1.02 h), MRT (2.04 0.67 h), CL (0.61 0.12 L/(h kg)) and AUC0 w N (28.29 1.29 mg h/ml). As control, the pharmacokinetic parameters of daidzein from oral administration were t1/2 (4.53 0.84 h), MRT (1.68 0.35 h), CL (0.86 0.31 L/(h kg)) and AUC0 w N (2.92 0.26 mg h/ml). The result indicated that SLNs with steric-stabilized lipid PEG-PE would prevent their rapid uptake by mononuclear phagocyte system (MPS) and improve their circulatory half-life, which had already been found in other delivery systems [19,20]. To evaluate in vivo uptake of daidzein, the distribution profiles of daidzein in heart, liver, spleen, lung, kidney and brain of rats were shown in Fig. 2B,C. At 6 h after intravenous administration, drug from SLNs was mainly distributed to blood, heart, liver and spleen. However, drug in DMSO solution by intravenous administration or suspension by oral administration mainly distributed to kidney, which indicated that they could be quickly eliminated through kidney. Compared with daidzein solution or oral
3.3.1. Measurement of MOC, MOU, CBF and CR In order to know the effect of SLNs on cardiovascular system, MOC, MOU, CBF and CR in dogs were measured. Fig. 3A demonstrated that the MOC of dogs injected SLNs dispersed in saline solution by intravenous administration decreased, and the change of MOC (%) showed significant difference at 10 min (p < 0.05), and very significant difference at 30 and 60 min (p < 0.01) compared with that of the group administrated orally. While the change of MOC (%) of dogs injected free daidzein solution showed the significant difference only after 30 min compared with that of the group administrated orally (p < 0.05). The rate of MOU was shown in Fig. 3B, the change of the rate of MOU (%) of dogs injected SLNs showed very significant difference from 30 to 60 min compared with those of the dogs administrated orally (p < 0.01). While the change of rate of MOU (%) of dogs injected free daidzein solution showed significant difference at 30 min (p < 0.05) and very significant difference at 60 min (p < 0.01) compared with that of the group administrated orally. Fig. 3C demonstrated that the CBF of dogs injected SLNs reduced sharply and the change of CBF (%) showed very significant difference from 30 to 60 min during experiment compared with that of the group administrated orally (p < 0.01). The change of CBF of dogs injected daidzein solution showed significant difference at 30 min compared with that of the group administrated orally (p < 0.05). The CR is also an important factor to evaluate the cardiac function and is closely related with CBF and MOC [21]. In present study, the effect of SLNs on CR were also measured, and the results were shown in Fig. 3D. The change of CR (%) of the animals treated with SLNs showed significant difference compared with that of the group administrated orally at 30 min (p < 0.05) and very significant difference at 60 min (p < 0.01). 3.3.2. Effects on rats with MCA occlusion/reperfusion model Rats subjected to 1 h of MCA occlusion were divided into five groups (n ¼ 16), sham-operation group and groups treated with free daidzein by oral administration at a dose of 12 mg/kg, saline solution, free daidzein solution or SLNs dispersed in saline solution (12 mg/kg) through intravenous route. At 24 h after perfusion following another 1 h of MCA occlusion, the neurological abnormality of rats was observed and scored. The results were shown in Fig. 4A. The sham-operation group showed no neurological abnormalities with the neurological score 0. The saline solution treated group showed severely neurological deficiencies such as circling to the right and circling to the left with the mean neurological score 2.4 0.53. The neurological deficiencies could be slightly alleviated by free daidzein administrated orally (2.1 0.43), and greatly alleviated by free daidzein injected intravenously (1.5 0.48). While SLNs group showed the best efficacy (0.9 0.25). Subsequently, the brains of the rats were excised to determine the cerebral water content and the infarct volume. Fig. 4B showed that the water content of the right damaged hemisphere was similar to the left normal hemisphere in sham-operation group (p > 0.05). While in the saline solution treated group, the water content of the right hemisphere was much higher than that of left hemisphere (p < 0.01). Treated with 12 mg/kg free drug intravenously could
Y. Gao et al. / Biomaterials 29 (2008) 4129–4136
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Fig. 3. Effect of SLNs on cardiovascular systems. Measurement of MOC (ml/min/100 g) (A), MOU (%) (B), CBF (ml/min) (C) and CR (Kpa min/ml) (D) in different time points. Free daidzein suspended in CMC-Na (0.5%, w/v) by oral administration (4 mg/kg) ( ), free daidzein dissolved in DMSO solution by intravenous administration (4 mg/kg) ( ), SLNs dispersed in saline solution by intravenous administration (4 mg/kg) ( ). Data were given as mean SD of four dogs.
reduce the brain edema compared with saline control group (p < 0.05), and SLNs group showed better efficacy at the same dose (p < 0.01), in particular, the water content of the right hemisphere showed no significant difference compared with that of the left hemisphere in this group (p > 0.05). On the contrary, treated with free drug administrated orally (12 mg/kg) could only slightly reduce the brain edema (p > 0.05). The infarct volume was measured by the TTC-staining in which the infarct tissue became pale and the normal tissue was red. In the saline solution-treating group, a well-defined region of ischemia was successfully induced (Fig. 5A). SLNs could significantly reduce the infarct volume compared with that of the control group. The group treated with free drug through intravenous route showed reduction of infarct ratio from 29.3% (control group) to 22.9%, while the infarct ratio of SLNs group reduce to 15.7% (Fig. 5B). As another control, the volume of reduced infarct in the group by free daidzein after oral administration was very limited. 3.3.3. Measurement of CeBF and CeR in dogs In order to know whether SLNs could improve the effect of daidzein on cerebrovascular system, the cerebrovascular blood supply and CeR in dogs were investigated. It has been reported that ischemic stroke is caused by decreased vital blood supply transiently or permanently resulted from embolism or hemorrhage in the brain, therefore, the improvement of blood supply can rescue the neuronal cells and even reverse the impairment in ischemic tissue [22]. The CeBF was measured pre and post administration of SLNs in anesthetic dogs. The result demonstrated that the CeBF of dogs increased from 145 to 186 ml/min within 60 min after intravenous administration of SLNs (4 mg/kg) (Fig. 6A). The CeBF of dogs administrated free drug solution also increased during the
initial phase of experiment and hold a little change until 60 min at a dose of 4 mg/kg. However, the CeBF of dogs administrated free drug suspension orally showed neglectable difference during experiment. Fig. 6B showed the changes of CeR of the animals treated with three formulations. SLNs could reduce the CeR of animals, and the change of CeR (%) from 15 to 60 min in this group demonstrated a very significant difference compared with that of dogs administrated orally (p < 0.01). Free daidzein also could reduce CeR of animals, and the change of CeR (%) in this group showed a significant difference compared with that of dogs administrated orally (p < 0.05). 4. Discussion To our knowledge, this is the first report about the solid lipid nanoparticle as an injectable delivery system for daidzein and applying SLNs loading daidzein for treatment of cardio-cerebrovascular disease. It has been reported that SLN is a particulate system for parenteral administration due to its solid property at body temperature and its slow drug release. In present work, the injectable daidzein-loaded SLNs were successfully prepared by the hot homogenization method. Compritol 888 ATO and ePC were selected as matrix material with PEG2000-PE as stabilizer. Considering that some serum proteins may destabilize the particles and accelerate drug release [23], meanwhile for a better mimic of in vivo environment and study on the release kinetics of SLNs, we chose the physiological saline solution with BSA (4%, w/v) as release media. Compared with the release kinetics of SLNs in media without BSA, SLNs showed more rapid release of daidzein in BSA solution, which suggested that the matrix was likely to destabilize
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Fig. 4. Effect of SLNs on rats with MCA occlusion/reperfusion model. Effect of SLNs on neurological abnormality in MCA occlusion/reperfusion model (A); Effect of SLNs on water content of different brain hemispheres (B). Sham-operation group ( ); saline control group ( ); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ); free daidzein dissolved in DMSO solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ). The dosage in each group was 12 mg/kg. Data were given as mean SD of four rats. *p < 0.05 and **p < 0.01 compared with saline control group. In saline control group, **p < 0.01 compared with left normal hemisphere.
in the presence of serum proteins or other biological factors in vivo. It has been reported that the lipid compritol might start to crystallize first and form an inner core of pure lipid during the solidification process of SLNs. As a result, a solid drug layer might be formed around this lipid core with an enrichment of drug in the outer particle region [12]. So, it was possible that the drug molecules in the outer region of SLNs moved out of the particles via short diffusion path and brought about a burst drug release. After burst release, the SLNs showed a good sustained release manner in the presence of BSA (Fig. 1B), which demonstrated that SLNs could be a promising system for long-term daidzein delivery. Due to poor solubility of daidzein, oral administration is one of the main routes, but these edible formulations presented poor bioavailability. Even if daidzein was intravenously injected by dissolving in DMSO solution, the quick elimination from blood without sufficient circulation time made daidzein be not able to cumulate in specific organs to exert curative effect. However, daidzein encapsulated in SLNs showed significant differences in terms of the pharmacokinetic behavior from daidzein suspension
or daidzein solution (Fig. 2A). In particular, some pharmacokinetic parameters of SLNs such as t1/2 were found to be much longer than that of the free drug. After 48 h, SLNs could still be detected in the plasma, whereas free daidzein formulations had almost disappeared from circulation system after 12 h. This is undoubtedly related to the solid state of the lipid, which allowed daidzein to release slowly from the particles, and the long PEG chain that stabilized SLNs by steric hindrance decreased macrophages recognition and increased blood circulation time [19,20]. Many clinical studies demonstrated that patients with myocardial ischemia showed very low ability to increase CBF in response to an increased myocardial oxygen demand [24]. It has been reported that the cardiovascular disease such as cardiac dysfunction, arrhythmias and infarction were caused by imbalance of myocardial oxygen demand and supply [25]. Therefore, it is very important to keep the myocardial oxygen demand and supply balance in cardiovascular system. In this work, we examined the change of MOC, MOU, CBF and CR in anesthetic dogs after administration of different daidzein formulations, and found that the SLNs could significantly reduce MOC, MOU and CR (Fig. 3). Compared with daidzein suspension and solution, SLNs showed the best effect on cardiovascular system to keep the myocardial oxygen demand and supply balance. This phenomenon could be explained by the pharmacokinetic behavior (Fig. 2A) and biodistribution (Fig. 2B,C) of different formulations. Daidzein suspension was not well absorbed and the drug concentration was very low. Although daidzein solution showed higher drug concentration than daidzein suspension, the daidzein was quickly eliminated and rapidly transported to tissues or organs from blood, which was accordant with other scholar’s reports [15,26]. According to the biodistribution data, we could find that the drug in DMSO solution by intravenous administration was mainly distributed to kidney not to the heart. So both suspension and solution could not exert the intrinsic effects of daidzein. However, SLNs showed much higher drug concentration in vivo, and daidzein could be detected even at 48 h after injection (Fig. 2A). Also, a relatively high drug accumulation in heart was detected (Fig. 2B,C), which indicated more drug could be carried into the cardiovascular systems. This property of SLNs should lead to higher efficacy of daidzein in cardiovascular systems. So far, there is no report about daidzein used for treatment of cerebrovascular disease, which could be due to its low bioavailability and the metabolism that occurs in the intestine via oral administration [11]. From the result of the tissue distribution, we found that daidzein in SLNs was apt to distribute to brain tissue, which could result from the brain targeting of SLN [13]. In addition, the SLNs prepared in the present study showed the long circulation property because of the lipid PEG-PE with long PEG chain [19,20] and the sustain-released characteristics resulted from the solid lipid. MCA occlusion/reperfusion model has been well characterized and frequently used to evaluate drug efficacy on cerebral ischemia and infarction [27,28]. Therefore, the protective effect of SLNs on rats with MCA occlusion/reperfusion model was studied in this work. The SLNs showed obvious superiority in alleviating the neurological deficiencies (Fig. 4A), reducing the cerebral water content (Fig. 4B) and the infarct volume (Fig. 5) compared with free drug suspension or solution. The aim of evaluating the cerebral water content was to study whether the SLNs treatment could alleviate the cerebral edema, which is the entry of plasma water into swollen cerebral tissue and has been calculated by the resultant change in cerebral tissue osmolality through the induction of cerebral ischemia-reperfusion [29]. The effect of SLNs on cerebrovascular system including the changes of CeBF and CeR in anesthetic dogs showed that the SLNs could significantly increase CeBF and reduce CeR compared with the other two daidzein formulations (Fig. 6). It has been reported that the presence of the
Y. Gao et al. / Biomaterials 29 (2008) 4129–4136
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Fig. 5. Effect of SLNs on rats with MCA occlusion/reperfusion model. TTC stains were shown from five treatment groups (A): sham-operation group (a); saline control group (b); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration (c); free daidzein dissolved in DMSO solution by intravenous administration (d); SLNs dispersed in saline solution by intravenous administration (e). The infarct ratios (%) of the four treatment groups (B), saline control group ( ); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ); free daidzein dissolved in DMSO solution by intravenous administration ( ); SLNs dispersed in saline solution by intravenous administration ( ). The drug dosage for each group was 12 mg/kg. Data were given as mean SD of four rats. **p < 0.01 compared with saline control group.
blood–brain barrier (BBB), which is the main obstacle for central nervous system (CNS), is the most critical issue encountered in brain drug delivery [13]. SLNs, with their well-defined particles and lipid formulation, have been reported to enhance the delivery across BBB and lead to drug accumulation in brain [13]. However, either daidzein suspension or daidzein solution was lack of this
brain delivery ability, so it is undoubted that free daidzein could not exert good effect when used in the treatment of cerebrovascular disease. These results indicated that SLNs could be a good candidate to protect the brain of rat from injury after cerebral ischemia and reperfusion and to keep the cerebral blood demand and supply balance.
Fig. 6. Effects of SLNs on cerebrovascular systems. Measurement of CeBF (ml/min) (A) and CeR 102 (Kpa min/ml) (B). Free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration (4 mg/kg) ( ), free daidzein dissolved in DMSO solution by intravenous administration (4 mg/kg) ( ), SLNs dispersed in saline solution by intravenous administration (4 mg/kg) ( ). Data were given as mean SD of four dogs.
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5. Conclusion The sterically stabilized daidzein SLNs showed mean particle size of 126 14 nm with entrapment efficiency of 82.5 3.7%, and a sustained release manner with cumulative release 90% within 120 h in BSA solution. The in vivo pharmacokinetic and biodistribution demonstrated that SLNs could significantly increase circulation time and target to brain. The in vivo efficacies of SLNs on cardio-cerebrovascular systems were very significant. SLNs (4 mg/ kg) could reduce the MOC, MOU and CR in the heart, increase CeBF and reduce CeR in the brain of anesthetized dogs. SLNs (12 mg/kg) also showed a good protective effect on rats with ischemia-reperfusion injury model. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases. Acknowledgements The National Basic Research Program of China (2007CB935804) and the Important Direction Program of CAS (KSCX2-YW-R-09) are gratefully acknowledged for financial support. References [1] Eisenberg MJ, Brox A, Bestawros AN. Calcium channel blockers: an update. Am J Med 2004;116:35–43. [2] Boumendil EF, Mugnier C. Risk of cardio-cerebrovascular accidents associated with nitrates and other cardiovascular drugs. Am J Hypertens 2000;13(Suppl 1):S264–5. [3] Murkies AL, Wilcox G, Davis SR. Clinical review 92: phytoestrogens. J Clin Endocrinol Metab 1998;83:297–303. [4] Wang HZ, Zhang Y, Xie LP, Yu XY, Zhang RQ. Effects of genistein and daidzein on the cell growth, cell cycle, and differentiation of human and murine melanoma cells. J Nutr Biochem 2002;13:421–6. [5] Lo FH, Mak NK, Leung KN. Studies on the anti-tumor activities of the soy isoflavone daidzein on murine neuroblastoma cells. Biomed Pharmacother 2007;61:591–5. [6] Choi MS, Jung UJ, Yeo J, Kim MJ, Lee MK. Genistein and daidzein prevent diabetes onset by elevating insulin level and altering hepatic gluconeogenic and lipogenic enzyme activities in non-obese diabetic (NOD) mice. Diabetes Metab Res Rev 2008;24:74–81. [7] Jia TL, Wang HZ, Xie LP, Zhang RQ. Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production. Biochem Pharmacol 2003;65:709–15. [8] Li M, Poon P, Woo J. A pilot study of phytoestrogen content of soy foods and traditional Chinese medicines for women’s health in Hong Kong. Int J Food Sci Nutr 2004;55:201–5. [9] Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr 1997;17:353–81.
[10] Rimbach G, Boesch-Saadatmandi C, Frank J, Fuchs D, Wenzel U, Daniel H, et al. Dietary isoflavones in the prevention of cardiovascular disease – a molecular perspective. Food Chem Toxicol 2008;46:1308–19. [11] Kulling SE, Honig DM, Metzler M. Oxidative metabolism of the soy isoflavones daidzein and genistein in vitro and in vivo. J Agric Food Chem 2001;49:3024– 33. [12] Mu¨ller RH, Ma¨der K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery d a review of the state of the art. Eur J Pharm Biopharm 2000; 50:161–77. [13] Blasi P, Giovagnoli S, Schoubben A, Ricci M, Rossi C. Solid lipid nanoparticles for targeted brain drug delivery. Adv Drug Deliv Rev 2007;59:454–77. [14] Zur Mu¨hlen A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery d Drug release and release mechanism. Eur J Pharm Biopharm 1998;45:149–55. [15] Janning P, Schuhmacher US, Upmeier A, Diel P, Michna H, Degen GH, et al. Toxicokinetics of the phytoestrogen daidzein in female DA/Han rats. Arch Toxicol 2000;74:421–30. [16] Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in the rats. Stroke 1989;20:84–91. [17] Mu¨ller RH, Jacobs C, Kayser O. Nanosuspensions as particulate drug formulations in therapy: rationale for development and what we can expect for the future. Adv Drug Deliv Rev 2001;47:3–19. [18] Qiu F, Chen X, Song B, Zhong D, Liu C. Influence of dosage forms on pharmacokinetics of daidzein and its main metabolite daidzein-7-O-glucuronide in rats. Acta Pharmacol Sinica 2005;26:1145–52. [19] Li YP, Pei YY, Zhou ZH, Zhang XY, Gu ZH, Ding J, et al. Stealth polycyanoacrylate nanoparticles as tumor necrosis factor-alpha carriers: pharmacokinetics and anti-tumor effect. Biol Pharm Bull 2001;24:662–5. [20] Li YP, Pei YY, Zhang XY, Gu ZH, Zhou ZH, Yuan WF, et al. PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and in vivo biodistribution in rats. J Controlled Release 2001;71:203–11. [21] Agnisola C. Role of nitric oxide in the control of coronary resistance in teleosts. Comp Biochem Physiol A Mol Integr Physiol 2005;142:178–87. [22] Bandera E, Botteri M, Minelli C, Sutton A, Abrams KR, Latronico N. Cerebral blood flow threshold of ischemic penumbra and infarct core in acute ischemic stroke: a systematic review. Stroke 2006;37:1334–9. [23] Mu¨ller RH, Ru¨hl D, Runge SA. Biodegradation of solid lipid nanoparticles as a function of lipase incubation time. Int J Pharm 1996;144:115–21. [24] Willigers HM, Prinzen FW, Roekaerts PM. Comparison of the effects of dexmedetomidine and esmolol on myocardial oxygen consumption in dogs. Eur J Anaesthesiol 2004;21:957–66. [25] Gerber BL, Wijns W, Vanoverschelde JL, Heyndrickx GR, De Bruyne B, Bartunek J, et al. Myocardial perfusion and oxygen consumption in reperfused noninfarcted dysfunctional myocardium after unstable angina: direct evidence for myocardial stunning in humans. J Am Coil Cardiol 1999;34:1939–46. [26] Ge YB, Chen DW, Xie LP, Zhang RQ. Optimized preparation of daidzein-loaded chitosan microspheres and in vivo evaluation after intramuscular injection in rats. Int J Pharm 2007;338:142–51. [27] Imai H, Masayasu H, Dewar D, Graham DI, Macrae IM. Ebselen protects both gray and white matter in a rodent model of focal cerebral ischemia. Stroke 2001;32:2149–54. [28] Yrja¨nheikki J, Tikka T, Keina¨nen R, Goldsteins G, Chan PH, Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 1999;96:13496–500. [29] Sinha J, Das N, Basu MK. Liposomal antioxidants in combating ischemiareperfusion injury in rat brain. Biochem Pharmacol 2001;55:264–71.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
The role of daidzein-loaded sterically stabilized solid lipid nanoparticles in therapy for cardio-cerebrovascular diseases Yu Gao 1, Wangwen Gu 1, Lingli Chen, Zhenghong Xu, Yaping Li* Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2008 Accepted 3 July 2008 Available online 29 July 2008
Daidzein is a very good candidate for treating cardio-cerebrovascular diseases, but its poor oral absorption and bioavailability limit its curative efficacy. In this work, daidzein-loaded solid lipid nanoparticles (SLNs) with PEGylated phospholipid as stabilizer were successfully prepared by hot homogenization method. SLNs showed the mean particle size 126 14 nm with entrapment efficiency 82.5 3.7%. In vitro release of SLNs demonstrated a sustained release manner with cumulative release over 90% within 120 h in bovine serum albumin solution (4%, w/v). The pharmacokinetic behavior showed that SLNs loading daidzein could significantly increase circulation time compared with orally administrated daidzein suspension or intravenously delivered daidzein solution. SLNs showed the better effect on cardiovascular system of the anesthetic dogs by reducing the myocardial oxygen consumption (MOC) and the coronary resistance (CR) in heart compared with oral suspension or intravenous solution. The SLNs demonstrated the best effect on cerebrovascular system by increasing cerebral blood flow (CeBF) and reducing cerebrovascular resistance (CeR) in anesthetized dogs, and the protective effect on rats with ischemia-reperfusion injury model among three formulations. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Daidzein Cardiovascular disease Cerebrovascular diseases Nanoparticles
1. Introduction It is well known that cardiovascular and cerebrovascular diseases including the coronary artery disease, heart failure, acute myocardial infarction, arrhythmias et al., are the common causes of morbidity and mortality in the world. Although there are many drugs for therapy of cardiovascular and cerebrovascular disease on the market, most of them show side effect and contraindication [1], and some drugs may even increase the risk of cardio-cerebrovascular accident [2]. Daidzein is one of the major soy isoflavones found in Leguminosae and certain Traditional Chinese Medicinal herbs such as Kudzu. Several studies have shown that daidzein has various biological activity such as a weak estrogenic or antiestrogenic effect by binding to the nuclear estrogen receptor [3], inhibition of the growth of cancer cells [4,5], prevention of diabetes onset [6], promotion of the proliferation of osteoblast cells [7], and alleviation of menopausal symptom [8]. Apart from these beneficial effects on human health, daidzein is also a good candidate for the treatment of cardiovascular disease, which is its most important
* Corresponding author. Tel./fax: þ86 21 5080 6820. E-mail address: [email protected] (Y. Li). 1 These authors contributed equally. 0142-9612/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2008.07.008
pharmacological activity and the primary reason being used as a drug entity in China [9,10]. Daidzein could reduce the low-density lipoprotein (LDL) cholesterol, inhibit pro-inflammatory cytokines, cell adhesion proteins and platelet aggregation, induce nitric oxide production, and improve vascular reactivity. Daidzein could be a good candidate in treating cardiovascular diseases in virtue of the advantages of low cost and abundance as well as its clinical safe, and the Food and Drugs Administration has approved a health claim for soy based clinical trials [10]. However, its poor oral absorption and bioavailability limit its curative efficacy, which could result from its poor solubility in water, low partition coefficient of oil/water, in particular, the strong metabolism that occurs in the intestine and liver [11]. In addition, these properties bring about difficulty for preparing its injection, which could be used for first aid in clinic. Therefore, it is of great importance to investigate a new safe delivery system for daidzein with high effect on cardio-cerebrovascular diseases. We are interested in designing and developing an injectable delivery system for daidzein. In the present work, a sterically stabilized solid lipid nanoparticle (SLN) as daidzein carrier was investigated because of its good handling properties, very low intrinsic toxicity toward normal tissues, manageable burst effect issue that are commonly associated with many other colloidal formulations [12], in particular, good effect on brain targeting [13]. The aim of this work was to prepare the SLNs loading daidzein,
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determine their physicochemical characteristics and evaluate their in vivo behavior and pharmacological effect including the myocardial oxygen consumption (MOC), myocardial oxygen uptake (MOU), coronary blood flow (CBF) and the coronary resistance (CR), cerebrovascular blood flow (CeBF), and cerebrovascular resistance (CeR) in anesthetized dogs and the protective effect on the ischemia-reperfusion injury model in rats. 2. Materials and methods 2.1. Materials Daidzein (purity >99%) was obtained from Huike Plants Exploiture Co. (Shanxi, China). CompritolÒ888 ATO and Carboxymethyl cellulose sodium (CMC-Na) were purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Egg phosphatidylcholine (ePC) and poly(ethylene glycol) phosphatidylethanolamine (PEG2000-PE) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). TR-FIA kit for daidzein was purchased from Labmaster Co. (Truku, Finland). b-glucuronidase was obtained from Roche Pharma, Ltd. (Switzerland). Sulfatase was purchased from Sigma (St. Louis, MO, USA). All other reagents and solvents were analytical grade. 2.2. Animals Sprague–Dawley rats (Grade II, 215 15 g) and male Beagle dogs (8–10 kg) were supplied by Shanghai Experimental Animal Center, CAS. The animals were acclimatized at 25 1 C and a relative humidity of 75 5%. All animal procedures were performed following the protocol approved by the Institutional Animal Care and Use Committee at the Second Military Medical University. 2.3. Preparation and characteristics of SLNs loading daidzein SLNs loading daidzein were prepared by the hot homogenization method as described previously [14]. Briefly, 20 mg daidzein was added into 500 mg melted lipid materials (CompritolÒ888 ATO:ePC:mPEG2000-PE ¼ 18:6:1, w/w) in 70 C water bath, and sonicated for 30 min to dissolve daidzein with a probe sonicator (250W, JY88-II, China). Then, the solution was quickly dispersed in 50 ml prewarmed distilled water and vigorously stirred to form hot, milky emulsion in 65 C water bath (RET, IKA, Germany). The resultant emulsion was cooled to obtain SLNs. Finally, SLNs were purified by gel filtration on Sephadex G-25 eluted with water and lyophilized. The particle size, size distribution and z potential of SLNs loading daidzein were measured by laser light scattering using a Nicomp 380/ZLS zeta potential analyzer (Particle Sizing System, USA). The morphology of SLNs was examined using a transmission electron microscope (TEM, CM12, Philips, Netherlands) after negative staining with sodium phosphotungstate solution (0.2%, w/v). In order to know drug entrapment efficiency, the amount of daidzein incorporated in SLNs was determined using HPLC. Briefly, 10 mg of SLNs was added to 20 ml of methanol. After lipids were dissolved when heated to 65 C, the solution was cooled to room temperature and centrifuged at 20,000 rpm for 10 min to remove CompritolÒ888 ATO. The supernatant (10 ml) was directly injected into HPLC with C18 column, the mobile phase: acetonitrile-water (65:35), detection wavelength: 249 nm, the flow rate: 1 ml/min. The drug entrapment efficiency was defined as the percentage of daidzein recovered from SLNs compared with the initial drug amount. The drug loading was calculated as the amount entrapped daidzein compared with the total amount of SLNs. In vitro release of daidzein from SLNs was measured using the dialysis method at 37 C. The SLNs were dispersed in 5 ml physiological saline solution containing bovine serum albumin (BSA, 4%, w/v), then surged vertically for 3 min and filled into dialysis bag (molecular cutoff of 5000 Da). The dialysis bag was put into a container with 100 ml solution with BSA (4%, w/v). The solution was stirred at 300 rpm at 37 C. Subsequently, 0.5 ml of the solution was withdrawn from the system at predetermined time point and analyzed by HPLC. As control, the release profile of daidzein in physiological saline solution without BSA was also performed. 2.4. Pharmacokinetics and biodistribution of SLNs The health adult Sprague–Dawley rats were randomly divided into three groups (n ¼ 20), one group received free daidzein suspended in CMC-Na solution (0.5%, w/ v) by oral administration at a dose of 20 mg/kg. Another two groups were administrated with daidzein in DMSO solution (20 mg/kg) or SLNs dispersed in saline solution at a dose of 20 mg/kg by intravenous route, respectively. At predetermined point, blood sample (0.2 ml) was collected via caudal vein with heparinized tubes. Then, the samples were centrifuged at 2500 g for 10 min. Plasma was transferred into microcentrifuge tubes and stored at 20 C until analysis. The daidzein in plasma was determined using the TR-FIA kit. After samples were treated with hydrolysis and extraction, the values of fluorescence were measured by a timeresolved fluorometry Victor3 1420 multilabel counter (PerkinElmer, USA) according
to the protocol recommended by the manufacturer. Pharmacokinetic parameters were obtained using the Practical Pharmacokinetic Program Version 97. For study on biodistribution of daidzein, the animals were sacrificed at predetermine time point, and then tissues including heart, liver, spleen, lung, kidney and brain were harvested and stored at 50 C until analysis. The daidzein in tissue was determined according to Janning’s report [15]. Briefly, tissue was cut into small pieces and triturated into powder under liquid nitrogen. To 100 mg tissue aliquots, 500 ml lysis buffer (NaCl 75 mM; EDTA 25 mM with 1% SDS, w/v) was added, and samples were allowed to lyse for 1.5–2 h at 4 C. The internal standard (1 mg/ml, 10 ml genistein) was then added. After addition of hydrolysis buffer (1 M sodium acetate containing 1% (w/v) ascorbic acid and 0.1% (w/v) EDTA), 8 ml b-glucuronidase and 4 ml sulfatase, the samples were kept at 37 C for at least 15 h. Then, methanol as extraction solvent was added and extracted three times. After centrifugation, the organic phase was combined and dried under a stream of nitrogen at 37 C. The concentration of daidzein after dissolved in 100 ml methanol was determined by HPLC analysis. 2.5. Measurement of MOC, MOU, CBF and CR Twelve male Beagle dogs were randomly divided into three groups (n ¼ 4): group 1: control group, treated with free daidzein (4 mg/kg) suspended in CMC-Na solution (0.5%, w/v) by oral administration; group 2: treated with free drug (4 mg/ kg) dissolved in DMSO solution, and group 3: treated with SLNs (4 mg/kg) dispersed in saline solution by intravenous route. The dogs were anesthetized with 3% sodium pentobarbital intravenously (30 mg/kg). The trachea was connected to a respirator (SC-M5, Shanghai) through a midline cervical incision with respiratory rate of 16– 18 times/min. The esophagus was separated and a trachea was inserted into gastric canal for oral administration of the daidzein suspension. The arterial blood pressure (ABP) was measured through femoral artery catheterization by pressure transducer (MPU20.5A, Nihon Khonden, Japan). Then, thoracotomy was performed in the fourth left intercostal space and the heart was suspended in a pericardial cradle. Subsequently, the left circumflex branch of coronary artery and the aortic arch were isolated, and then the probes of electromagnetic flowmeter (MFV-1100/1200, Nihon Kohden, Japan) were placed to determine CBF and myocardial blood flow (MBF). Through the cervical incision, the right common carotid artery and internal jugular vein were isolated, and two catheters were inserted separately. The blood samples were extracted synchronously to determine PO2 and pH (DH-1830, Blood Gas Analyzer, China). Finally, the blood oxygen content in coronary venous sinus (VO2) and in artery (AO2) was calculated. After the operation was completed and the observation parameters displayed stable, the free drug (4 mg/kg) dissolved in DMSO solution and SLNs dispersed in saline solution were infused through femoral vein with an electro-driven constant flow pump (SH-88AB, Quanzhou, China) for 30 min. Free daidzein suspension (4 mg/ kg) was delivered though the gastric canal. The above parameters were recorded at preadministration (0) and 10, 30 and 60 min post administration. After the record finished, the heart was taken out, washed with normal saline solution and weighed. The MOC, MOU and CR were calculated from (Eqs. (1)–(3)). MOCðml=min=100gÞ ¼ MBFðml=minÞ ðAO2 ðml=LÞ VO2 ðml=LÞÞ=100g
(1)
Rate of MOUð%Þ ¼ ðAO2 ðml=LÞ VO2 ðml=LÞÞ=AO2 ðml=LÞ
(2)
CRðKpa=ml=minÞ ¼ ABPðKpaÞ=CBFðml=minÞ
(3)
2.6. Effect on rats with middle cerebral artery (MCA) occlusion and reperfusion model The protection against cerebral ischemia of SLNs was investigated by a MCA occlusion/reperfusion model in rats according to Longa’s method [16]. Briefly, eighty male Sprague–Dawley rats were divided into five groups (n ¼ 16): group 1: sham control group; group 2: control group, treated with saline solution by intravenous administration; group 3: oral group, treated with free daidzein suspension (12 mg/kg) by oral administration; group 4: treated with free daidzein solution (12 mg/kg) and group 5: treated with SLNs (12 mg/kg) by intravenous route. Rats were anesthetized with 10% chloral hydrate (3 ml/kg). The right carotid artery bifurcation was exposed, and the right common carotid artery (CCA) and internal carotid artery (ICA) were isolated and ligated transiently. The external carotid artery (ECA) was isolated and an 18 mm length of 4-0 nylon suture was introduced into the ECA lumen and advanced into the ICA to block the origin of MCA. Saline solution, free daidzein solution or SLNs were intravenously administered just 1 h after occlusion. Free daidzein suspension was administrated by direct gastric gavage at 1 h after occlusion. The suture was left in place for another 1 h, and then restoration of MCA blood flow was achieved by withdrawing the suture to the ECA. The sham control rats received the same surgery procedures but had not the suture inserted. Twenty-three hours after the reperfusion, the neurological symptom score was determined according to Longa’s five score regulation [16], which consists of
Y. Gao et al. / Biomaterials 29 (2008) 4129–4136 0 grade: no neurological defect; 1 grade: failure to extend right forepaw fully; 2 grade: circling to the right; 3 grade: circling to the reverse of the right-the left; 4 grade: rats did not walk spontaneously and had a depressed level of consciousness. After the evaluation of neurological symptoms, rats were sacrificed to excise their brains. In each group, one half of the brain (n ¼ 8) was used to detect the brain water content, and the other half was used to measure the infarct volume. The water content was assessed by comparing the wet weight with the dry weight, which were determined immediately after excision and after staying in an oven at 105 C for 24 h, respectively. The water content were calculated from (Eq. (4)): Water content ð%Þ ¼ ðwet weight dry weightÞ 100=wet weight:
(4)
The infarct volume measurement was performed by slicing the brain into seven sections (2-mm thick) and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 C for 30 min with shaking, then immersed in 10% formaldehyde neutral buffer solution for preservation. Photograph of each section was taken with a digital camera (Fujifilm, Tokyo, Japan) and the infarct area was measured with a software (Medbrain 2.0). The percentage of infarction (infarct ratio) was calculated from the data of infarct volume and total coronal section according to (Eq. (5)): Ratioð%Þ ¼ infarct volume mm3 100=total coronal section mm3
(5)
2.7. Measurement of CeBF and CeR in anesthetic dogs Twelve male Beagle dogs were divided into three groups (n ¼ 4): group 1: control group, treated with free daidzein suspension (4 mg/kg) by oral administration; group 2: treated with free drug solution (4 mg/kg); and group 3: treated with SLNs (4 mg/kg) dispersed in saline solution. The dogs were anesthetized with 3% sodium pentobarbital 30 mg/kg intravenously. After a midline cervical incision, the esophagus was separated and a trachea was inserted into the gastric canal for oral administration of the daidzein suspension. Then, CCA and ECA were isolated, and ECA were ligated transiently. A probe of electromagnetic flowmeter with diameter of 2.0 w 2.5 mm was placed into CCA to determine the blood flow, which was denoted as ICA blood flow. Then, the femoral artery was isolated, and ABP was measured through femoral artery catheterization by pressure transducer. The catheter was also implanted into the femoral vein for infusion of daidzein solution and SLNs. After the surgical, the animals were placed to stabilize for 30 min, and the above parameters were recorded at preadministration (0 min). After the free drug solution or SLNs infused through femoral vein with an electro-driven constant flow pump for 30 min, and the free daidzein suspension was administrated though the gastric canal, the parameters were recorded at 5, 15, 30 and 60 min. When the record finished, the brain was taken out, washed with normal saline solution and weighed. The CeBF and CeR were calculated from Eqs. (6) and (7). CeBFðml=min=100gÞ ¼ 2 CCA blood flow ðml=min=100gÞ
(6)
CeR ¼ ABPðkPaÞ=½CBFðml=minÞ,100g brain
(7)
2.8. Statistical analysis Data were presented as the mean SD of each group. Statistical analysis of the data from the physicochemical studies was performed by applying the Student’s t-test. A1-way ANOVA followed by the Bonferroni correction was used to assess
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statistical differences for the physiological variables. Analysis of variance (ANVOA) followed by Duncan’s multiple-range test was used for the scores obtained in the neurological symptom. Values of p < 0.05 were indicative of significant differences, and p < 0.01 indicative of a very significant difference.
3. Results 3.1. Physicochemical characteristics of SLNs In order to improve the pharmacological activity of daidzein on cardio-cerebrovascular diseases, SLNs loading daidzein were successfully prepared by the hot homogenization method with CompritolÒ888 ATO and ePC as solid core, and PEG2000-PE as stabilizer to increase the circulation time and drug accumulation in brain [13]. SLNs were round and uniform with the mean particle size of 126 14 nm (Fig. 1A), polydispersity indexes of 0.226 0.023 and zeta potentials of 34.5 3.4 mV. It has been reported that the zeta potential is a very important factor to evaluate the stability of colloidal dispersion because particles could be dispersed stably when absolute value of zeta potential is over 30 mV due to the electric repulsion between particles [17]. In addition, SLNs showed the drug entrapment efficiency of 82.5 3.7% with the drug loading 3.5 0.4%. In order to know the release behavior of daidzein from SLNs, we performed the release experiment in physiological saline solution with or without BSA (4%, w/v). The drug release profiles from SLNs were shown in Fig. 1B. The SLNs showed a burst drug release (45%) within the initial 12 h in BSA solution. Subsequently, the release rate gradually slowed, and the cumulative release of drug was over 90% within 120 h. As control, the release of daidzein from SLNs in physiological saline solution without BSA showed a sustained release manner without obvious burst drug release, and the cumulative release of daidzein was about 40% within 120 h. 3.2. Pharmacokinetics and biodistribution The pharmacokinetic behavior of daidzein encapsulated in SLNs was investigated in healthy Sprague–Dawley rats after intravenous administration. The drug concentration-time curves of daidzein were shown in Fig. 2A. After a single dose intravenous administration, SLNs showed a prolonged circulation time of daidzein in blood, which could still be measured in plasma at 48 h. However, the free drug in DMSO solution was quickly removed from the circulation system. As control, daidzein by oral route showed very low drug concentration and bioavailability, which was accordant with Qiu’s report [18]. The pharmacokinetic profiles for daidzein in rats were best described by a non-compartmental model. The main pharmacokinetic parameters of SLNs were t1/2 (12.62 1.36 h),
Fig. 1. Transmission electron microscopy of SLNs (A) (Bar ¼ 200 nm); release profiles of daidzein from SLNs in saline solution (
) or BSA solution (
) at 37 C (B).
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Daidzein concentration (ng/mL)
1000000
suspension, SLNs exhibited a bias to brain with more drug accumulation in brain. At 12 h, the SLNs showed an increasing accumulation in brain, which could result from the brain targeting of SLNs [13]. In addition, SLNs still kept higher drug level in plasma after 12 h than that of other two formulations, which should attribute to the PEG chains on the surface of SLNs that increased the circulatory half-life time and decreased the drug uptake in reticuloendothelial system (RES) sites.
A
10000
3.3. In vivo pharmacological efficacy
100
1
Daidzein amount (µg/g tissue)
Daidzein amount µg/g tissue) (µ
25
0
12
24 Time (h)
36
48
B
20 15 10 5 0 25 20
*
*
blood heart liver spleen lung kidney brain
C
15 10 5 0
*
*
blood heart liver spleen lung kidney brain
Fig. 2. Pharmacokinetics and biodistribution of SLNs loading daidzein. (A) Blood clearance curves of daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ), free daidzein dissolved in DMSO solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ) at a dose of 20 mg/kg. Data were given as mean SD of six to eight rats per group. (B) Tissue distribution of daidzein in rat at 6 and 12 h (C). Free daidzein suspension by oral administration ( ), free daidzein solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ) at a dose of 20 mg/kg. Data were given as mean SD of four to five rats per group. *p < 0.01 compared with oral suspension.
MRT (5.59 0.76 h), CL (0.24 0.13 L/(h kg)) and AUC0 w N (83.62 1.89 mg h/ml), which showed significant difference with free drug in DMSO solution with t1/2 (5.62 1.02 h), MRT (2.04 0.67 h), CL (0.61 0.12 L/(h kg)) and AUC0 w N (28.29 1.29 mg h/ml). As control, the pharmacokinetic parameters of daidzein from oral administration were t1/2 (4.53 0.84 h), MRT (1.68 0.35 h), CL (0.86 0.31 L/(h kg)) and AUC0 w N (2.92 0.26 mg h/ml). The result indicated that SLNs with steric-stabilized lipid PEG-PE would prevent their rapid uptake by mononuclear phagocyte system (MPS) and improve their circulatory half-life, which had already been found in other delivery systems [19,20]. To evaluate in vivo uptake of daidzein, the distribution profiles of daidzein in heart, liver, spleen, lung, kidney and brain of rats were shown in Fig. 2B,C. At 6 h after intravenous administration, drug from SLNs was mainly distributed to blood, heart, liver and spleen. However, drug in DMSO solution by intravenous administration or suspension by oral administration mainly distributed to kidney, which indicated that they could be quickly eliminated through kidney. Compared with daidzein solution or oral
3.3.1. Measurement of MOC, MOU, CBF and CR In order to know the effect of SLNs on cardiovascular system, MOC, MOU, CBF and CR in dogs were measured. Fig. 3A demonstrated that the MOC of dogs injected SLNs dispersed in saline solution by intravenous administration decreased, and the change of MOC (%) showed significant difference at 10 min (p < 0.05), and very significant difference at 30 and 60 min (p < 0.01) compared with that of the group administrated orally. While the change of MOC (%) of dogs injected free daidzein solution showed the significant difference only after 30 min compared with that of the group administrated orally (p < 0.05). The rate of MOU was shown in Fig. 3B, the change of the rate of MOU (%) of dogs injected SLNs showed very significant difference from 30 to 60 min compared with those of the dogs administrated orally (p < 0.01). While the change of rate of MOU (%) of dogs injected free daidzein solution showed significant difference at 30 min (p < 0.05) and very significant difference at 60 min (p < 0.01) compared with that of the group administrated orally. Fig. 3C demonstrated that the CBF of dogs injected SLNs reduced sharply and the change of CBF (%) showed very significant difference from 30 to 60 min during experiment compared with that of the group administrated orally (p < 0.01). The change of CBF of dogs injected daidzein solution showed significant difference at 30 min compared with that of the group administrated orally (p < 0.05). The CR is also an important factor to evaluate the cardiac function and is closely related with CBF and MOC [21]. In present study, the effect of SLNs on CR were also measured, and the results were shown in Fig. 3D. The change of CR (%) of the animals treated with SLNs showed significant difference compared with that of the group administrated orally at 30 min (p < 0.05) and very significant difference at 60 min (p < 0.01). 3.3.2. Effects on rats with MCA occlusion/reperfusion model Rats subjected to 1 h of MCA occlusion were divided into five groups (n ¼ 16), sham-operation group and groups treated with free daidzein by oral administration at a dose of 12 mg/kg, saline solution, free daidzein solution or SLNs dispersed in saline solution (12 mg/kg) through intravenous route. At 24 h after perfusion following another 1 h of MCA occlusion, the neurological abnormality of rats was observed and scored. The results were shown in Fig. 4A. The sham-operation group showed no neurological abnormalities with the neurological score 0. The saline solution treated group showed severely neurological deficiencies such as circling to the right and circling to the left with the mean neurological score 2.4 0.53. The neurological deficiencies could be slightly alleviated by free daidzein administrated orally (2.1 0.43), and greatly alleviated by free daidzein injected intravenously (1.5 0.48). While SLNs group showed the best efficacy (0.9 0.25). Subsequently, the brains of the rats were excised to determine the cerebral water content and the infarct volume. Fig. 4B showed that the water content of the right damaged hemisphere was similar to the left normal hemisphere in sham-operation group (p > 0.05). While in the saline solution treated group, the water content of the right hemisphere was much higher than that of left hemisphere (p < 0.01). Treated with 12 mg/kg free drug intravenously could
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Fig. 3. Effect of SLNs on cardiovascular systems. Measurement of MOC (ml/min/100 g) (A), MOU (%) (B), CBF (ml/min) (C) and CR (Kpa min/ml) (D) in different time points. Free daidzein suspended in CMC-Na (0.5%, w/v) by oral administration (4 mg/kg) ( ), free daidzein dissolved in DMSO solution by intravenous administration (4 mg/kg) ( ), SLNs dispersed in saline solution by intravenous administration (4 mg/kg) ( ). Data were given as mean SD of four dogs.
reduce the brain edema compared with saline control group (p < 0.05), and SLNs group showed better efficacy at the same dose (p < 0.01), in particular, the water content of the right hemisphere showed no significant difference compared with that of the left hemisphere in this group (p > 0.05). On the contrary, treated with free drug administrated orally (12 mg/kg) could only slightly reduce the brain edema (p > 0.05). The infarct volume was measured by the TTC-staining in which the infarct tissue became pale and the normal tissue was red. In the saline solution-treating group, a well-defined region of ischemia was successfully induced (Fig. 5A). SLNs could significantly reduce the infarct volume compared with that of the control group. The group treated with free drug through intravenous route showed reduction of infarct ratio from 29.3% (control group) to 22.9%, while the infarct ratio of SLNs group reduce to 15.7% (Fig. 5B). As another control, the volume of reduced infarct in the group by free daidzein after oral administration was very limited. 3.3.3. Measurement of CeBF and CeR in dogs In order to know whether SLNs could improve the effect of daidzein on cerebrovascular system, the cerebrovascular blood supply and CeR in dogs were investigated. It has been reported that ischemic stroke is caused by decreased vital blood supply transiently or permanently resulted from embolism or hemorrhage in the brain, therefore, the improvement of blood supply can rescue the neuronal cells and even reverse the impairment in ischemic tissue [22]. The CeBF was measured pre and post administration of SLNs in anesthetic dogs. The result demonstrated that the CeBF of dogs increased from 145 to 186 ml/min within 60 min after intravenous administration of SLNs (4 mg/kg) (Fig. 6A). The CeBF of dogs administrated free drug solution also increased during the
initial phase of experiment and hold a little change until 60 min at a dose of 4 mg/kg. However, the CeBF of dogs administrated free drug suspension orally showed neglectable difference during experiment. Fig. 6B showed the changes of CeR of the animals treated with three formulations. SLNs could reduce the CeR of animals, and the change of CeR (%) from 15 to 60 min in this group demonstrated a very significant difference compared with that of dogs administrated orally (p < 0.01). Free daidzein also could reduce CeR of animals, and the change of CeR (%) in this group showed a significant difference compared with that of dogs administrated orally (p < 0.05). 4. Discussion To our knowledge, this is the first report about the solid lipid nanoparticle as an injectable delivery system for daidzein and applying SLNs loading daidzein for treatment of cardio-cerebrovascular disease. It has been reported that SLN is a particulate system for parenteral administration due to its solid property at body temperature and its slow drug release. In present work, the injectable daidzein-loaded SLNs were successfully prepared by the hot homogenization method. Compritol 888 ATO and ePC were selected as matrix material with PEG2000-PE as stabilizer. Considering that some serum proteins may destabilize the particles and accelerate drug release [23], meanwhile for a better mimic of in vivo environment and study on the release kinetics of SLNs, we chose the physiological saline solution with BSA (4%, w/v) as release media. Compared with the release kinetics of SLNs in media without BSA, SLNs showed more rapid release of daidzein in BSA solution, which suggested that the matrix was likely to destabilize
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Fig. 4. Effect of SLNs on rats with MCA occlusion/reperfusion model. Effect of SLNs on neurological abnormality in MCA occlusion/reperfusion model (A); Effect of SLNs on water content of different brain hemispheres (B). Sham-operation group ( ); saline control group ( ); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ); free daidzein dissolved in DMSO solution by intravenous administration ( ), SLNs dispersed in saline solution by intravenous administration ( ). The dosage in each group was 12 mg/kg. Data were given as mean SD of four rats. *p < 0.05 and **p < 0.01 compared with saline control group. In saline control group, **p < 0.01 compared with left normal hemisphere.
in the presence of serum proteins or other biological factors in vivo. It has been reported that the lipid compritol might start to crystallize first and form an inner core of pure lipid during the solidification process of SLNs. As a result, a solid drug layer might be formed around this lipid core with an enrichment of drug in the outer particle region [12]. So, it was possible that the drug molecules in the outer region of SLNs moved out of the particles via short diffusion path and brought about a burst drug release. After burst release, the SLNs showed a good sustained release manner in the presence of BSA (Fig. 1B), which demonstrated that SLNs could be a promising system for long-term daidzein delivery. Due to poor solubility of daidzein, oral administration is one of the main routes, but these edible formulations presented poor bioavailability. Even if daidzein was intravenously injected by dissolving in DMSO solution, the quick elimination from blood without sufficient circulation time made daidzein be not able to cumulate in specific organs to exert curative effect. However, daidzein encapsulated in SLNs showed significant differences in terms of the pharmacokinetic behavior from daidzein suspension
or daidzein solution (Fig. 2A). In particular, some pharmacokinetic parameters of SLNs such as t1/2 were found to be much longer than that of the free drug. After 48 h, SLNs could still be detected in the plasma, whereas free daidzein formulations had almost disappeared from circulation system after 12 h. This is undoubtedly related to the solid state of the lipid, which allowed daidzein to release slowly from the particles, and the long PEG chain that stabilized SLNs by steric hindrance decreased macrophages recognition and increased blood circulation time [19,20]. Many clinical studies demonstrated that patients with myocardial ischemia showed very low ability to increase CBF in response to an increased myocardial oxygen demand [24]. It has been reported that the cardiovascular disease such as cardiac dysfunction, arrhythmias and infarction were caused by imbalance of myocardial oxygen demand and supply [25]. Therefore, it is very important to keep the myocardial oxygen demand and supply balance in cardiovascular system. In this work, we examined the change of MOC, MOU, CBF and CR in anesthetic dogs after administration of different daidzein formulations, and found that the SLNs could significantly reduce MOC, MOU and CR (Fig. 3). Compared with daidzein suspension and solution, SLNs showed the best effect on cardiovascular system to keep the myocardial oxygen demand and supply balance. This phenomenon could be explained by the pharmacokinetic behavior (Fig. 2A) and biodistribution (Fig. 2B,C) of different formulations. Daidzein suspension was not well absorbed and the drug concentration was very low. Although daidzein solution showed higher drug concentration than daidzein suspension, the daidzein was quickly eliminated and rapidly transported to tissues or organs from blood, which was accordant with other scholar’s reports [15,26]. According to the biodistribution data, we could find that the drug in DMSO solution by intravenous administration was mainly distributed to kidney not to the heart. So both suspension and solution could not exert the intrinsic effects of daidzein. However, SLNs showed much higher drug concentration in vivo, and daidzein could be detected even at 48 h after injection (Fig. 2A). Also, a relatively high drug accumulation in heart was detected (Fig. 2B,C), which indicated more drug could be carried into the cardiovascular systems. This property of SLNs should lead to higher efficacy of daidzein in cardiovascular systems. So far, there is no report about daidzein used for treatment of cerebrovascular disease, which could be due to its low bioavailability and the metabolism that occurs in the intestine via oral administration [11]. From the result of the tissue distribution, we found that daidzein in SLNs was apt to distribute to brain tissue, which could result from the brain targeting of SLN [13]. In addition, the SLNs prepared in the present study showed the long circulation property because of the lipid PEG-PE with long PEG chain [19,20] and the sustain-released characteristics resulted from the solid lipid. MCA occlusion/reperfusion model has been well characterized and frequently used to evaluate drug efficacy on cerebral ischemia and infarction [27,28]. Therefore, the protective effect of SLNs on rats with MCA occlusion/reperfusion model was studied in this work. The SLNs showed obvious superiority in alleviating the neurological deficiencies (Fig. 4A), reducing the cerebral water content (Fig. 4B) and the infarct volume (Fig. 5) compared with free drug suspension or solution. The aim of evaluating the cerebral water content was to study whether the SLNs treatment could alleviate the cerebral edema, which is the entry of plasma water into swollen cerebral tissue and has been calculated by the resultant change in cerebral tissue osmolality through the induction of cerebral ischemia-reperfusion [29]. The effect of SLNs on cerebrovascular system including the changes of CeBF and CeR in anesthetic dogs showed that the SLNs could significantly increase CeBF and reduce CeR compared with the other two daidzein formulations (Fig. 6). It has been reported that the presence of the
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Fig. 5. Effect of SLNs on rats with MCA occlusion/reperfusion model. TTC stains were shown from five treatment groups (A): sham-operation group (a); saline control group (b); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration (c); free daidzein dissolved in DMSO solution by intravenous administration (d); SLNs dispersed in saline solution by intravenous administration (e). The infarct ratios (%) of the four treatment groups (B), saline control group ( ); free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration ( ); free daidzein dissolved in DMSO solution by intravenous administration ( ); SLNs dispersed in saline solution by intravenous administration ( ). The drug dosage for each group was 12 mg/kg. Data were given as mean SD of four rats. **p < 0.01 compared with saline control group.
blood–brain barrier (BBB), which is the main obstacle for central nervous system (CNS), is the most critical issue encountered in brain drug delivery [13]. SLNs, with their well-defined particles and lipid formulation, have been reported to enhance the delivery across BBB and lead to drug accumulation in brain [13]. However, either daidzein suspension or daidzein solution was lack of this
brain delivery ability, so it is undoubted that free daidzein could not exert good effect when used in the treatment of cerebrovascular disease. These results indicated that SLNs could be a good candidate to protect the brain of rat from injury after cerebral ischemia and reperfusion and to keep the cerebral blood demand and supply balance.
Fig. 6. Effects of SLNs on cerebrovascular systems. Measurement of CeBF (ml/min) (A) and CeR 102 (Kpa min/ml) (B). Free daidzein suspended in CMC-Na solution (0.5%, w/v) by oral administration (4 mg/kg) ( ), free daidzein dissolved in DMSO solution by intravenous administration (4 mg/kg) ( ), SLNs dispersed in saline solution by intravenous administration (4 mg/kg) ( ). Data were given as mean SD of four dogs.
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5. Conclusion The sterically stabilized daidzein SLNs showed mean particle size of 126 14 nm with entrapment efficiency of 82.5 3.7%, and a sustained release manner with cumulative release 90% within 120 h in BSA solution. The in vivo pharmacokinetic and biodistribution demonstrated that SLNs could significantly increase circulation time and target to brain. The in vivo efficacies of SLNs on cardio-cerebrovascular systems were very significant. SLNs (4 mg/ kg) could reduce the MOC, MOU and CR in the heart, increase CeBF and reduce CeR in the brain of anesthetized dogs. SLNs (12 mg/kg) also showed a good protective effect on rats with ischemia-reperfusion injury model. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases. Acknowledgements The National Basic Research Program of China (2007CB935804) and the Important Direction Program of CAS (KSCX2-YW-R-09) are gratefully acknowledged for financial support. References [1] Eisenberg MJ, Brox A, Bestawros AN. Calcium channel blockers: an update. Am J Med 2004;116:35–43. [2] Boumendil EF, Mugnier C. Risk of cardio-cerebrovascular accidents associated with nitrates and other cardiovascular drugs. Am J Hypertens 2000;13(Suppl 1):S264–5. [3] Murkies AL, Wilcox G, Davis SR. Clinical review 92: phytoestrogens. J Clin Endocrinol Metab 1998;83:297–303. [4] Wang HZ, Zhang Y, Xie LP, Yu XY, Zhang RQ. Effects of genistein and daidzein on the cell growth, cell cycle, and differentiation of human and murine melanoma cells. J Nutr Biochem 2002;13:421–6. [5] Lo FH, Mak NK, Leung KN. Studies on the anti-tumor activities of the soy isoflavone daidzein on murine neuroblastoma cells. Biomed Pharmacother 2007;61:591–5. [6] Choi MS, Jung UJ, Yeo J, Kim MJ, Lee MK. Genistein and daidzein prevent diabetes onset by elevating insulin level and altering hepatic gluconeogenic and lipogenic enzyme activities in non-obese diabetic (NOD) mice. Diabetes Metab Res Rev 2008;24:74–81. [7] Jia TL, Wang HZ, Xie LP, Zhang RQ. Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production. Biochem Pharmacol 2003;65:709–15. [8] Li M, Poon P, Woo J. A pilot study of phytoestrogen content of soy foods and traditional Chinese medicines for women’s health in Hong Kong. Int J Food Sci Nutr 2004;55:201–5. [9] Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr 1997;17:353–81.
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