Improved tumor targeting and antitumor activity of camptothecin loaded solid lipid nanoparticles by preinjection of blank solid lipid nanoparticles

Biomedicine & Pharmacotherapy 80 (2016) 162–172

ScienceDirect

Improved tumor targeting and antitumor activity of camptothecin loaded solid lipid nanoparticles by preinjection of blank solid lipid nanoparticles Dong-Jin Janga , Cheol Moonb , Euichaul Ohc,* a

Department of Pharmaceutical Engineering, Inje University, Gimhae 50834, Republic of Korea College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do 14662, Republic of Korea b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 February 2016 Received in revised form 17 March 2016 Accepted 17 March 2016

This study aimed to enhance the in vivo antitumor effects of camptothecin (CPT), a strong antitumor agent whose delivery is limited by poor aqueous solubility and instability of the active lactone form. CPT was loaded into sterically stabilized, solid lipid nanoparticles (CPT-SLNs) formulated for intravenous administration. The influence of preinjected blank SLNs on the tumor targeting, pharmacokinetics and antitumor activity of CPT-SLNs was investigated. The CPT-SLNs composed of trilaurin-based lipid matrix containing poloxamer188 and pegylated phospholipid as stabilizers were prepared by hot homogenization method and evaluated for in vitro characteristics and in vivo performance. The CPT-SLNs showed an in vitro long-term sustained release pattern and effectively protected the CPT lactone form from hydrolysis under physiological conditions. Notable tumor targeting and tumor growth inhibition were observed after intravenous administration of CPT-SLNs to mice with subcutaneous transplants of CT26 carcinoma cells. In pharmacokinetic studies in rats, CPT-SLNs markedly elevated plasma CPT level and prolonged blood circulation compared to free CPT. Nonetheless, high uptake of CPT-SLNs by reticuloendothelial system (RES)-rich tissues resulted in limited tumor targeting of CPT-SLNs and plasma CPT levels. Preinjection of blank SLNs before administration of CPT-SLNs to tumor-bearing mice substantially reduced the accumulation of CPT-SLNs in RES organs. This led to significantly enhanced tumor targeting, improved pharmacokinetic parameters and increased antitumor efficacy of CPT-SLNs. These results suggested that the in vivo antitumor effects of CPT-SLNs could be further enhanced by preinjection of blank SLNs. Therefore, CPT-SLNs with preinjected blank SLNs could be a potential approach for stable and effective CPT-based cancer therapy. ã 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Camptothecin Solid lipid nanoparticles Reticuloendothelial system blocking Tumor targeting Pharmacokinetics Antitumor activity

1. Introduction Camptothecin (CPT) is a potent broad-spectrum anticancer drug that inhibits topoisomerase I during the S-phase of cell cycle [1–3]. CPT is poorly water soluble and its active lactone form rapidly hydrolyzes to the inactive carboxylate form under physiological conditions [4,5], limiting the delivery and clinical application of CPT in cancer therapy [6,7]. CPT also requires a prolonged schedule of multiple, low-dose administrations to show antitumor efficacy in humans [8]. Among various delivery approaches to improve the solubility and lactone ring stability

* Corresponding author. E-mail address: [email protected] (E. Oh). http://dx.doi.org/10.1016/j.biopha.2016.03.018 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.

of CPT, nanoparticulate delivery systems that incorporate CPT have been extensively investigated for their drug-loading capacity, controlled release and tumor-targeting ability [7,9,10]. In particular, solid lipid nanoparticles (SLNs) consisted of physiological lipids and biocompatible stabilizers have received considerable attention as injectable and targetable nanosized CPT carriers that have low cytotoxicity relative to polymeric nanoparticles [9,11,12]. In the previous researches with CPT-loaded SLNs, encapsulation of CPT into the lipid core matrix of SLNs solubilized the hydrophobic CPT molecules and protected them from rapid hydrolysis before their sustained release [13–15]. The nanosize and lipophilic nature of CPT-loaded SLNs resulted in their prolonged circulation in blood, which led to CPT accumulation in tumor tissues via the enhanced permeation and retention (EPR) effects and consequently improved the antitumor activity of CPT [13,16,17].

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Nanoparticles in the bloodstream are usually recognized as foreign substances and quickly removed from blood by the reticuloendothelial system (RES) of macrophages, particularly liver Kupffer cells [11,18,19]. The rapid and extensive uptake of nanoparticles by the RES is problematic for the systemic delivery of drugs to non-RES tumors or tissues, although RES accumulation is useful for treating tumors or diseases in which RES-containing cells are the target [11,18,19]. Attempts to reduce the phagocytic uptake of nanoparticles and increase their blood circulation time have modified the nanoparticle size, surface hydrophobicity or charge [11,18,19]. The surface of nanoparticles has often been modified with hydrophilic polymers such as polyethylene glycol (PEG) or poloxamer [18,20–24]. The decreased uptake by the RES of surface modified (stealth) nanoparticles encapsulating antitumor agents can enhance the targeting of non-RES tumor tissues by leaky vascularization of longer-circulating nanoparticles [18,22,24]. However, even for stealth nanoparticles, considerable uptake by the RES occurs after long circulation times, limiting their ability to effectively target tumors [13,25,26]. Some PEGylated liposomes, polymeric nanoparticles, or SLNs also induce accelerated blood clearance and lose their long-circulating characteristics upon repeated administration [27–30]. Thus, other strategies besides surface modification are needed to reduce the RES clearance of nanoparticles carrying antitumor drugs to maximize tumor targeting and to improve antitumor efficacy with minimal adverse effects [10,31]. Rapid uptake of liposomes or lipid microemulsions intravenously administered to animals by the RES can be effectively inhibited by blocking the RES with blank colloidal carriers or RES blocking agents such as dextran sulfate, aminomannose-cholesterol, or latex particles [32–38]. Temporary, reversible RES blocking may cause the remainder of the injected drug-carrying particles in circulation to increase their accumulation at non-RES target sites; this procedure is referred to as inverse targeting [32,34–36,39]. Recently, inverse targeting was demonstrated by Liu et al. [40] by pretreating with intralipid that significantly decreased initial RES uptake of superparamagnetic iron-oxide nanoparticles. For applications with SLNs, the same principle could be applied to suppress RES function without permanent or irreversible impairment by predosing safe blocking agents. The initial RES accumulation of antitumor drug-loaded SLNs intravenously administered without pretreatment of blocking agents might have toxic or adverse effects in non-tumor RES-rich tissues or organs such as the liver or spleen [32,38]. It could be expected that blank, drug-free SLNs might be effective as nontoxic, transient RES blocking agents if they were composed of a biocompatible and biodegradable lipid matrix that could be readily cleared from the blood into the RES [32,38,39]. In this study, we postulated that the phagocytic clearance of CPT-loaded SLNs by the RES would be effectively reduced by a transient RES blocking with preinjection of blank SLNs. We further hypothesized that the RES blocking by blank SLNs would result in prolonged blood circulation of CPT-loaded SLNs with enhanced tumor targeting. To investigate these hypotheses, we studied the influence of preinjected blank SLNs on in vivo tissue distribution, tumor targeting, pharmacokinetics and antitumor activity of sterically stabilized CPT-loaded SLNs (CPT-SLNs) designed using physiologically safe components. The stealth CPT-SLNs were employed for a combination effect of RES avoidance with preinjection of blank SLNs. CPT-SLNs were physicochemically characterized and examined for in vitro cytotoxicity, release and stability. In vivo performance studies on the CPT-SLNs were performed in the presence and absence of preinjected blank SLNs.

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2. Materials and methods 2.1. Materials CPT, trilaurin (TL), poloxamer188, n-hexyl-p-hydroxybenzoate and 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Egg yolk phosphatidylcholine (ePC) and distearoylphosphatidyl-ethanolamine-n-poly(ethylene glycol)2000 (PEG2000PE) were from Avanti Polar Lipid Inc. (Alabaster, AL, USA). The purity degree of all lipids was over 99%. All solvents were of high performance liquid chromatography (HPLC) grade and other chemicals were of highest reagent grade. 2.2. Animals and cell lines Male BALB/c mice (6–8 weeks old, 20–25 g) and male SpragueDawley (SD) rats (8 weeks old, 280–300 g) were purchased from Orient Bio (Sungnam, Korea). All animal care and procedures were conducted in accordance with the ARRIVE guidelines, and the animal study protocol was approved (Approval No. 2014-047) by the Institutional Animal Care and Use Committee on the Sungsim Campus of the Catholic University of Korea (Bucheon, Korea). The mice and rats were housed in an air-conditioned room with free access to water and fasted for 12 h before drug administration. The CT26 human colon carcinoma cell line was obtained from Korean Cell Line Bank (Seoul, Korea). Roswell Park Memorial Institute Medium (RPMI 1640), pH 7.4 phosphate-buffered saline (PBS), fetal bovine serum (FBS) and other materials for cell culture were from Gibco Life Technologies (Rockville, MD, USA). 2.3. Preparation of CPT-SLNs CPT-SLNs were prepared by hot melt homogenization method, as described previously [41,42]. CPT (0.1 g) was dispensed into a mixture of TL (0.9 g), ePC (0.25 g), DSPE-PEG2000 (0.25 g) and poloxamer188 (1.0 g) melted in a pear-shaped glass tube maintained at 65
C in a bath-type Branson 3210R-DTH ultrasonic sonicator (Danbury, CT, USA), and homogenously mixed by sonicating for 1 h to completely dissolve CPT. Glycerin (2.5 g) as an isotonic agent was dissolved in 95 mL of distilled water and preheated to 65
C. The entire water phase was added to the tube containing the lipid phase. The resultant mixture was sonicated for 3 h to yield a hot, milky, crude emulsion. The emulsion was homogenized for 10 cycles at 65–70
C with 100 MPa using an Avestin Emulsiflex1 EF-B3 high pressure homogenizer (Ottawa, ON, Canada) wired with Barnstead Thermolyne1 heating tape (Dubuque, IA, USA). The fine emulsion was rapidly dipped into liquid nitrogen and thawed in a water-bath at room temperature to make a CPT-SLN suspension, followed by mixing with the cryoprotectants, mannitol (0.5 g) and dextrose (0.25 g). The final suspension was immediately cooled at 70
C, and lyophilized at 25
C under a vacuum (5 mm Hg) for 48 h using an Ilshin Bondiro freeze-dryer (Yangju, Korea). The soft, crumbly lyophilate was gently pulverized with a rubber spatula below 40% relative humidity. Lyophilized CPT-SLNs were easily reconstituted in distilled water. Blank SLNs were prepared in the absence of CPT only by employing the same components and procedures as those used for preparing CPT-SLNs. 2.4. Characterization of CPT-SLNs The morphology of CPT-SLNs dispersed in water was examined using a Carl Zeiss LIBRA 120 energy-filtered transmission electron

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microscope (TEM; Oberkochen, Germany) after negative staining samples with uranyl acetate (1%, w/v) to enhance the contrast. The mean particle size and distribution were determined by photon correlation spectroscopy using a Nicomb 370 submicron particle sizer (Santa Barbara, CA, USA). The zeta potential was measured using an Otsuka ELS-8000 electrophoretic light scattering spectrophotometer (Osaka, Japan). Encapsulation efficiency and drug loading were estimated by weighing lyophilized CPT-SLNs before dispersion in water and determining the amount of unencapsulated CPT in the dispersion. CPT-SLN dispersion was ultrafiltered using a Millipore Microcon YM-10 ultrafilter (MW cut-off of 10 kDa; Bedford, MA, USA) to obtain unencapsulated CPT. Amounts of total and unencapsulated CPT in CPT-SLN dispersions were measured by an HPLC analysis. 2.5. HPLC analysis of CPT The concentrations of CPT lactone and carboxylate form were simultaneously determined by a reverse-phase HPLC method as previously described [43,44]. Assays were performed using a Shiseido Capcell Pak C18 reverse-phase column (4.6  250 mm, 5 mm; Tokyo, Japan) on an HPLC system equipped with a Shimadzu LC-9A solvent delivery pump and SCL-6B system controller (Kyoto, Japan), and a Hitachi F-1050 fluorescence detector (Tokyo, Japan). The isocratic mobile phase, 20% (v/v) acetonitrile and 80% (v/v) pH 6.0 aqueous buffer (0.1 M potassium phosphate monobasic, 0.5 mM tetrabutylammonium dihydrogen phosphate and 0.4 mM triethylamine), was run at a flow rate of 1 mL/min. Eluents were monitored at an excitation wavelength of 360 nm and an emission wavelength of 430 nm. The internal standard was n-hexyl-phydroxybenzoate. 2.6. In vitro CPT release from CPT-SLNs and stability of CPT lactone form in CPT-SLNs The CPT release from CPT-SLNs was tested at 37
C by a dialysis method. One milliliter of CPT-SLN dispersion was added to a Spectra/Por1 molecular dialysis tubing (MW cut-off of 3.5 kDa; Los Angeles, CA, USA) immersed in 500 mL of pH 7.4 PBS and placed in the dissolution vessel of a Varian VK 7000 dissolution tester (Cary, NC, USA) set at a paddle speed of 50 rpm. At designated times, samples were removed from the PBS and filtered through a membrane filter (0.20 mm). Samples were acidified with a drop of glacial acetic acid to convert all CPT released to its lactone form before assaying by the HLPC method. To examine the protective effect of SLNs against hydrolysis of the CPT lactone ring over time, CPT lactone form in CPT-SLNs incubated in pH 7.4 PBS at 37
C was quantified for 120 h. CPT concentration of each sample was adjusted to 2.5 mg/mL. After removal at designated times, samples were immediately analyzed by the HPLC method to determine the amounts of CPT lactone and carboxylate form. Stability of CPT in solution dissolved in methanol (2.5 mg/mL) was analyzed for comparison using the same procedures used for CPT-SLNs.

an aqueous dispersion of blank SLNs was represented in terms of the concentration of CPT in an aqueous dispersion of CPT-SLNs having the same amount of blank SLNs. The cells attached to the wells were washed with PBS and incubated with varied concentrations (0–200 nM) of blank SLNs, CPT-SLNs and CPT solution for 24 h. The cells were then washed again with PBS, treated with MTT solution (5 mg/mL in PBS) and incubated for 5 h at 37
C. Ultraviolet absorbance was measured at 570 nm using a Spectrofluor microplate reader (Tecan, Austria). Cell viability (%) was calculated and compared with untreated controls. 2.8. In vivo tissue distribution of CPT-SLNs in tumor-bearing mice The tissue distribution of CPT-SLNs was studied in male BALB/c mice with subcutaneous transplants of CT26 cells (1 106 cells) in the thigh of the left hind leg. Free CPT formulation as a suspension was prepared by suspending CPT powder in water containing DMSO (10%, v/v) and Tween 80 (5%, v/v) and CPT-SLNs were dispersed in water for all animal experiments. After tumor volumes reached approximately 100 mm3, free CPT or CPT-SLNs formulation (CPT dose of 3 mg/kg body weight) was intravenously administered for 30 s via the tail vein (n = 10 per each CPT formulation). Mice were anaesthetized with light diethylether before surgical procedures for tissue collection. At 1 and 12 h postadministration (n = 5 at each time point), blood samples were collected from the ocular artery after eyeball removal and put into test tubes containing 10 mL heparin solution. Blood samples were centrifuged to obtain plasma samples. At the same time points, tumor (left hind) and non-tumor (right hind) thighs, brain, lung, liver, kidney and spleen were excised. Each tissue or organ was weighed, blotted dry with tissue paper, acidified with 0.2 N HCl and homogenized using an IKA Ultra-Turrax Homogenizer (Staufen, Germany). Supernatants from centrifugation of tissue homogenates or plasma samples were vortexed with tert-butyl ether to extract CPT, and centrifuged again to separate the organic phase. The organic phases were evaporated under a gentle stream of nitrogen gas, and dried extract was reconstituted in the mobile phase. The amount of CPT in plasma, tissue or organ samples was determined by the HPLC analysis described above. 2.9. In vivo antitumor activity of CPT-SLNs in tumor-bearing mice The tumor-bearing BALB/c mice were also used to study the antitumor activity of CPT-SLNs. When tumor volumes were approximately 50–60 mm3 (8 days after CT26 cell injection), normal saline, free CPT or CPT-SLN formulation (CPT dose of 3 mg/kg body weight) was injected for 30 s via the tail vein in a single dose, and on 8 and 11 days for double dosing after inoculation of CT26 cells (n = 10 per group). Normal saline was used as a control. Tumor size was measured every 2–3 days for 32 days after single or double dosing of each CPT formulation using a vernier caliper across the longest (m) and shortest diameters (n) of the tumor. Tumor volume (V) was calculated using the formula V = 0.5 mn2. Survival rate (%) was recorded every 2–3 days for 72 days after inoculation of CT26 cells.

2.7. In vitro cytotoxicity assay 2.10. In vivo pharmacokinetic study of CPT-SLNs in rats The cell viability of blank (CPT free) SLNs, CPT-SLNs and CPT solution was measured using the CT26 human colon carcinoma cell line by MTT assay. RPMI 1640 medium supplemented with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin was used for culturing CT26 cells at 37
C under 5% CO2. Cultured cells were placed at a density of 1 104 cells/well in 96-well microplates and allowed to adhere onto the wells. CPT solutions in methanol and aqueous dispersions of blank SLNs and CPT-SLNs were prepared at different concentrations of CPT. The concentration of blank SLNs in

Male SD rats were fasted overnight and had free access to food and water before surgery. The carotid arteries were cannulated with a Clay Adams polyethylene tube (Franklin Lakes, NJ, USA) under light anesthesia with diethylether for blood collection. Rats were injected for 30 s via the tail vein with free CPT or CPT-SLN formulation at a CPT dose of 3.0 mg/kg (5 rats/group). Blood samples (0.12 mL) were collected at 0 (control), 1, 2, 3, 4, 6, 8, 10 and 24 h. The cannula was immediately flushed with a

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heparinized saline to prevent blood clotting. After centrifugation of blood samples at 13,000 rpm for 10 min at 4
C, plasma samples (50 mL) were collected and analyzed to determine CPT concentrations in plasma by the same HPLC method as described above. The following pharmacokinetic parameters were calculated using a noncompartmental analysis (WinNonlin1 program, Scientific Consulting Inc.; Cary, NC, USA): the total area under the plasma concentration-time curve from time zero to infinity (AUC0!1); the time-averaged total body clearance (CL); the mean residence time (MRT); the apparent volume of distribution at steady state (Vss); and the terminal half life (t1/2). 2.11. Effect of preinjected blank SLNs on in vivo performance of CPTSLNs Lyophilized blank SLNs were dispersed in water (19.1 mg/mL solid content) and slowly injected for 30 s via the tail vein of mice (0.4 mL) or rats (4.8 mL) 30 min before administration of CPT-SLNs. The in vivo studies on the tissue distribution and antitumor activity of CPT-SLNs in tumor-bearing BALB/c mice as well as the pharmacokinetics of CPT-SLNs in SD rats were duplicated to monitor the effects of preinjected blank SLNs. 2.12. Statistical analysis All data were expressed as means  standard deviations. A p value less than 0.05 was considered to be statistically significant using an unpaired Student’s t-test or one-way analysis of variance test (Jandel Scientific Sigmaplot; Corte Madera, CA, USA). 3. Results 3.1. Characteristics of CPT-SLNs CPT-SLNs dispersed in water were nearly spherical by TEM (Fig. 1A). The mean particle size of CPT-SLNs measured by photon correlation spectroscopy was 156  16 nm in a diameter (n = 3) with a narrow size distribution (in the range of 36–531 nm), as shown in Fig. 1B. The mean zeta potential of CPT-SLN was 43.9  4.3 mV (n = 3), which was high enough to prevent aggregation. No significant changes were observed in a mean diameter or an integrity of CPT-SLNs kept under pH 7.4 PBS for 4 weeks. The CPT loading in CPT-SLNs was 5.0  0.2% (n = 3) and encapsulation efficiency was 96.5  0.4% (n = 3). The results for particle size, zeta potential and encapsulation efficiency corresponded with characteristics that are widely accepted as appropriate for an intravenous formulation and passive targeting of nanoparticles [31,45,46]. 3.2. In vitro CPT release from CPT-SLNs and stability of CPT lactone form in CPT-SLNs The CPT release-time profile from CPT-SLNs in pH 7.4 PBS at 37
C is in Fig. 2. CPT-SLNs exhibited sustained release of CPT with an initial rapid-release phase followed by gradually slower release pattern. The cumulative release of CPT was around 35% up to 12 h and about 85% for 120 h. This result suggests that CPT-SLNs are capable of sustaining the release of CPT over at least 5 days. The amounts (%) of the CPT lactone form remaining over time in CPT solution and CPT-SLNs incubated in pH 7.4 PBS at 37
C were determined (Fig. 2). In the CPT solution, the lactone form was rapidly hydrolyzed to the carboxylate form and only 15% of the lactone form remained after 12 h. In contrast, hydrolysis of the CPT lactone form occurred slowly in CPT-SLNs and about 67% of CPT was in the active lactone form even after 12 h. This difference in the lactone form remaining between in CPT solution and in CPT-SLNs

Fig. 1. (A) Morphological image and (B) particle size distribution of CPT-SLNs. Microscopic images were observed using transmission electron microscopy and particle size distributions were measured using photon correlation spectroscopy in distilled water.

should be due to the protection of lactone form by SLNs against its hydrolysis in pH 7.4 PBS. The protection lasted over 120 h, resulting in the preservation of around 17% of the lactone form. In the CPTSLNs, the amount (%) of CPT lactone form remaining was nearly the same as the portion (%) of unreleased CPT at each time point, as shown in Fig. 2. This observation implies that almost all CPT molecules entrapped in SLNs were unhydrolyzed and remained in the lactone form, while CPT molecules released from the CPT-SLNs were hydrolyzed to the carboxylate form as rapidly as those in the CPT solution. 3.3. In vitro cytotoxicity of CPT-SLNs Results on the in vitro cell viability (%) of CPT solution (in methanol), blank SLNs and CPT-SLNs against CT26 human colon carcinoma cells are in Fig. 3. Blank SLNs exhibited almost no cytotoxic effects (<3% cell death at 200 nM). This indicates that the blank SLNs are noncytotoxic. Unlike blank SLNs, CPT-SLNs and CPT solution showed a significantly cytotoxic effect. As the

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100

CPT re le ase from CPT-SLNs % of CPT lactone form in CPT-SLNs % of CPT lactone form in CPT solution

80

80

60

60

40

40

20

20

0

CPT lactone form (%)

Cummulative CPT release (%)

100

0 0

20

40

60

80

100

120

Time (hours) Fig. 2. In vitro CPT release-time profile from CPT-SLNs in pH 7.4 phosphate-buffered saline (PBS) at 37
C (n = 3), and percentages of CPT lactone form remaining in CPT solution (in methanol) and CPT-SLNs incubated in PBS at 37
C over time (n = 3). CPT release test was conducted across a dialysis membrane (MW cutoff of 3500 Da) contained in a dissolution apparatus vessel (paddle speed = 50 rpm). Amounts of CPT released or CPT lactone form remaining were determined by a HPLC analysis.

100

Cell viability (%)

80

60

blank SLNs CPT-SLNs CPT solution

40

20

0 0

50

10 0

150

20 0

Concentration of CPT (nM) Fig. 3. In vitro cell viability of blank SLN (CPT free), CPT solution (in methanol) and CPT-SLNs against CT26 human colon carcinoma cells with the concentrations of CPT used. Cell viability (%) for 24 h was determined by a MTT assay (n = 3) is in the y-axis.

concentration of CPT in CPT-SLNs and CPT solution increased, the cell viability gradually decreased (56.5% and 25.3% cell death at 200 nM, respectively). The in vitro cytotoxic efficacy of CPT-SLNs was greater than that of CPT solution.

3.4. In vivo tissue distribution of CPT-SLNs in tumor-bearing mice The in vivo tissue distribution and tumor targeting ability of CPT-SLNs were estimated by determining CPT concentrations in

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Fig. 4. (A) CPT concentrations in plasma, tumor (left hind) and non-tumor (right hind) thighs, and (B) comparative tissue distribution of CPT at 1 and 12 h after intravenous administration of free CPT and CPT-SLN formulations to tumor-bearing BALB/c mice (n = 5). CPT concentrations in non-tumor thigh were blow the limit of quantification (LOQ = 0.1 ng/mL) with treatment of CPT-SLNs. *p < 0.01 and **p < 0.001 compared with free CPT; #p < 0.001 compared with non-tumor thigh.

the tissues and organs studied after intravenous administration of free CPT or CPT-SLNs formulation to tumor-bearing BALB/c mice. As shown in Fig. 4A, the CPT concentrations in plasma and tumor (left hind) thigh were significantly higher in CPT-SLNs than in free CPT at 1 and 12 h. High CPT concentrations in CPT-SLNs were maintained in tumor thigh for 12 h. In contrast, the CPT concentration in non-tumor (right hind) thigh was undetectable

in CPT-SLNs at 1 and 12 h. These results suggest that CPT-SLNs have a high tumor targeting and retention ability with their increased concentration in plasma. The CPT concentrations in spleen, liver, lung, kidney and brain were higher than those in plasma and tumor thigh for both free CPT and CPT-SLNs formulation (Fig. 4A and B). The accumulation of CPT-SLNs in RES organs (spleen, liver and lung) and brain was

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higher than those of free CPT (Fig. 4B). These observations are consistent with previous findings [13]. However, the 2- to 4-fold elevated uptake of CPT-SLNs by RES organs relative to tumor thigh was considerably less than the 4- to 10-fold increase in uptake of free CPT. In addition, CPT was rapidly eliminated from individual organs at a rate that was similar for both free CPT and CPT-SLNs formulation over 12 h postadministration: only about 10–20% of the CPT concentration at 1 h remained at 12 h (Fig. 4B), in accordance with previous studies [25,26,31]. In contrast, almost half of the CPT was maintained in tumor tissues over 12 h after injection of CPT-SLNs (Fig. 4A). These results support the high tumor targeting and retention of CPT-SLNs seen above. 3.5. Effect of preinjected blank SLNs on in vivo tissue distribution of CPT-SLNs in tumor-bearing mice Although CPT-SLNs were much more distributed into tumor thigh than free CPT, the high uptake of CPT-SLNs by the RES organs resulted in limited tumor targeting and plasma levels in tumorbearing BALB/c mice. Blank SLNs were preinjected before administration of CPT-SLNs to tumor-bearing mice and changes in tissue distribution of CPT-SLNs were monitored. As shown in Fig. 5, preinjection of blank SLNs significantly reduced the accumulation of CPT-SLNs in the RES organs (by 67.2, 45.0 and 50.7% for spleen, liver and lung, respectively, at 1 h; by 38.8, 51.7 and 47.6% for spleen, liver and lung, respectively, at 12 h) but significantly increased that in plasma (by 74.7 and 117% at 1 h and 12 h, respectively) and tumor thigh (by 86.4 and 106% at 1 h and 12 h, respectively). This result could be interpreted to be due to partial and temporary blocking of the RES organs in tumor-bearing mice by preinjecting blank SLNs. This could lead to less RES uptake of CPT-SLNs by RES organs, which is similar to the results obtained from the previous studies on blank colloidal carriers as RES blocking agents for liposomes or lipid emulsions [32,35,37,39]. Thus, the transient RES blocking by blank SLNs resulted in significantly higher plasma level, greater tumor uptake and longer retention of CPT-SLNs.

3.6. Effect of preinjected blank SLNs on in vivo pharmacokinetics of CPT-SLNs in rats Plasma concentration-time curves of CPT after intravenous administration of free CPT, CPT-SLNs or CPT-SLNs with preinjection of blank SLNs to SD rats are in Fig. 6. In all cases, the curves declined in a poly-exponential manner. At all times, the plasma concentrations of CPT in CPT-SLNs with preinjection of blank SLNs were significantly higher than those in CPT-SLNs, which were also significantly higher than those in free CPT. The relevant pharmacokinetic parameters are listed in Table 1. In CPT-SLNs with preinjection of blank SLNs, the AUC0!1, CL, MRT and Vss of CPT were significantly greater, slower, longer and smaller, respectively, than those in CPT-SLNs (by 202, 64.8, 119 and 22.7%, respectively). In CPT-SLNs, the AUC0!1, CL, MRT and t1/2 of CPT were also significantly greater, slower longer and longer, respectively, than those in free CPT (by 77.3, 46.7 100 and 57.1%, respectively). From these results, it can be stated that intravenously administered CPT-SLNs elevated the plasma levels and prolonged the blood circulation of CPT as well as preinjection of blank SLNs significantly further improved pharmacokinetic parameters of CPT-SLNs. In particular, the initial (0–2 h) CPT clearance rate was considerably slowed by preinjection of blank SLNs compared to free CPT or CPT-SLNs without preinjection of blank SLNs. 3.7. Effect of preinjected blank SLNs on in vivo antitumor activity of CPT-SLNs in tumor-bearing mice Antitumor activities of free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs were evaluated by determining tumor volume and survival rate in tumor-bearing BALB/c mice injected at single and double dosing of 3 mg CPT/kg. No acute toxicity-induced death before 21 days was observed in any experimental group. The tumor volumes in CPT-SLNs with preinjection of blank SLNs were still significantly smaller than those in CPT-SLNs which were significantly smaller than those in free CPT (Fig. 7A and C). At

Fig. 5. Effects of preinjected blank SLNs on the tissue distributions of CPT-SLNs at 1 and 12 h after intravenous administration to tumor-bearing BALB/c mice (n = 5). Blank SLNs were preinjected 30 min before administration of CPT-SLNs. *p < 0.05 and **p < 0.01 compared with CPT-SLNs without preinjection of blank SLNs.

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Fig. 6. Plasma concentration-time profiles of CPT in male SD rats after intravenous administration of free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs at dose of 3 mg CPT/kg (n = 5). Blank SLNs were preinjected 30 min before administration of CPT-SLNs. *p < 0.05 and **p < 0.01 compared with free CPT; #p < 0.01 compared with CPTSLNs.

Table 1 Mean pharmacokinetic parameters following intravenous administration of free CPT, CPT-SLNs or CPT-SLNs with preinjection of blank SLN in male SD rats (n = 5 per each). *p < 0.05 and **p < 0.01 compared with free CPT; #p < 0.05 and ##p < 0.01 compared with free CPT or CPT-SLNs. Parameters

Formulation Free CPT

AUC0!1a (ng h/mL) CLb (L/h/kg) MRTc (h) Vssd (L/h/kg) t1/2e (h)

90.8 33.0 1.8 59.4 5.6

    

CPT-SLNs

33.4 7.6 0.5 6.8 0.8

161 17.6 3.6 63.4 8.8

    

31.1** 3.4* 0.6** 7.3 1.0**

CPT-SLNs with preinjection of SLNs 486 6.2 7.9 49.0 8.8

    

68.4## 1.2## 1.3## 2.6# 1.1

Data are expressed as mean  standard deviation. a AUC0!1: area under the plasma concentration-time curve from time zero to infinity. b CL: total body clearance. c MRT: mean residence time. d Vss: apparent volume of distribution at steady state. e t1/2: terminal half-life.

32 days after single dose injection of CPT formulations, the tumor volume in free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs were 4363, 3382 and 2295 mm3, respectively (Fig. 7A). Likewise, the survival rates in CPT-SLNs with preinjection of blank SLNs were even higher than those in CPT-SLNs which were higher than those in free CPT (Fig. 7B). At 60 days postinoculation (day mice were all dead in normal saline), the survival rate in free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs were 20, 40 and 50%, respectively. The effects of tumor regression and survival extension became stronger following injection of CPT formulations at double doses (Fig. 7C and D). When double dosing of CPT formulations, the tumor volume in free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs at 32 days postinjection were 3980, 2658 and 1101 mm3, respectively and the survival rate in free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs at 60 days postinoculation were 30, 60 and 70%, respectively. The differences in tumor growth inhibition and survival rate in

between free CPT and CPT-SLNs or between CPT-SLNs and CPTSLNs with preinjection of blank SLNs appeared greater for double dosing than for single dosing as days passed. The stronger suppression of tumor growth and higher survival rate following administration of CPT-SLNs seem to correspond to their improved tumor targeting and prolonged blood circulation compared to free CPT. The significantly superior antitumor effects in CPT-SLNs with preinjection of blank SLNs are likely due to significantly further improved tumor targeting and prolonged blood circulation that resulted from transient RES blocking by blank SLNs. 4. Discussion SLNs can effectively solubilize poorly water soluble CPT and stabilize its lactone ring against hydrolysis. The SLN system containing CPT is regarded as a promising delivery system because it can prolong blood circulation of CPT and enhance its tumor

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Fig. 7. Antitumor effects of free CPT, CPT-SLNs and CPT-SLNs with preinjection of blank SLNs in tumor-bearing BALB/c mice (n = 10). Changes in (A) tumor volume and (B) survival rate with a single dose (3 mg CPT/kg); changes in (C) tumor volume and (D) survival rate with double doses. Individual CPT formulations were injected on 8 days for single dosing, and on 8 and 11 days for double dosing after inoculation of CT26 human colon carcinoma cells, as indicated by the arrows. Blank SLNs were preinjected 30 min before administration of CPT-SLNs. *p < 0.05 and **p < 0.01 compared with free CPT; #p < 0.05 and ##p < 0.01 compared with CPT-SLNs.

targeting [7,13–15,17]. In this work, sterically stabilized (stealth) SLNs were prepared using TL, ePC, poloxamer 188 and PEG2000-PE to promote tumor targeting potential of CPT. TL (mp 46.5
C) is a medium-chain triglyceride compatible for hot melt homogenization and was used as the core lipid component of the SLN matrix with ePC as the emulsifier [24,27]. Poloxamer188 (co-emulsifier) and PEG2000-PE are widely employed as steric stabilizers and were incorporated into the lipid matrix for surface modification [47–49]. The use of TL and excipients which are biocompatible for parenteral administration might be a clinical advantage since they potentially reduce the risk of acute or chronic toxicity [18,24,47,50,51]. The in vitro safety of blank SLNs was confirmed by a MTT assay, in which they showed almost no cytotoxic effects on CT26 human colon carcinoma cells (Fig. 3). Also, no acute toxicityinduced death was observed up to 21 h after intravenous injection of blank SLNs into tumor-bearing BALB/c mice.

We demonstrated that CPT was efficiently encapsulated into spherical SLNs and was preserved in the active lactone form inside the hydrophobic inner core of SLNs before release (Fig. 2). CPTSLNs were stable under simulated physiological conditions (pH 7.4 PBS at 37
C), showing no significant changes in particle size or surface charge over 4 weeks. Additionally, CPT-SLNs showed a sustained CPT release pattern for longer than 5 days (Fig. 2). Around 15% of CPT were still remained in CPT-SLNs and released in a gradually sustained manner even after 5days. In pharmacokinetic studies in SD rats, CPT-SLNs exhibited markedly elevated plasma levels and prolonged circulation of CPT compared to free CPT (Fig. 6 and Table 1). The longer circulating CPT in CPT-SLNs was almost cleared within 24 h and its plasma level was greatly decreased even at 2 h (remaining at 4.2%). Since it was observed in the release study of CPT-SLNs that only a small portion (13.7%) of CPT was released from CPT-SLNs at 2 h but the rest of CPT were still

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remained in CPT-SLNs with being protected from hydrolysis (Fig. 2), however, the majority of the released CPT and CPT-SLNs cleared from the blood circulation at 2 h must have been distributed to rat tissues and organs. This interpretation is supported by that the CPT concentrations in tumor (lest hind) thigh (2.4 times), brain (7.6 times), lung (9.4 times), liver (8.5 times), kidney (4.0 times) and spleen (10 times) were much higher than that in plasma at 1 h after administration of CPT-SLNs to tumor-bearing BALB/c mice (Fig. 4). Tissue distribution experiments also showed that intravenously injected CPT-SLNs were significantly more concentrated in tumor tissue (left hind thigh) or retained in plasma than free CPT, and accumulated significantly greater in tumor tissue than in non-tumor tissue (left hind thigh) (Fig. 4A). A large fraction of CPT molecules remained in the active lactone form and circulated systemically before CPT-SLNs were distributed in tumor tissues. CPT-SLNs acted as a depot, providing a sustained supply of CPT for extended periods within tumors. The prolonged circulation and increased tissue distribution of CPT-SLNs could increase tumor uptake via the EPR effect, known as passive targeting, with consequently enhanced antitumor activity. A distinct difference in tumor growth inhibition in free CPT and CPT-SLNs could be observed at 5 days and the tumor volumes in CPT-SLNs were significantly smaller than those in free CPT over 32 days following administration of both CPT formulations to tumorbearing BALB/c mice (Fig. 7A and C). Also, a distinct difference in survival rate in between free CPT and CPT-SLNs could be observed at 31 days and the survival rates which were higher than those in free CPT over 52 days after injection of both CPT formulations (Fig. 7B and D). The suppression of tumor growth and survival rate became increased with repeated dosing of CPT-SLNs. Nonetheless, CPT-SLNs intravenously injected into tumor-bearing BALB/c mice were significantly more uptaken by RES organs than by tumor tissue (left hind thigh), as seen in Fig. 4, even if they were surface modified with poloxamer and PEG. Significant RES uptake has been previously reported in stealth or nonstealth nanoparticles incorporating anticancer drugs [13,17,22,26,52]. Extensive accumulation of nanoparticles in the RES organs would be beneficial for treating tumors or diseases with the RES as a target site, but would be problematic if non-RES tissues were the target [11,18,19]. In this work, increased uptake of CPT-SLNs by RES organs resulted in limited tumor targeting and plasma levels in tumor-bearing mice. Accordingly, only limited RES avoidance could be achieved with the stealth strategy of modifying the surface properties of SLNs containing CPT. Decreasing RES uptake would be needed for retaining CPT-SLNs in the bloodstream to promote passive tumor targeting by SLNs. Fig. 5 shows significantly reduced RES uptake, increased accumulation in tumor tissue (left hind thigh), and increased retention of CPT-SLNs following intravenous administration with preinjection of blank SLNs into tumor-bearing mice. Differences compared with the administration of CPT-SLNs alone seem to be attributed to the transient RES blocking by preinjected blank SLNs, because the RES in animals can be temporarily blocked by injection of blank colloidal carriers or RES blocking agents [32,35,38,39]. Preinjected blank SLNs markedly elevated the plasma level of CPT-SLNs and prolonged their blood circulation time in rats by temporarily depleting RES uptake (Fig. 6 and Table 1). This allowed extravasation of encapsulated CPT and accumulation into tumor tissue through the EPR effect or enhanced passive tumor targeting in tumor-bearing mice pretreated with blank SLNs. The increased accumulation of CPT-SLNs in tumor tissue contributed to the significantly improved antitumor efficacy seen in a human colon carcinoma xenograft model (Fig. 7). The suppression of tumor growth and survival extension in CPT-SLNs with preinjection of blank SLNs were significantly enhanced compared to CPT-SLNs without preinjection of blank SLNs. The reduced level of CPT-SLNs in RES organs might mitigate possible systemic toxic effects.

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