http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.974001
RESEARCH ARTICLE
Formulation, characterization, and in vitro/vivo studies of aclacinomycin A-loaded solid lipid nanoparticles Youpeng Jia1, Jun Ji2, Feng Wang3, Liangang Shi1, Jingbo Yu1, and Dong Wang1
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1
Department of General Surgery, Dalian Municipal Center Hospital, Liaoning, China, 2Department of Central Laboratory, Dalian Municipal Center Hospital, Liaoning, China, and 3Department of Pharmaceutics, Shanghai Institute of Pharmaceutical Industry, Shanghai, China
Abstract
Keywords
Objective: The aim of this study was to prepare aclacinomycin A (ACM)-loaded solid lipid nanoparticles (SLNs) and to evaluate their in vitro and in vivo characteristics. Methods: SLNs were prepared using an emulsion evaporation–solidification method, and characterized in accordance with the morphological examination, particle size distribution, entrapment efficiency, drug-loading, and in vitro release. Pharmacokinetic and biodistribution studies were employed to evaluate the in vivo of SLNs. Results: The SLNs were spherical in shape, uniform in size, and appropriate for administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The SLNs were produced with stable physical properties and demonstrated significantly sustained release. The pharmacokinetic behavior of ACM was greatly improved by lyophilized injection of SLN with sustained drug release and high bioavailability. In addition, the results obtained from tissue distribution showed that ACM-SLNs were hepatic targeting in vivo. Conclusions: The present work demonstrated the feasibility of liver-targeted delivery of ACM utilizing SLNs.
Aclacinomycin A, biodistribution, in vitro release, pharmacokinetics, solid lipid nanoparticles
Introduction Aclacinomycin A (ACM) hydrochloride is an anthracycline antitumor antibiotic isolated from Streptomyces galilaeus MA-144-M1. ACM inhibits the synthesis of nucleic acid, especially RNA, by acting as an inhibitor of not only topoisomerase II but also topoisomerase I (Nitiss et al., 1997; Li et al., 2006; Chen et al., 2006; Liu et al., 2007; Griaznova et al., 1992; Shiokawa et al., 2005; Wei et al., 2011). This drug is widely used for the treatment of liver, stomach, lung, and ovarian carcinoma; malignant lymphoma and acute leukemia, but its clinical use is still limited by its toxicity. To reduce the toxic responses and increase the antitumor effect of ACM, it should be effectively targeted to the tumor. Targeting agents are usually divided into two categories: active and passive (Torchilin, 2010; Hirsja¨rvi et al., 2011; Danhier et al., 2010). Active targeting preparation uses modified pharmaceutical carriers which act like missiles to deliver specifically targeting drugs and then play a pharmacodynamic concentration. After surface modification, if the
Address for correspondence: Youpeng Jia, Department of General Surgery, Dalian Municipal Center Hospital, Dalian, China. E-mail: [email protected]
History Received 12 August 2014 Revised 3 October 2014 Accepted 3 October 2014
drug-loaded particles are not recognized by macrophages, or they can connect with receptors and target cells bound up with a specific ligand, or they develop immunity due to attachment with monoclonal antibodies, then the modification can avoid macrophage intake, prevent the concentration in the liver, and change the distribution of particles in the body naturally to reach specific targets. The drug can also be modified into prodrug, which can be activated as pharmacologically inert substance and be effective in certain target areas. If the particles, through active targeting, intend to reach the target site without being intercepted (diameter 4–7 mm), usually the particle size should be no larger than 4 mm. Passive targeting happens when drug-loaded particles are absorbed by macrophages of the monocyte–macrophage system (especially the kupffer cells of liver), and then delivered to liver, spleen and other organs by normal physiological process but difficult to reach other target areas. After intravenous injection, the distribution of passive targeting particles within body is primarily decided by their size. When the particle diameter is between 50 and 200 nm, the majority enters the circulatory system. But instead of being released into the systemic circulation directly, they are absorbed by the reticulo endothelial system (Res), leading to greatest drug accumulation in the liver and thus achieving the targeting effect. In addition to particle size, the nature of the surfaces of the particles also plays an
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important role in the distribution. Currently, many reports have touched the ACM targeting agents, for instance, liposomes (Griaznova et al., 1992), microemulsion (Shiokawa et al., 2005), nanoparticles (Gao et al., 2004) and others. Solid lipid nanoparticles (SLNs) are one of the new nanoparticle drug-delivery systems (Mu¨ller et al., 2000; Wissing et al., 2004). SLNs are a solid colloidal drugdelivery system with particle size ranging from 50 to 1000 nm, in which the drugs are wrapped into the lipid core with solid natural or synthetic lipids. As a nanoparticle drug-delivery system is newer than emulsions, Liposomes, microparticles, SLNs have been used to control drug release (Li et al., 2006). It has many advantages, for example, good physiological compatibility, well-controlled drug release, good targeting (Chen et al., 2006; Liu et al., 2007), high physical stability similar to polymer nanoparticles, low toxicity, and large-scale production comparable with liposomes and emulsions. The many administration routes for SLNs include intravenous administration to achieve a targeted effect or controlled release, oral administration to control the drug release in the gastrointestinal tract, and local administration. In this study, SLNs were prepared using emulsion evaporation–solidification at a low temperature, and SLNs lyophilized powder were produced with a refrigerated air dryer. Some preliminary studies were done on the quality of the SLNs morphology, its physical and chemical properties, and effect of in vitro release. Moreover, pharmacokinetics and biodistribution studies of ACM solid lipid nanoparticles (ACM-SLNs) were investigated to provide a reference for clinical application.
Materials and methods Materials ACM (Figure 1A, purity 495%) was purchased from Jiangsu Yangzhou Pharma Ltd, China. Egg-phosphatidyl choline (EPC) and high purity cholesterol (CHOL) were donated by Phospholipid Tech Ltd, Dalian, China. Quercetin (Figure 1B, purity 499.3%) was given by the National Institute for Food and Drug control (Beijing, China). Pluronic F-68 was purchased from Beijing Bayerd Biopharm Ltd, Beijing, China. All the chemicals and solvents used in the present study were of analytic grade. Deionized water was used throughout the experiment. The experiments were performed on rats weighing between 220 ± 20 g. The animals were kept in cages in a room at a temperature of 25 ± 2 C, with a 12:12 light-dark cycle. Food and water were accessible. All experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the China National Institutes of Health. Preparation of ACM-SLNs ACM (50 mg), CHOL (400 mg), and EPC (750 mg) were weighed accurately and dissolved in chloroform (10 mL), forming an organic phase at 45 C ± 1 C water bath. The aqueous phase consisted of pluronic F-68 solution (5%, 40 mL) at 45 C ± 1 C water bath. The organic phase was
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Figure 1. The structure of ACM (A) and Quercetin (B, internal standard).
slowly added to the water phase under mechanical stirring (600 rpm) at 45 C ± 1 C. After 4 h, the volume was condensed by approximately half to obtain a semitransparent solution. The solution was quickly dispersed in the water (75 mL, temperatures ranging from 0 to 2 C) by stirring for another 2 h. Thus, a solid lipid nanoparticle suspension was obtained. Two substances were chosen as the cryoprotectant: 5% (w/v) glucose and 5% (w/v) mannitol. About 1.0 mL cryoprotectant and 1.0 mL suspension of ACM-SLNs at a ratio of 1:1(v/v) were added to the vial (10 mL). The vial was pre-frozen for 8 h at 60 C and, after the solution was added, it was kept in the refrigerated air dryer for another 24 h to obtain the ACM-SLNs lyophilized powder. Morphological characterization and particle sizing The morphological examination of the ACM-SLNs was performed using a transmission electron microscope (TEM) (Philips CM120, Philips, Amsterdam, The Netherlands)). In practice, 10 mg of ACM-SLNs lyophilized powder containing 0.5% (w/v) phosphotungstic acids were placed on a carbon film coated on a copper grid and observed at 80 kV in the electron microscope. Particle size distribution and mean diameter of the prepared ACM-SLN were determined by dynamic light scattering (DLS) using a NICOMP 380 Submicron Particle Sizer (PSS, Santa Barbara, CA) equipped with a 5 mW heliumneon laser at 632.8 nm. Sample solutions were transferred into the light scattering cells. The intensity autocorrelation was measured at a scattering angle of 90 at room temperature. Data were analyzed in terms of intensityweighted NICOMP distributions. Each reported experimental result is the average of at least three dh values obtained from analysis of the autocorrelation function accumulated for at
ACM A-loaded SNPs
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least 20 min. Zeta potential was measured on the same samples prepared for size analysis. Drug content, entrapment efficiency, and drug loading The drug content analysis was conducted on various batches of the SLNs. Briefly, the ACM-SLNs lyophilized powders were dissolved in an adequate quantity of phosphate buffer pH 6.8 and then filtered. The drug content of ACM was detected by high-performance liquid chromatography (HPLC) (described as follows). The drug entrapment efficiency and drug loading were calculated by the following equations:
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DL% ¼
WACM WACMSLNs
EE% ¼
100%
WACM 100% WTotal
ð1Þ
3
Stability The stability of the protocol was designed based on the ICH Guidelines. The ACM-SLNs were stored at a temperature of 25 ± 2 C and 60 ± 5% relative humidity (RH) for a period of 6 months and at 5 ± 3 C for 12 months. Both represent the accelerated storage temperature and long-term storage temperature respectively for products which are intended to be refrigerated. The stored samples were tested for their drug content, particle size distribution, and for any physical change. The drug content was determined by the HPLC method. The testing was carried out at 0, 2, 4, and 6 months for accelerated storage condition and at a 3-month interval for a period of 12 months for long-term storage condition as per ICH guideline. Pharmacokinetics evaluation
ð2Þ
Here WACM represents the amount of ACM loaded in the SLNs, WTotal represents the total ACM amount added during preparation of the SLNs and WACM-SLN represents the weight of the ACM-SLNs. Thermal analysis Differential scanning calorimetry (DSC) was performed on ACM, blank SLNs, physical mixture of ACM and blank SLNs as well as ACM-SLNs. For the structural, crystal, and physical state characterizations of the drug, DSC studies were conducted. The DSC measurements were carried out on a Shimadzu DSC (DSC 60, Shimadzu Corporation, Kyoto, Japan) with thermal analyzer. Accurately weighed samples (about 5 mg) were placed in a sealed aluminum pan before being heated under nitrogen flow (50 mL/min) at a scanning rate of 10 per min from 100 to 400 . An empty aluminum pan was used as a reference (Tadwee et al., 2011). Powder X-ray diffraction The crystallinities of ACM and ACM-SLNs were evaluated by XRD measurements recorded for ACM, blank SLNs, physical mixture of ACM, and blank SLNs and ACM-SLNs using X-ray diffractometer (BRUKER, Karlsruhe, Germany D8 Advance). Scanning was done up to 2 range between 2 and 90 using Ni-filtered (Mahajan & Gattani, 2010). In vitro drug release studies The in vitro release of ACM-SLNs was measured in phosphate buffered saline (PBS, pH 7.4) at a temperature of 37 ± 0.5 C by the dialysis method. ACM-SLNs samples (ACM 5 mg) were placed into and suspended in 100 mL release media and stirred at 50 rpm using the USP paddle method. At the predetermined time of 0.25, 0.5, 1, 2, 4, 6, 8, 10, 16, 24, and 48 h, samples (2 mL) were withdrawn with a syringe filter (0.45 mm pore size) from the release media and replaced by an equal volume of fresh media to maintain a constant volume. The test solution was analyzed by the HPLC method as follows. Triplicates were conducted and the results were averaged.
Twelve rats were used to investigate the effect of SLNs formulation on the pharmacokinetics of ACM after i.v. administration. Rats were divided into two groups at random and given a single dose of the ACM-SLNs or free ACM (both contain ACM 10 mg/kg) by vein injection. An appropriate amount of ACM-SLNs was diluted with water for administration. Blood samples (0.5 mL) were collected by heparinized tubes from the caudal vein at 2, 5, 15, 45 min, 1, 2, 4, 8, 12, and 24 h after i.v. administration. Blood was immediately processed to obtain plasma by centrifugation at 3000 g for 10 min. Plasma samples were frozen and maintained at 70 C for upcoming analysis. Pharmacokinetic parameters were calculated against the plasma concentration–time data. The elimination half-life (t1/2) was determined by linear regression of the terminal portion of the plasma concentration–time data. The areaunder-the-plasma concentration–time curve from zero to the last measurable plasma concentration point (AUC0t) was calculated by the linear trapezoidal method. Extrapolation to time infinity (AUC0 1) was calculated as follows: AUC0 1 ¼ AUC0 t + Ct/ke, where Ct is the last measurable plasma concentration and ke is the terminal elimination rate constant. The results were expressed as mean±standard deviation (SD). Biodistribution studies Forty-eight rats were used in the experiment to assess the effect of SLNs formulation on the biodistribution of ACM after i.v. administration. The rats were divided into two groups at random and given a single dose (10 mg/kg) of either the ACM-SLNs or free ACM by tail-vein injection. At the 1st, 6th, 12th, and 24th hour after drug injection, each animal (n ¼ 6 for each time point) was euthanized to collect its heart, liver, spleen, lung, and kidney. Tissue samples were washed in ice-cold saline, blotted with paper towel to remove excess fluid, weighed, and stored at 70 C until assessed for drug concentration by HPLC. HPLC analysis The amount of ACM in each sample was determined by HPLC (LC-10A, Shimadzu Co Ltd, Kyoto, Japan) equipped with a UV detector. Chromatographic separation was
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achieved using a Dikma Diamonsil TM C18 column (Dikma Co Ltd, Beijing, China, 5 mm, 200 mm 4.6 mm) and a precolumn (Nova-Pak, 10 mm, C18, Waters, Milford, MA). About 0.2% N ammonium acetate:acetonitrile:methanol ¼ 32:28:40 (V/V/V, pH 3.0, phosphoric acid) was used as the mobile phase at a flow rate of 1.0 mL/min. The column was maintained at an ambient temperature (35 C). The UV detector was operated at a wavelength of 430 nm. About 100 mL of the plasma sample was transferred to a 10 mL plastic test tube together with 10 mL of I.S. solution (1 mg/mL). After vortex shaking for 30 s (Eppendorf, 5432 vortex mixer, Eppendorf, Hamburg, Germany), 3 mL of ethyl acetate was added and the mixture was vortexed for another 2 min. After centrifugation at 3000 g for 10 min (Thermo IEC, Micromax, Thermo Electron Corporation, Milford, MA), the upper organic layer was quantitatively transferred to a 10-mL glass tube and evaporated to dryness using an evaporator at 40 C. The residue was reconstituted in 100 mL of methanol, and then vortex-mixed. After centrifugation at 2000 g for 5 min, 20 mL aliquot of the solution was injected into the HPLC system for analysis. Tissue samples were homogenized in a mixed solution of acetonitrile and water (50:50, v/v). About 10 mL of I.S. solution (1 mg/mL) was added to 200 mL of tissue samples and vortexed for 1 min. The drug and internal standard were then extracted into 3 mL of ethyl acetate by vortex mixing for 2 min. After centrifugation at 3000 g for 10 min, the clear supernatant was removed and evaporated under a gentle stream of nitrogen. The residue was then dissolved by 100 mL of methanol, and centrifuged at 2000 g for 5 min, 20 mL aliquot of the solution was injected into the HPLC system for analysis. Histology studies Twelve rats were used to investigate the histopathological change of ACM-SLNs after 7 d of continuous i.v. administration of the formulation (5 mg/kg). Animals (including a control group) were anesthetized and their livers, spleens, and kidneys were dissected and washed with cold saline. The organs were pressed between filter pads, weighed, and then fixed in 10% neutral formalin using standard techniques and stained with hematoxylin and eosin for histopathological examination. All tissue samples were examined and graded under light microscopy with 500 magnification (Nanji et al., 2002). Statistical analysis Values were expressed as mean ± standard deviation (SD) for each group. Statistical evaluation of the experimental data was performed using Student’s t-test. A p50.05 was considered statistically significant.
Results and discussion Preparation of ACM-SLNs As is shown in Figure 2, the surface morphology of ACMSLNs was observed by transmission electron microscope (TEM). The SLNs were spherical in shape with a smooth surface and the size was uniform and appropriate for
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Figure 2. Transmission ACM-SLNs. 3000.
electron
microscope
photograph
of
administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The poly disperse index (PDI) was 0.16 ± 0.05 and the zeta potential was 10.2 ± 0.67 mV. Particle size is an important factor in evaluating drugloaded nanoparticles in vivo. Generally speaking, a small nanoparticle has a small curvature radius and is weak at adsorption of protein within the systemic circulation. Therefore, reducing the particle size could prolong the retention time. When the particle size is less than 70 nm, the aggregation in the liver is obvious, which is trapped by the sinusoidal tubules in the liver and spleen (Service, 2003). Particle size is pivotal for preparation of ACM-SLNs. In this experiment, we used the CHOL, EPC, and pluronic F-68 to prepare SLNs. The obtained particle size is small at 70 nm with a good size distribution. Stirring speed was another factor affecting the particle size. If the speed was too low, the particle size tended to increase and its stability undermined; if the speed was too high, a large amount of foam would be produced, which directly affected the emulsifying effect of surfactant. The optimum stirring speed was proved to be 600 rpm. Thermal analysis Thermal analysis DSC was performed on ACM, blank SLNs, physical mixture of ACM and blank SLNs as well as ACMSLNs. For the structural, crystal, and physical state characterizations of the drug, DSC studies were employed. Figure 3 of DSC thermogram shows that ACM had a particular peak at about 151 C, which also appeared in the physical mixture of
DOI: 10.3109/10717544.2014.974001
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Figure 3. DSC analyses of the samples: (A) ACM, (B) blank SLNs, (C) physical mixture of ACM and blank SLNs, (D) ACM-SLNs.
Figure 4. X-ray diffraction spectra of (A) ACM, (B) physical mixture of ACM and blank SLNs, (C) blank SLNs, and (D) ACM-SLNs.
Figure 5. In vitro release profiles of ACM-SLNs from three batches. Release experiments were carried out in phosphate buffer solution (PBS) (pH 7.4), at 37 ± 0.5 C. Each point represents the mean value of three different experiments ± SD ^, free ACM; 4, ACM-SLNs; *p50.05 free ACM versus ACM-SLNs.
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blank SLNs and ACM, indicating that ACM existed as crystal in its natural state and did not have chemical interaction with the blank SLNs. Whereas the disappearance of the particular peak in ACM-SLNs revealed that ACM was successfully encapsulated in SLNs. Reduction of peak height and disappearance of peaks pointed toward a positive interaction. The decrease in enthalpy change represented a reduction in crystallinity, indicating a successful entrapment of respective drugs in SLNs which in turn ensured a decreased drug expulsion and a better controlled release from the SLNs with a minimal burst effect.
released versus the square root of time depicted the release following the Higuchi model are shown in Table 1. In vitro data showed that in comparison with ACM-SLNs, the free ACM released ACM much faster. ACM-SLNs demonstrated a well-sustained release efficacy. The sustained release of ACM by SLNs revealed its applicability as a drug delivery system which could minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in tumor sites.
Powder X-ray diffraction The X-ray diffraction spectra for pure drug, blank SLNs, physical mixture of ACM and blank SLNs as well as ACMSLNs are depicted in Figure 4. ACM had shown distinctly intense peaks between 2 of 6 and 22 , but in the case of blank SLNs and drug-loaded SLNs, the intensity of peaks decreased, indicating an amorphous nature of the drug after entrapment into SLNs by freeze-drying.
The drug content in the long-term storage conditions did not vary to a large extent in the SLN formulations; a maximum variation of 2.5% was seen in SLNs 12 months after the date of manufacture. The formulations were stable for 6 months under accelerated storage conditions at 25 ± 2 C and 60 ± 5% RH. A maximum decrease of 5.8% was observed. The average particle size did not vary appreciably and the physical– chemical characteristic changes were found to be negligible and had no impact on the quality of the formulations.
In vitro drug release behavior
Pharmacokinetic studies
In vitro drug release behavior of ACM-SLNs was studied using a dialysis method. The release profiles of free ACM and ACM-SLNs are shown in Figure 5. A very fast release behavior of free ACM was observed, whereas the cumulative release of ACM-SLNs was much slower followed by a sustained release. In the free ACM group, 95% of ACM were released in the first 2 h. In contrast, only 33% and 75% of ACM were released from SLNs in the first 2 h and 48 h (p50.05), respectively. The in vitro release was kinetically analyzed according to the zero-order, first-order, Higuchi, and Weibull model. The relatively high correlation coefficient values obtained from the analysis of the amount of the drug
The analytical peaks of ACM and internal standard were resolved with good symmetry and no endogenous sources of interference were observed at the retention time of the
Table 1. Correlation coefficients for kinetic analysis of release data for ACM-SLNs. Correlation coefficient (r) Formulation
Zero order
First order
Higuchi
Weibull
ACM-SLNs
0.9612
0.9733
0.9993
0.9826
Figure 6. Mean plasma concentration–time profiles of ACM after i.v. administration of a single 10 mg/kg dose of free ACM and ACMSLNs to rats. The values are expressed as mean ± SD (n ¼ 6).
Stability
Table 2. Pharmacokinetic parameters of the two formulations. Formulations Parameter t1/2a (h) t1/2b (h) Cmax (ng/mL) AUC0 t (ngh/mL) AUC0 1 (ngh/mL) MRT (h) CL (L/h)
Injection
SLNs
0.18 ± 0.3 2.6 ± 1.1 3950.5 ± 334.3 3015.6 ± 318.1 4231.7 ± 353.6 11.2 ± 3.1 27.7 ± 4.7
0.94 ± 0.5* 12.9 ± 2.8* 2019.3 ± 232.5* 15 376.2 ± 1021.4* 20 542.6 ± 1207.9* 23.9 ± 4.3* 14.8 ± 3.1*
Cmax, maximum plasma concentration; AUC0 t, area under the concentration–time curve from time 0 to the last measurable concentration; AUC0 1, area under the concentration–time curve extrapolated to infinity; MRT, mean residence time; CL, plasma clearance. *p50.05: ACM-SLNs versus ACM injection.
DOI: 10.3109/10717544.2014.974001
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analyte. The LLOQ was 0.01 mg/mL (S/N ¼ 5). Good linearity was observed over the concentration range of 0.01–10 mg/mL plasma (r ¼ 0.9995). The intra-/inter-day precision and accuracy were determined. The RSD of ACM ranged from 4.6% to 6.4% for intra-day and 3.4% to 7.5% for inter-day. The relative error of ACM ranged from 1.1% to 2.2% and 1.3% to 3.1% for intra-day and inter-day, respectively. Freshly prepared solutions showed no evidence of degradation for either ACM or the internal standard. No significant degradation was observed for any analytes during the sample processing and
Figure 7. Distribution in tissue in rats after following i.v. administration of a single 10 mg/kg dose of ACM injection tissues (ng/g) (each point represents the mean ± SD of six rats). Figure 8. Distribution in tissue in rats after following i.v. administration of a single 10 mg/kg dose of ACM-SLNs tissues (ng/g) (each point represents the mean ± SD of six rats).
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extraction, including the dry-down procedure. The stability of the sample solution in the auto sampler at 4 C was also assessed. ACM in the sample solution was found to be stable for approximately 24 h according to the fact that the obtained concentrations were between 97% and 102% of the initial. Plasma samples collected from studies of ACM were evaluated before and after storage at 18 C for stability and found to be stable for at least 3 months. The mean absolute recoveries for ACM were 89.3%, 90.6%, and 91.4% at the high, middle, and low concentrations respectively (n ¼ 3). Pharmacokinetic studies were carried out in rats using the SLNs prepared from CHOL and EPC. The time course of the plasma concentrations of ACM and SLNs is summarized in Figure 6. The pharmacokinetic parameters calculated from the plasma drug concentration versus time profiles are listed in Table 2. The 3P97 software (Tedia Company Inc., Fairfield, OH) was used to fit the compartment model of the average plasma drug concentration–time curve. As regards the pharmacokinetic parameters, the AUC of ACM-SLNs and of free ACM was 20 542.6 (ngh/mL) and 4231.7 (ngh/mL), respectively, indicating a great improvement in the bioavailability of SLN preparation compared with the control group. The half-life was 12.9 h for the SLNs group but 2.6 h for the control group, which may be because the drug was wrapped into the solid lipid matrix to avoid the metabolism action of CYP enzymes, or that ACM was released slowly from the SLNs and stayed in the systemic circulation for a long time (Hancock & Zografi, 1997). Moreover, the prepared ACMSLNs were administered by intravenous injection to avoid adverse reactions happening after oral administration in the gastrointestinal tract (Tsukagoshi et al., 1980). There were significant differences in t1/2a, t1/2b, Cmax, AUC0 t, AUC0 1, MRT, and CL between the ACM-SLNs and free ACM (p50.05).
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Biodistribution studies The in vivo biodistribution behavior of ACM after i.v. administration of the ACM-SLNs was investigated with free ACM as a control. The amounts of drug distributed in unit mass of heart, liver, spleen, lung, and kidney at various times were measured. Figures 7 and 8 present the mean concentration–time profiles of ACM in unit mass
of each organ in rats. The total amount of drug accumulated in each organ within 24 h (AUC0 t) was calculated, and the results are shown in Table 3. The ACM AUC0 t of the SLNs was lower in heart, lung, kidney, spleen, and higher in liver compared with the free ACM. There were significant differences in all organs between the ACM-SLNs and free ACM (p50.05).
Table 3. The AUC0 24 h of ACM in tissues after i.v. administration of injection and SLNs to rats.
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Formulation ACM injection (ngh/g) ACM-SLNs (ngh/g) Ratioa
Heart
Liver
Spleen
Lung
Kidney
2612.3 ± 221.4 317.1 ± 29.6 0.12*
1920.5 ± 132.5 4002.1 ± 378.7 2.08*
2522.5 ± 232.7 905.2 ± 101.2 0.36*
2658.2 ± 224.3 632.5 ± 73.8 0.24*
3987.5 ± 365.9 373.9 ± 43.8 0.09*
a
The ratio was AUC (ACM-SLNs) /AUC (ACM injection). *p50.05: ACM-SLNs versus ACM injection.
Figure 9. Histopathological studies of liver, spleen, and kidney. (A) Free ACM and (B) ACM-SLNs.
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DOI: 10.3109/10717544.2014.974001
According to Tables 2 and 3, one can see that in comparison with free ACM, ACM-SLNs altered the distribution of ACM in vivo and the half-life periods after i.v. injection of ACM-SLNs (t1/2a ¼ 0.94 h, t1/2b ¼ 12.9 h) were prolonged remarkably than those (t1/2a ¼ 0.18 h, t1/2b ¼ 2.6 h) after i.v. injection of free ACM. This indicated that the ACM-SLNs had sustained release efficacy. The results showed that the SLNs could deliver ACM mainly to liver after i.v. injection and the concentration of ACM in liver was significantly higher than in other tissues. Compared with free ACM, the drug concentration of ACM in liver after i.v. injection of ACM-SLNs increased from 67.5 to 453.2 ng/g (1 h). ACM-SLNs might be preferentially taken up by Hepatocytes because the RES mainly existed in the liver, spleen, bone marrow, and some other organs. The SLNs were thus easily to be detected and swallowed by RES. Therefore, the drug made to be the nanoparticle carrier was more easily targeted to the above tissues. In addition, the tissue distribution behavior of intravenously injected nanoparticles was greatly influenced by their size. When the nanoparticles with a diameter of 50–100 nm enter the circulatory system, they are not released into the systemic circulation directly. Instead, they are absorbed by the RES, leading to greatest drug accumulation in the liver and thus achieving the targeting effect (He et al., 2007; Lu et al., 2008; Hu et al., 2013). Histology studies The histopathological examination of liver, spleen, and kidney was carried out to identify any damage done to the tissue. The microphotographs of liver, spleen, and kidney were taken following their incubation with SLNs formulations for more than 7 d (Figure 9). Free ACM was the control. No sign of damage such as the appearance of epithelial necrosis and sloughing of epithelial cells was detected.
Conclusion A single-factor experiment was performed for the exploration of ACM-SLNs. The SLNs were spherical in shape, uniform in size, and appropriate for administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The poly disperse index (PDI) was 0.16 ± 0.05 and the zeta potential was 10.2 ± 0.67 mV. The SLNs were produced with stable physical properties and demonstrated significantly sustained release. The pharmacokinetic behavior of ACM was greatly improved by lyophilized injection of SLN with sustained drug release and high bioavailability. In addition, the results obtained from tissue distribution showed that ACM-SLNs were hepatic targeting in vivo.
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Declaration of interest The authors declare that they have no conflicts of interest.
References Chen H, Chang X, Du D, et al. (2006). Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting. J Control Release 110: 296–306. Danhier F1, Feron O, Pre´at V. (2010). To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anticancer drug delivery. J Control Release 148:135–46. Gao H, Wang JY, Shen XZ, et al. (2004). Preparation of magnetic polybutylcyanoacrylate nanospheres encapsulated with aclacinomycin A and its effect on gastric tumor. World J Gastroenterol 10:2010–13. Griaznova NS, Petiushenko RM, Belianskaia IV, et al. (1992). Incorporation of an antineoplastic drug aclarubicin into liposomes in relation to the conditions of its encapsulation. Antibiot Khimioter 37: 22–5. Hancock BC, Zografi G. (1997). Characteristics and significance of the amorphous state in pharmaceutical systems. J Pharm Sci 86:1–12. He J, Hou S, Lu W, et al. (2007). Preparation, pharmacokinetics and body distribution of silymarin-loaded solid lipid nanoparticles after oral administration. J Biomed Nanotechnol 3:195–203. Hirsja¨rvi S1, Passirani C, Benoit JP. (2011). Passive and active tumour targeting with nanocarriers. Curr Drug Discov Technol 8:188–96. Hu H, Liu D, Zhao X, et al. (2013). Preparation, characterization, cellular uptake and evaluation in vivo of solid lipid nanoparticles loaded with cucurbitacin B. Drug Dev Ind Pharm 39:770–9. Li YC, Dong L, Jia A, et al. (2006). Preparation of solid lipid nanoparticles loaded with traditional Chinese medicine by highpressure homogenization. South Med Univ 26:541–4. Liu J, Hu W, Chen H, et al. (2007). Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery. Int J Pharm 328: 191–5. Lu W, He LC, Wang CH, et al. (2008). The use of solid lipid nanoparticles to target a lipophilic molecule to the liver after intravenous administration to mice. Int J Biol Macromol 43:320–4. Mahajan HS, Gattani SG. (2010). Nasal administration of ondansetron using a novel microspheres delivery system Part II: ex vivo and in vivo. Pharm Dev Technol 15:653–7. Mu¨ller RH1, Ma¨der K, Gohla S. (2000). Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. Eur J Pharm Biopharm 50:161–77. Nanji AA, Su GL, Laposata M, et al. (2002). Pathogenesis of alcoholic liver disease – recent advances. Alcohol Clin Exp Res 26:731–6. Nitiss JL, Pourquier P, Pommier Y. (1997). Aclacinomycin A stabilizes topoisomerase I covalent complexes. Cancer Res 57:4564–9. Service RF. (2003). Nanomaterials show signs of toxicity. Science 300: 243. Shiokawa T1, Hattori Y, Kawano K, et al. (2005). Effect of polyethylene glycol linker chain length of folate-linked microemulsions loading aclacinomycin A on targeting ability and antitumor effect in vitro and in vivo. Clin Cancer Res 11:2018–25. Tadwee I, Shahi SR, Thube MW. (2011). Spray dried nasal mucoadhesive microspheres of carbamazepine: preparation and in vitro/ex vivo evaluation. Int J Sci Publ Res Pharm 2:23–32. Torchilin VP. (2010). Passive and active drug targeting: drug delivery to tumors as an example. Handb Exp Pharmacol 197:3–53. Tsukagoshi S, Tsuruo T, Yamori T, et al. (1980). Antitumor efficacies of aclacinomycin A by oral administration. J Pharmacobiodyn 10:532–6. Wei G, Ni W, Chiao JW, et al. (2011). A meta-analysis of CAG (cytarabine, aclarubicin, G-CSF) regimen for the treatment of 1029 patients with acute myeloid leukemia and myelodysplastic syndrome. J Hematol Oncol 4:46–58. Wissing SA, Kayser O, Mu¨ller RH. (2004). Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev 56:1257–72.
RESEARCH ARTICLE
Formulation, characterization, and in vitro/vivo studies of aclacinomycin A-loaded solid lipid nanoparticles Youpeng Jia1, Jun Ji2, Feng Wang3, Liangang Shi1, Jingbo Yu1, and Dong Wang1
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1
Department of General Surgery, Dalian Municipal Center Hospital, Liaoning, China, 2Department of Central Laboratory, Dalian Municipal Center Hospital, Liaoning, China, and 3Department of Pharmaceutics, Shanghai Institute of Pharmaceutical Industry, Shanghai, China
Abstract
Keywords
Objective: The aim of this study was to prepare aclacinomycin A (ACM)-loaded solid lipid nanoparticles (SLNs) and to evaluate their in vitro and in vivo characteristics. Methods: SLNs were prepared using an emulsion evaporation–solidification method, and characterized in accordance with the morphological examination, particle size distribution, entrapment efficiency, drug-loading, and in vitro release. Pharmacokinetic and biodistribution studies were employed to evaluate the in vivo of SLNs. Results: The SLNs were spherical in shape, uniform in size, and appropriate for administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The SLNs were produced with stable physical properties and demonstrated significantly sustained release. The pharmacokinetic behavior of ACM was greatly improved by lyophilized injection of SLN with sustained drug release and high bioavailability. In addition, the results obtained from tissue distribution showed that ACM-SLNs were hepatic targeting in vivo. Conclusions: The present work demonstrated the feasibility of liver-targeted delivery of ACM utilizing SLNs.
Aclacinomycin A, biodistribution, in vitro release, pharmacokinetics, solid lipid nanoparticles
Introduction Aclacinomycin A (ACM) hydrochloride is an anthracycline antitumor antibiotic isolated from Streptomyces galilaeus MA-144-M1. ACM inhibits the synthesis of nucleic acid, especially RNA, by acting as an inhibitor of not only topoisomerase II but also topoisomerase I (Nitiss et al., 1997; Li et al., 2006; Chen et al., 2006; Liu et al., 2007; Griaznova et al., 1992; Shiokawa et al., 2005; Wei et al., 2011). This drug is widely used for the treatment of liver, stomach, lung, and ovarian carcinoma; malignant lymphoma and acute leukemia, but its clinical use is still limited by its toxicity. To reduce the toxic responses and increase the antitumor effect of ACM, it should be effectively targeted to the tumor. Targeting agents are usually divided into two categories: active and passive (Torchilin, 2010; Hirsja¨rvi et al., 2011; Danhier et al., 2010). Active targeting preparation uses modified pharmaceutical carriers which act like missiles to deliver specifically targeting drugs and then play a pharmacodynamic concentration. After surface modification, if the
Address for correspondence: Youpeng Jia, Department of General Surgery, Dalian Municipal Center Hospital, Dalian, China. E-mail: [email protected]
History Received 12 August 2014 Revised 3 October 2014 Accepted 3 October 2014
drug-loaded particles are not recognized by macrophages, or they can connect with receptors and target cells bound up with a specific ligand, or they develop immunity due to attachment with monoclonal antibodies, then the modification can avoid macrophage intake, prevent the concentration in the liver, and change the distribution of particles in the body naturally to reach specific targets. The drug can also be modified into prodrug, which can be activated as pharmacologically inert substance and be effective in certain target areas. If the particles, through active targeting, intend to reach the target site without being intercepted (diameter 4–7 mm), usually the particle size should be no larger than 4 mm. Passive targeting happens when drug-loaded particles are absorbed by macrophages of the monocyte–macrophage system (especially the kupffer cells of liver), and then delivered to liver, spleen and other organs by normal physiological process but difficult to reach other target areas. After intravenous injection, the distribution of passive targeting particles within body is primarily decided by their size. When the particle diameter is between 50 and 200 nm, the majority enters the circulatory system. But instead of being released into the systemic circulation directly, they are absorbed by the reticulo endothelial system (Res), leading to greatest drug accumulation in the liver and thus achieving the targeting effect. In addition to particle size, the nature of the surfaces of the particles also plays an
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important role in the distribution. Currently, many reports have touched the ACM targeting agents, for instance, liposomes (Griaznova et al., 1992), microemulsion (Shiokawa et al., 2005), nanoparticles (Gao et al., 2004) and others. Solid lipid nanoparticles (SLNs) are one of the new nanoparticle drug-delivery systems (Mu¨ller et al., 2000; Wissing et al., 2004). SLNs are a solid colloidal drugdelivery system with particle size ranging from 50 to 1000 nm, in which the drugs are wrapped into the lipid core with solid natural or synthetic lipids. As a nanoparticle drug-delivery system is newer than emulsions, Liposomes, microparticles, SLNs have been used to control drug release (Li et al., 2006). It has many advantages, for example, good physiological compatibility, well-controlled drug release, good targeting (Chen et al., 2006; Liu et al., 2007), high physical stability similar to polymer nanoparticles, low toxicity, and large-scale production comparable with liposomes and emulsions. The many administration routes for SLNs include intravenous administration to achieve a targeted effect or controlled release, oral administration to control the drug release in the gastrointestinal tract, and local administration. In this study, SLNs were prepared using emulsion evaporation–solidification at a low temperature, and SLNs lyophilized powder were produced with a refrigerated air dryer. Some preliminary studies were done on the quality of the SLNs morphology, its physical and chemical properties, and effect of in vitro release. Moreover, pharmacokinetics and biodistribution studies of ACM solid lipid nanoparticles (ACM-SLNs) were investigated to provide a reference for clinical application.
Materials and methods Materials ACM (Figure 1A, purity 495%) was purchased from Jiangsu Yangzhou Pharma Ltd, China. Egg-phosphatidyl choline (EPC) and high purity cholesterol (CHOL) were donated by Phospholipid Tech Ltd, Dalian, China. Quercetin (Figure 1B, purity 499.3%) was given by the National Institute for Food and Drug control (Beijing, China). Pluronic F-68 was purchased from Beijing Bayerd Biopharm Ltd, Beijing, China. All the chemicals and solvents used in the present study were of analytic grade. Deionized water was used throughout the experiment. The experiments were performed on rats weighing between 220 ± 20 g. The animals were kept in cages in a room at a temperature of 25 ± 2 C, with a 12:12 light-dark cycle. Food and water were accessible. All experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the China National Institutes of Health. Preparation of ACM-SLNs ACM (50 mg), CHOL (400 mg), and EPC (750 mg) were weighed accurately and dissolved in chloroform (10 mL), forming an organic phase at 45 C ± 1 C water bath. The aqueous phase consisted of pluronic F-68 solution (5%, 40 mL) at 45 C ± 1 C water bath. The organic phase was
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Figure 1. The structure of ACM (A) and Quercetin (B, internal standard).
slowly added to the water phase under mechanical stirring (600 rpm) at 45 C ± 1 C. After 4 h, the volume was condensed by approximately half to obtain a semitransparent solution. The solution was quickly dispersed in the water (75 mL, temperatures ranging from 0 to 2 C) by stirring for another 2 h. Thus, a solid lipid nanoparticle suspension was obtained. Two substances were chosen as the cryoprotectant: 5% (w/v) glucose and 5% (w/v) mannitol. About 1.0 mL cryoprotectant and 1.0 mL suspension of ACM-SLNs at a ratio of 1:1(v/v) were added to the vial (10 mL). The vial was pre-frozen for 8 h at 60 C and, after the solution was added, it was kept in the refrigerated air dryer for another 24 h to obtain the ACM-SLNs lyophilized powder. Morphological characterization and particle sizing The morphological examination of the ACM-SLNs was performed using a transmission electron microscope (TEM) (Philips CM120, Philips, Amsterdam, The Netherlands)). In practice, 10 mg of ACM-SLNs lyophilized powder containing 0.5% (w/v) phosphotungstic acids were placed on a carbon film coated on a copper grid and observed at 80 kV in the electron microscope. Particle size distribution and mean diameter of the prepared ACM-SLN were determined by dynamic light scattering (DLS) using a NICOMP 380 Submicron Particle Sizer (PSS, Santa Barbara, CA) equipped with a 5 mW heliumneon laser at 632.8 nm. Sample solutions were transferred into the light scattering cells. The intensity autocorrelation was measured at a scattering angle of 90 at room temperature. Data were analyzed in terms of intensityweighted NICOMP distributions. Each reported experimental result is the average of at least three dh values obtained from analysis of the autocorrelation function accumulated for at
ACM A-loaded SNPs
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least 20 min. Zeta potential was measured on the same samples prepared for size analysis. Drug content, entrapment efficiency, and drug loading The drug content analysis was conducted on various batches of the SLNs. Briefly, the ACM-SLNs lyophilized powders were dissolved in an adequate quantity of phosphate buffer pH 6.8 and then filtered. The drug content of ACM was detected by high-performance liquid chromatography (HPLC) (described as follows). The drug entrapment efficiency and drug loading were calculated by the following equations:
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DL% ¼
WACM WACMSLNs
EE% ¼
100%
WACM 100% WTotal
ð1Þ
3
Stability The stability of the protocol was designed based on the ICH Guidelines. The ACM-SLNs were stored at a temperature of 25 ± 2 C and 60 ± 5% relative humidity (RH) for a period of 6 months and at 5 ± 3 C for 12 months. Both represent the accelerated storage temperature and long-term storage temperature respectively for products which are intended to be refrigerated. The stored samples were tested for their drug content, particle size distribution, and for any physical change. The drug content was determined by the HPLC method. The testing was carried out at 0, 2, 4, and 6 months for accelerated storage condition and at a 3-month interval for a period of 12 months for long-term storage condition as per ICH guideline. Pharmacokinetics evaluation
ð2Þ
Here WACM represents the amount of ACM loaded in the SLNs, WTotal represents the total ACM amount added during preparation of the SLNs and WACM-SLN represents the weight of the ACM-SLNs. Thermal analysis Differential scanning calorimetry (DSC) was performed on ACM, blank SLNs, physical mixture of ACM and blank SLNs as well as ACM-SLNs. For the structural, crystal, and physical state characterizations of the drug, DSC studies were conducted. The DSC measurements were carried out on a Shimadzu DSC (DSC 60, Shimadzu Corporation, Kyoto, Japan) with thermal analyzer. Accurately weighed samples (about 5 mg) were placed in a sealed aluminum pan before being heated under nitrogen flow (50 mL/min) at a scanning rate of 10 per min from 100 to 400 . An empty aluminum pan was used as a reference (Tadwee et al., 2011). Powder X-ray diffraction The crystallinities of ACM and ACM-SLNs were evaluated by XRD measurements recorded for ACM, blank SLNs, physical mixture of ACM, and blank SLNs and ACM-SLNs using X-ray diffractometer (BRUKER, Karlsruhe, Germany D8 Advance). Scanning was done up to 2 range between 2 and 90 using Ni-filtered (Mahajan & Gattani, 2010). In vitro drug release studies The in vitro release of ACM-SLNs was measured in phosphate buffered saline (PBS, pH 7.4) at a temperature of 37 ± 0.5 C by the dialysis method. ACM-SLNs samples (ACM 5 mg) were placed into and suspended in 100 mL release media and stirred at 50 rpm using the USP paddle method. At the predetermined time of 0.25, 0.5, 1, 2, 4, 6, 8, 10, 16, 24, and 48 h, samples (2 mL) were withdrawn with a syringe filter (0.45 mm pore size) from the release media and replaced by an equal volume of fresh media to maintain a constant volume. The test solution was analyzed by the HPLC method as follows. Triplicates were conducted and the results were averaged.
Twelve rats were used to investigate the effect of SLNs formulation on the pharmacokinetics of ACM after i.v. administration. Rats were divided into two groups at random and given a single dose of the ACM-SLNs or free ACM (both contain ACM 10 mg/kg) by vein injection. An appropriate amount of ACM-SLNs was diluted with water for administration. Blood samples (0.5 mL) were collected by heparinized tubes from the caudal vein at 2, 5, 15, 45 min, 1, 2, 4, 8, 12, and 24 h after i.v. administration. Blood was immediately processed to obtain plasma by centrifugation at 3000 g for 10 min. Plasma samples were frozen and maintained at 70 C for upcoming analysis. Pharmacokinetic parameters were calculated against the plasma concentration–time data. The elimination half-life (t1/2) was determined by linear regression of the terminal portion of the plasma concentration–time data. The areaunder-the-plasma concentration–time curve from zero to the last measurable plasma concentration point (AUC0t) was calculated by the linear trapezoidal method. Extrapolation to time infinity (AUC0 1) was calculated as follows: AUC0 1 ¼ AUC0 t + Ct/ke, where Ct is the last measurable plasma concentration and ke is the terminal elimination rate constant. The results were expressed as mean±standard deviation (SD). Biodistribution studies Forty-eight rats were used in the experiment to assess the effect of SLNs formulation on the biodistribution of ACM after i.v. administration. The rats were divided into two groups at random and given a single dose (10 mg/kg) of either the ACM-SLNs or free ACM by tail-vein injection. At the 1st, 6th, 12th, and 24th hour after drug injection, each animal (n ¼ 6 for each time point) was euthanized to collect its heart, liver, spleen, lung, and kidney. Tissue samples were washed in ice-cold saline, blotted with paper towel to remove excess fluid, weighed, and stored at 70 C until assessed for drug concentration by HPLC. HPLC analysis The amount of ACM in each sample was determined by HPLC (LC-10A, Shimadzu Co Ltd, Kyoto, Japan) equipped with a UV detector. Chromatographic separation was
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achieved using a Dikma Diamonsil TM C18 column (Dikma Co Ltd, Beijing, China, 5 mm, 200 mm 4.6 mm) and a precolumn (Nova-Pak, 10 mm, C18, Waters, Milford, MA). About 0.2% N ammonium acetate:acetonitrile:methanol ¼ 32:28:40 (V/V/V, pH 3.0, phosphoric acid) was used as the mobile phase at a flow rate of 1.0 mL/min. The column was maintained at an ambient temperature (35 C). The UV detector was operated at a wavelength of 430 nm. About 100 mL of the plasma sample was transferred to a 10 mL plastic test tube together with 10 mL of I.S. solution (1 mg/mL). After vortex shaking for 30 s (Eppendorf, 5432 vortex mixer, Eppendorf, Hamburg, Germany), 3 mL of ethyl acetate was added and the mixture was vortexed for another 2 min. After centrifugation at 3000 g for 10 min (Thermo IEC, Micromax, Thermo Electron Corporation, Milford, MA), the upper organic layer was quantitatively transferred to a 10-mL glass tube and evaporated to dryness using an evaporator at 40 C. The residue was reconstituted in 100 mL of methanol, and then vortex-mixed. After centrifugation at 2000 g for 5 min, 20 mL aliquot of the solution was injected into the HPLC system for analysis. Tissue samples were homogenized in a mixed solution of acetonitrile and water (50:50, v/v). About 10 mL of I.S. solution (1 mg/mL) was added to 200 mL of tissue samples and vortexed for 1 min. The drug and internal standard were then extracted into 3 mL of ethyl acetate by vortex mixing for 2 min. After centrifugation at 3000 g for 10 min, the clear supernatant was removed and evaporated under a gentle stream of nitrogen. The residue was then dissolved by 100 mL of methanol, and centrifuged at 2000 g for 5 min, 20 mL aliquot of the solution was injected into the HPLC system for analysis. Histology studies Twelve rats were used to investigate the histopathological change of ACM-SLNs after 7 d of continuous i.v. administration of the formulation (5 mg/kg). Animals (including a control group) were anesthetized and their livers, spleens, and kidneys were dissected and washed with cold saline. The organs were pressed between filter pads, weighed, and then fixed in 10% neutral formalin using standard techniques and stained with hematoxylin and eosin for histopathological examination. All tissue samples were examined and graded under light microscopy with 500 magnification (Nanji et al., 2002). Statistical analysis Values were expressed as mean ± standard deviation (SD) for each group. Statistical evaluation of the experimental data was performed using Student’s t-test. A p50.05 was considered statistically significant.
Results and discussion Preparation of ACM-SLNs As is shown in Figure 2, the surface morphology of ACMSLNs was observed by transmission electron microscope (TEM). The SLNs were spherical in shape with a smooth surface and the size was uniform and appropriate for
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Figure 2. Transmission ACM-SLNs. 3000.
electron
microscope
photograph
of
administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The poly disperse index (PDI) was 0.16 ± 0.05 and the zeta potential was 10.2 ± 0.67 mV. Particle size is an important factor in evaluating drugloaded nanoparticles in vivo. Generally speaking, a small nanoparticle has a small curvature radius and is weak at adsorption of protein within the systemic circulation. Therefore, reducing the particle size could prolong the retention time. When the particle size is less than 70 nm, the aggregation in the liver is obvious, which is trapped by the sinusoidal tubules in the liver and spleen (Service, 2003). Particle size is pivotal for preparation of ACM-SLNs. In this experiment, we used the CHOL, EPC, and pluronic F-68 to prepare SLNs. The obtained particle size is small at 70 nm with a good size distribution. Stirring speed was another factor affecting the particle size. If the speed was too low, the particle size tended to increase and its stability undermined; if the speed was too high, a large amount of foam would be produced, which directly affected the emulsifying effect of surfactant. The optimum stirring speed was proved to be 600 rpm. Thermal analysis Thermal analysis DSC was performed on ACM, blank SLNs, physical mixture of ACM and blank SLNs as well as ACMSLNs. For the structural, crystal, and physical state characterizations of the drug, DSC studies were employed. Figure 3 of DSC thermogram shows that ACM had a particular peak at about 151 C, which also appeared in the physical mixture of
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Figure 3. DSC analyses of the samples: (A) ACM, (B) blank SLNs, (C) physical mixture of ACM and blank SLNs, (D) ACM-SLNs.
Figure 4. X-ray diffraction spectra of (A) ACM, (B) physical mixture of ACM and blank SLNs, (C) blank SLNs, and (D) ACM-SLNs.
Figure 5. In vitro release profiles of ACM-SLNs from three batches. Release experiments were carried out in phosphate buffer solution (PBS) (pH 7.4), at 37 ± 0.5 C. Each point represents the mean value of three different experiments ± SD ^, free ACM; 4, ACM-SLNs; *p50.05 free ACM versus ACM-SLNs.
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blank SLNs and ACM, indicating that ACM existed as crystal in its natural state and did not have chemical interaction with the blank SLNs. Whereas the disappearance of the particular peak in ACM-SLNs revealed that ACM was successfully encapsulated in SLNs. Reduction of peak height and disappearance of peaks pointed toward a positive interaction. The decrease in enthalpy change represented a reduction in crystallinity, indicating a successful entrapment of respective drugs in SLNs which in turn ensured a decreased drug expulsion and a better controlled release from the SLNs with a minimal burst effect.
released versus the square root of time depicted the release following the Higuchi model are shown in Table 1. In vitro data showed that in comparison with ACM-SLNs, the free ACM released ACM much faster. ACM-SLNs demonstrated a well-sustained release efficacy. The sustained release of ACM by SLNs revealed its applicability as a drug delivery system which could minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in tumor sites.
Powder X-ray diffraction The X-ray diffraction spectra for pure drug, blank SLNs, physical mixture of ACM and blank SLNs as well as ACMSLNs are depicted in Figure 4. ACM had shown distinctly intense peaks between 2 of 6 and 22 , but in the case of blank SLNs and drug-loaded SLNs, the intensity of peaks decreased, indicating an amorphous nature of the drug after entrapment into SLNs by freeze-drying.
The drug content in the long-term storage conditions did not vary to a large extent in the SLN formulations; a maximum variation of 2.5% was seen in SLNs 12 months after the date of manufacture. The formulations were stable for 6 months under accelerated storage conditions at 25 ± 2 C and 60 ± 5% RH. A maximum decrease of 5.8% was observed. The average particle size did not vary appreciably and the physical– chemical characteristic changes were found to be negligible and had no impact on the quality of the formulations.
In vitro drug release behavior
Pharmacokinetic studies
In vitro drug release behavior of ACM-SLNs was studied using a dialysis method. The release profiles of free ACM and ACM-SLNs are shown in Figure 5. A very fast release behavior of free ACM was observed, whereas the cumulative release of ACM-SLNs was much slower followed by a sustained release. In the free ACM group, 95% of ACM were released in the first 2 h. In contrast, only 33% and 75% of ACM were released from SLNs in the first 2 h and 48 h (p50.05), respectively. The in vitro release was kinetically analyzed according to the zero-order, first-order, Higuchi, and Weibull model. The relatively high correlation coefficient values obtained from the analysis of the amount of the drug
The analytical peaks of ACM and internal standard were resolved with good symmetry and no endogenous sources of interference were observed at the retention time of the
Table 1. Correlation coefficients for kinetic analysis of release data for ACM-SLNs. Correlation coefficient (r) Formulation
Zero order
First order
Higuchi
Weibull
ACM-SLNs
0.9612
0.9733
0.9993
0.9826
Figure 6. Mean plasma concentration–time profiles of ACM after i.v. administration of a single 10 mg/kg dose of free ACM and ACMSLNs to rats. The values are expressed as mean ± SD (n ¼ 6).
Stability
Table 2. Pharmacokinetic parameters of the two formulations. Formulations Parameter t1/2a (h) t1/2b (h) Cmax (ng/mL) AUC0 t (ngh/mL) AUC0 1 (ngh/mL) MRT (h) CL (L/h)
Injection
SLNs
0.18 ± 0.3 2.6 ± 1.1 3950.5 ± 334.3 3015.6 ± 318.1 4231.7 ± 353.6 11.2 ± 3.1 27.7 ± 4.7
0.94 ± 0.5* 12.9 ± 2.8* 2019.3 ± 232.5* 15 376.2 ± 1021.4* 20 542.6 ± 1207.9* 23.9 ± 4.3* 14.8 ± 3.1*
Cmax, maximum plasma concentration; AUC0 t, area under the concentration–time curve from time 0 to the last measurable concentration; AUC0 1, area under the concentration–time curve extrapolated to infinity; MRT, mean residence time; CL, plasma clearance. *p50.05: ACM-SLNs versus ACM injection.
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analyte. The LLOQ was 0.01 mg/mL (S/N ¼ 5). Good linearity was observed over the concentration range of 0.01–10 mg/mL plasma (r ¼ 0.9995). The intra-/inter-day precision and accuracy were determined. The RSD of ACM ranged from 4.6% to 6.4% for intra-day and 3.4% to 7.5% for inter-day. The relative error of ACM ranged from 1.1% to 2.2% and 1.3% to 3.1% for intra-day and inter-day, respectively. Freshly prepared solutions showed no evidence of degradation for either ACM or the internal standard. No significant degradation was observed for any analytes during the sample processing and
Figure 7. Distribution in tissue in rats after following i.v. administration of a single 10 mg/kg dose of ACM injection tissues (ng/g) (each point represents the mean ± SD of six rats). Figure 8. Distribution in tissue in rats after following i.v. administration of a single 10 mg/kg dose of ACM-SLNs tissues (ng/g) (each point represents the mean ± SD of six rats).
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extraction, including the dry-down procedure. The stability of the sample solution in the auto sampler at 4 C was also assessed. ACM in the sample solution was found to be stable for approximately 24 h according to the fact that the obtained concentrations were between 97% and 102% of the initial. Plasma samples collected from studies of ACM were evaluated before and after storage at 18 C for stability and found to be stable for at least 3 months. The mean absolute recoveries for ACM were 89.3%, 90.6%, and 91.4% at the high, middle, and low concentrations respectively (n ¼ 3). Pharmacokinetic studies were carried out in rats using the SLNs prepared from CHOL and EPC. The time course of the plasma concentrations of ACM and SLNs is summarized in Figure 6. The pharmacokinetic parameters calculated from the plasma drug concentration versus time profiles are listed in Table 2. The 3P97 software (Tedia Company Inc., Fairfield, OH) was used to fit the compartment model of the average plasma drug concentration–time curve. As regards the pharmacokinetic parameters, the AUC of ACM-SLNs and of free ACM was 20 542.6 (ngh/mL) and 4231.7 (ngh/mL), respectively, indicating a great improvement in the bioavailability of SLN preparation compared with the control group. The half-life was 12.9 h for the SLNs group but 2.6 h for the control group, which may be because the drug was wrapped into the solid lipid matrix to avoid the metabolism action of CYP enzymes, or that ACM was released slowly from the SLNs and stayed in the systemic circulation for a long time (Hancock & Zografi, 1997). Moreover, the prepared ACMSLNs were administered by intravenous injection to avoid adverse reactions happening after oral administration in the gastrointestinal tract (Tsukagoshi et al., 1980). There were significant differences in t1/2a, t1/2b, Cmax, AUC0 t, AUC0 1, MRT, and CL between the ACM-SLNs and free ACM (p50.05).
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Biodistribution studies The in vivo biodistribution behavior of ACM after i.v. administration of the ACM-SLNs was investigated with free ACM as a control. The amounts of drug distributed in unit mass of heart, liver, spleen, lung, and kidney at various times were measured. Figures 7 and 8 present the mean concentration–time profiles of ACM in unit mass
of each organ in rats. The total amount of drug accumulated in each organ within 24 h (AUC0 t) was calculated, and the results are shown in Table 3. The ACM AUC0 t of the SLNs was lower in heart, lung, kidney, spleen, and higher in liver compared with the free ACM. There were significant differences in all organs between the ACM-SLNs and free ACM (p50.05).
Table 3. The AUC0 24 h of ACM in tissues after i.v. administration of injection and SLNs to rats.
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Formulation ACM injection (ngh/g) ACM-SLNs (ngh/g) Ratioa
Heart
Liver
Spleen
Lung
Kidney
2612.3 ± 221.4 317.1 ± 29.6 0.12*
1920.5 ± 132.5 4002.1 ± 378.7 2.08*
2522.5 ± 232.7 905.2 ± 101.2 0.36*
2658.2 ± 224.3 632.5 ± 73.8 0.24*
3987.5 ± 365.9 373.9 ± 43.8 0.09*
a
The ratio was AUC (ACM-SLNs) /AUC (ACM injection). *p50.05: ACM-SLNs versus ACM injection.
Figure 9. Histopathological studies of liver, spleen, and kidney. (A) Free ACM and (B) ACM-SLNs.
ACM A-loaded SNPs
Drug Delivery Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/21/14 For personal use only.
DOI: 10.3109/10717544.2014.974001
According to Tables 2 and 3, one can see that in comparison with free ACM, ACM-SLNs altered the distribution of ACM in vivo and the half-life periods after i.v. injection of ACM-SLNs (t1/2a ¼ 0.94 h, t1/2b ¼ 12.9 h) were prolonged remarkably than those (t1/2a ¼ 0.18 h, t1/2b ¼ 2.6 h) after i.v. injection of free ACM. This indicated that the ACM-SLNs had sustained release efficacy. The results showed that the SLNs could deliver ACM mainly to liver after i.v. injection and the concentration of ACM in liver was significantly higher than in other tissues. Compared with free ACM, the drug concentration of ACM in liver after i.v. injection of ACM-SLNs increased from 67.5 to 453.2 ng/g (1 h). ACM-SLNs might be preferentially taken up by Hepatocytes because the RES mainly existed in the liver, spleen, bone marrow, and some other organs. The SLNs were thus easily to be detected and swallowed by RES. Therefore, the drug made to be the nanoparticle carrier was more easily targeted to the above tissues. In addition, the tissue distribution behavior of intravenously injected nanoparticles was greatly influenced by their size. When the nanoparticles with a diameter of 50–100 nm enter the circulatory system, they are not released into the systemic circulation directly. Instead, they are absorbed by the RES, leading to greatest drug accumulation in the liver and thus achieving the targeting effect (He et al., 2007; Lu et al., 2008; Hu et al., 2013). Histology studies The histopathological examination of liver, spleen, and kidney was carried out to identify any damage done to the tissue. The microphotographs of liver, spleen, and kidney were taken following their incubation with SLNs formulations for more than 7 d (Figure 9). Free ACM was the control. No sign of damage such as the appearance of epithelial necrosis and sloughing of epithelial cells was detected.
Conclusion A single-factor experiment was performed for the exploration of ACM-SLNs. The SLNs were spherical in shape, uniform in size, and appropriate for administration via intravenous injection. The drug content, encapsulation efficiency, and drug loading of prepared SLNs were 96.4% ± 4.6%, 86.7% ± 2.3%, and 4.8% ± 0.7% (n ¼ 3), respectively, and the mean diameter was 68.2 ± 5.6 nm from three batches. The poly disperse index (PDI) was 0.16 ± 0.05 and the zeta potential was 10.2 ± 0.67 mV. The SLNs were produced with stable physical properties and demonstrated significantly sustained release. The pharmacokinetic behavior of ACM was greatly improved by lyophilized injection of SLN with sustained drug release and high bioavailability. In addition, the results obtained from tissue distribution showed that ACM-SLNs were hepatic targeting in vivo.
9
Declaration of interest The authors declare that they have no conflicts of interest.
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