http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(6): 509–517 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2014.897708

ORIGINAL ARTICLE

Preparation of vincristine sulfate-loaded poly (butylcyanoacrylate) nanoparticles modified with pluronic F127 and evaluation of their lymphatic tissue targeting

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Rong Tan*, Mengmeng Niu, Jihui Zhao, Ying Liu, and Nianping Feng Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China

Abstract

Keywords

In order to improve the lymphatic targeting efficiency of anti-cancer agent vincristine sulfate (VCR), the poly (butylcyanoacrylate) nanoparticles (VCR-PBCA-NPs) were prepared by emulsion polymerization and modified superficially with Pluronic F127. These prepared nanoparticles with (F127-VCR-PBCA-NPs) and without surface modification (VCR-PBCA-NPs) were characterized and their lymphatic targeting efficiencies were evaluated in vitro and in vivo. The results showed that VCR was released more sustained from both kinds of VCR-loaded nanoparticles, compared with the VCR solution. The up-taking efficiency of VCR into raji cells was enhanced by F127-VCR-PBCA-NPs, compared with the VCR-PBCA-NPs or VCR solution. Lower clearance (CL) of VCR from the systemic circulation and higher lymphatic targeting efficiency of VCR were observed for F127-VCR-PBCA-NPs than the VCR-PBCA-NPs or VCR solution, and F127-VCR-PBCANPs showed greater antitumor efficacy than the VCR-PBCA-NPs or VCR solution in the human Burkitt’s lymphoma (raji)-bearing nude mice. These findings suggest that superficially modified nanoscale carriers might be promising vehicles for chemotherapeutic agents in the treatments of metastatic tumors and malignant lymphoma.

Lymphatic targeting, pharmacokinetics, Pluronic F127, polybutylcyanoacrylate nanoparticles, tissue distribution, vincristine sulfate

Introduction In many carcinomas, including breast, lung and neck squamous cell cancers, the lymphatic system and sentinel lymph node play a significant role in early-stage diagnostics and subsequent therapies [1]. On invasion by tumor cells, the regional lymph nodes act as a reservoir from where cancer cells can seed other parts of the body [2]. Lymphatic metastasis is believed to be one of the most important pathways for the spread of many types of solid tumors. The inability to remove all the lymph metastases remains the primary cause of death from cancer [3–5]. To overcome this, many approaches, including surgery [6], radiotherapy [7], and chemotherapy [8,9], have been used. Among these, conventional chemotherapy is the most commonly used method. However, systemic delivery of chemotherapeutic agents often fails to achieve drug concentrations high enough to eradicate all metastatic lesions [10]. Moreover, targeting drugs to the lymphatic system is difficult because of its

*Dr Rong Tan currently works at Hangzhou Tea Research Institute, CHINA COOP, Hangzhou 310016, China. Address for correspondence: Nianping Feng, Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Cailun Road 1200, Shanghai 201203, PR China. Tel: +86 21 51322198. Fax: +86 21 51322197. E-mail: [email protected]

History Received 2 January 2014 Revised 19 February 2014 Accepted 20 February 2014 Published online 13 March 2014

complexity, peculiarity, and anatomy [11,12]. Accordingly, an effective treatment for metastatic tumors is urgently required. Vincristine sulfate (VCR) is an effective chemotherapeutic agent that has been extensively used to treat various cancers, including malignant lymphoma [13]. Unfortunately, many tumor cells are not sensitive to VCR because of its Pglycoprotein-mediated efflux from the cells [14,15]. Increasing plasma VCR concentration may improve the effectiveness of antitumor therapy, but systemic administration may result in severe cardiovascular and nervous system toxicity [16]. One way of decreasing VCR’s toxicity and improving its antitumor efficacy is to encapsulate it into nanoparticles capable of targeting tumor tissues or cells and changing their in vivo distributive characteristics. Among the nanotechnologies developed to address these issues, poly (butylcyanoacrylate) (PBCA) nanoparticles are of particular interest [17–19]. They are believed to be excellent candidates for medical application because of their excellent biodegradability, high biocompatibility, drug compatibility, and permeability. Many drugs have been efficiently loaded into PBCA nanoparticles, either through adsorption onto nanospheres in the presence of hydrophobic cations or by encapsulation into the internal cavity of aqueous-core nanocapsules. Drugloaded PBCA nanoparticles have been shown to be promising in the treatment of both non-resistant and resistant cancers, achieving appropriate active drug concentrations in the tumor tissue [20]. Moreover, when polymeric degradation occurs,

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degradates, and the drug can form a drug-poly (cyanoacrylic acid) ion pair, which can cross the cell membranes without being recognized by P-glycoprotein (P-gp) [21,22]. Thus, PBCA nanoparticles show promise for overcoming drug resistance in tumors and may improve VCR delivery in the treatment of tumors, including malignant lymphoma. However, systemic administration of drug-loaded PBCA nanoparticles is disadvantageous, since nanoparticles of approximately 100 nm are susceptible to phagocytosis by reticuloendothelial system (RES), leading to an inadequate drug concentration in the tumor site. To achieve a longer circulation time and better bioavailability, PBCA nanoparticles are often coated with polymeric materials [23,24]. One representative of such a material is Pluronic F 127, an amphiphilic synthetic polymer containing a hydrophilic poly (ethylene oxide) (PEO) block and a hydrophobic poly (propylene oxide) (PPO) block arranged in a triblock structure: PEO-PPO-PEO. Coating F127 onto the surface of PBCA nanoparticles reduces their susceptibility to macrophage endocytosis. Recent research indicated that F127 inhibited the P-gp efflux system by ATP depletion in multidrug-resistant (MDR) cancer cells, as well as by inhibiting the glutathione/glutathione-S-transferase detoxification system and altering apoptotic signal transduction [25]. Therefore, we took advantage of the beneficial influences of Pluronic F127 and PBCA nanoparticles on the pharmacokinetic and tissue distribution behaviors of VCR, and F127VCR-PBCA-NPs were prepared and evaluated in vivo and in vitro.

Materials and methods Materials VCR was obtained from the Shanghai Antikang Phytochemistry Co. Ltd. (Shanghai, China). Butylcyanoacrylate was purchased from Beijing Component Medical Adhesive Co. Ltd (Beijing, China). Pluronic F127 was kindly supplied by BASF Ltd. (Shanghai, China). Penicillin–streptomycin, RPMI 1640, and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersberg, MD). Hematoxylin–eosin (H & E) staining kit was provided by the Department of Experimental Animals, Traditional Chinese Medicine of Shanghai University (Shanghai, China). Purified deionized water was prepared using the Milli-Q plus system (Millipore Co., Billerica, MA). All other reagents and solvents were of analytical grade. Human Burkitt’s lymphoma B-cell NHL Raji cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Male Sprague–Dawley (SD) rats (weight: 200 ± 10 g) and male BALB/c nude mice (weight: 20 ± 2 g) were supplied by the Department of Experimental Animals, Traditional Chinese Medicine of Shanghai University (Shanghai, China). They were acclimated at 25  C and 55% humidity under natural light/dark conditions for 1 week before dosing. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of the Traditional Chinese Medicine of Shanghai University (Shanghai, China).

J Drug Target, 2014; 22(6): 509–517

Preparation of VCR-loaded PBCA nanoparticles VCR-loaded PBCA nanoparticles (VCR-PBCA-NPs) were prepared using an emulsion polymerization method reported previously [26]. Briefly, 50 mg of Pluronic F68, 50 mg of dextran 70, and 4 mg of VCR were dissolved in 10 mL of hydrochloric acid (pH 2.5) Then 100 mL of butylcyanoacrylate (BCA) was added under gentle stirring, and the mixture was kept under continuous stirring for 3 h. The pH of the mixture was adjusted to 7.4 with 0.1 N NaOH. After extrusion through a 0.8-mm microfilter, the suspension of VCRPBCA-NPs was obtained. To further modify VCR-PBCA-NPs, the suspension of VCR-PBCA-NPs was mixed with an appropriate amount of Pluronic F127 (1%) and incubated at 25  C under gentle agitation for 1 h. After extrusion through the 0.8-mm microfilter, the suspension of F127-VCR-PBCA-NPs was obtained. Physicochemical characterization of nanoparticles The contents of VCR in nanoparticles were determined by using an HPLC method reported previously, which was modified slightly [27]. High-performance liquid chromatography (HPLC) separations were acquired on a C18 column (dimensions: 250 mm  4.6 mm, 5 mm; Ultimate, Palo Alto, CA) with a mobile phase composed of methanol and 1.6% diethylamine solution (78:22, v/v). The pH value of the mobile phase was adjusted to 7.0 by phosphoric acid. The amount of VCR incorporated into the nanoparticles was determined after separating free VCR from the nanoparticles by centrifugal ultrafiltration (10 kDa MWCO) at 1000 rpm for 10 min. VCR entrapment efficiency was expressed as a percentage of the difference between the initial amount of VCR and the free amount in the wash-out buffer relative to the total amount used for the nanoparticle preparation. Particle size was determined by a Nicomp 380/ZLS dynamic light scattering instrument (PSS, Inc, Pleasanton, CA) and analyzed by the ZPW388 software program (Davisco Foods International Inc, Sueur, MN) and expressed with intensity-based Gaussian distribution. Morphological evaluation of the nanoparticles was performed using transmission electron microscopy (TEM, Philips Tecnai 12, Eindhoven, The Netherlands). In vitro release studies The release of VCR from F127-VCR-PBCA-NPs and VCRPBCA-NPs was studied according to the method described by Zhang et al. [27]. Suspensions of nanoparticles containing 14.5 mg of VCR were placed in a cellulose dialysis bag (cut-off, 10–15 kDa, Viskase), which was then placed in 98 mL of saline solution, and stirred at 100 rpm. The temperature was maintained at 37.0 ± 0.5  C during the experiments. At predetermined time points, samples (1 mL) were withdrew and filtered through a 0.22-mm millipore syringe filter. The concentrations of VCR were determined by using the HPLC mentioned above. An equal volume of saline solution, maintained at the same temperature, was added after each sampling.

DOI: 10.3109/1061186X.2014.897708

VCR solution (VCR was dissolved in distilled water to get the VCR solution with a drug concentration of 0.4 mg/mL) was used as a control. The in vitro studies were performed under sink conditions, and obtained data were analyzed and fitted to kinetic equations. Cellular uptake experiments Cell culture

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Raji cells were cultured in RPMI-1640 medium containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37  C, 90% relative humidity, and 5% CO2. Cells were sub-cultured regularly (by trypsin/EDTA) on reaching 70–80% confluence. All experiments were performed on cells in the logarithmic phase of growth. Cellular accumulation of VCR Raji cells were seeded onto 6-well plates at a density of 1  106 and kept there for 24 h, then incubated with suspensions of VCR-PBCA-NPs, F127-VCR-PBCA-NPs, or VCR solution at 37  C for 4, 8, and 12 h, respectively. The cells were washed carefully with PBS (pH 7.4) for three times, observed under microscopy, and then lysed by using an ultrasonic probe at 4  C. After centrifugation at 2000 rpm for 10 min, the supernatants were collected and stored separately at 20  C until analysis. The concentrations of VCR in the supernatants were determined by using the HPLC method mentioned above. In vivo pharmacokinetics and biodistribution Eighteen male SD rats (200 ± 10 g) were randomly divided into VCR-PBCA-NPs, F127-VCR-PBCA-NPs, and VCR groups (n ¼ 6 for each group). Suspensions of VCR-PBCA-NPs, F127-VCR-PBCA-NPs, or saline solution of free VCR were given to each rat in corresponding groups via tail vein as a single injection (1 mg/ kg). Serial 300 -mL blood samples were collected into heparinized tubes by puncturing the retro-orbital sinus according to the following time schedule: 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after dosing. The heparinized blood samples were centrifuged immediately at 5000 rpm for 10 min. A volume of 150 mL plasma was withdrawn from each supernatant. To each plasma sample, 300 mL of methanol:acetonitrile solution (1;1, V/V) was added. After mixing for 5 min by using a vortex mixer and centrifugation at 12 000 rpm for 10 min, the organic solvent layer from each plasma sample was collected and dried under gentle nitrogen gas stream at room temperature. The residues were kept separately at 20  C until analysis. The VCR concentrations in rat plasma were determined using the same HPLC methods as described in Physicochemical characterization of nanoparticles section. The analytical methods were validated, and the linearity range was 41.4–1380.0 ng/mL (r40.9900 in plasma and all tissue homogenates), and LOD was 13.7 ng/mL. Before analysis, each residue was reconstituted with 100 mL of mobile phase. Compartmental pharmacokinetic analysis was performed using Practical Pharmacokinetic Program 3P97 (Mathpharmacology Committee, Chinese Academy of

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Pharmacology, Beijing, China). The pharmacokinetic parameters were calculated according to the plasma drug concentration–time curves. The area under the plasma concentration–time curve (AUC) was calculated using the trapezoid method. The clearance (CL) was calculated by dividing the dose by AUC and adjusting for body weight. To investigate the biodistribution of the VCR-PBCA-NPs and F127-VCR-PBCA-NPs, 90 male SD rats were randomly divided into three groups and treated as described in the pharmacokinetic study. At 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after the administration, rats were sacrificed (n ¼ 3 for each time point per group). Heart, liver, spleen, lung, kidney, and lymph nodes (cervical, axillary, and mesenteric lymph nodes) from each rat were removed, cut into small pieces and diluted with saline (4 mL of saline per gram), separately. After homogenization and centrifugation at 12 000 rpm for 10 min at 4  C, 150 mL of supernatant was withdrew from each organ homogenate sample and processed following the same procedures of plasma sample preparation as mentioned above. Similarly, the VCR levels in different rat organs and tissues were determined by using the same HPLC method mentioned above. Targeting efficiency (TeC ) was used to express the targeting effect of VCR nanoparticles for the target organs [28], and calculated using the following equations: AUC01 TeC ¼ P ðAUC01 Þ where AUC0–1 is the AUC of VCR in the target tissues, and is the AUC of VCR in non-target tissues. Antitumor efficacy evaluation The human Burkitt’s lymphoma (Raji) model in nude mice was established as reported previously with slight modification [29]. Briefly, 0.2 mL of Raji cells suspension (2.57  107 cells/mL) was inoculated subcutaneously on the left axilla of a 5-week-old nude BALB/c mouse. After inoculation, the mice were maintained at standard conditions, and tumor volumes were calculated using the formula: (length  width2)  0.5, until the mean volume of the tumors is around 100 mm3. Twenty tumor-bearing mice were randomly divided into three treatment groups and one control group (n ¼ 5 for each group). The suspensions of VCR-PBCA-NPs, F127-VCRPBCA-NPs, or the saline solution of free VCR were injected to mice in the corresponding treatment groups via tail vein every 3 d in consecutive 9 d, at a dosage of 1 mg/kg. Similarly, 0.2 mL of normal saline was injected to rats in the control group following the same dosage regimen. The tumor volume and weight were measured twice a week in consecutive 3 weeks. The antitumor efficiencies of VCR-loaded nanoparticles were compared with free VCR by using the relative tumor volume (RTV), the relative proliferation rate of the tumor (T/C), and tumor inhibition rate (TIR) as indices [30,31]. RTV, T/C, and TIR were calculated by using the following equations:   Vt RTV ¼  100% V0

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Figure 1. Transmission electron micrographs of VCR-PBCA-NPs (A) and F127-VCR-PBCA-NPs (B) (80 000).

where Vt is the tumor volume calculated at each measurement time point, and V0 is the tumor volume prior to the treatment   TRTV T=C ¼  100% CRTV where TRTV and CRTV are the mean RTVs of the treatment and control groups, respectively TIR ¼ ðWc  Wt Þ=Wc  100% where Wc and Wt are the mean tumor weights of the treatment and control groups, respectively. After the experiment, surviving mice were sacrificed, and the tumors were harvested, weighed, and photographed, separately. To evaluate the changes in cell viability and morphology, paraffin-embedded formalin fixed tumor sections were stained with hematoxylin and eosin (H & E). The slices were observed under a microscope and pictures were taken. Statistical analysis Data were expressed as the mean ± standard error, unless otherwise stated. The results were examined for statistically significant differences by using a one-way analysis of variance (ANOVA), considering p50.05 as statistically significant.

Results and discussion Nanoparticle characterization The prepared VCR-loaded nanoparticles with or without surface modification exhibited similar physical characteristics. As indicated by the transmission electron micrographs (Figure 1), both VCR-PBCA-NPs and F127-VCR-PBCA-NPs were almost spherical in shape. A discernable outer lamella, supposed to be the surface coating layer, was observed from the micrograph of F127-VCR-PBCA-NPs. The mean particle sizes of VCR-PBCA-NPs and F127-VCR-PBCA-NPs were 98.9 ± 3.05 nm and 115.6 ± 2.24 nm, respectively, separately, indicating a slight size increase after the surface coating. The entrapment efficiencies (EE) and loadings of VCR in

Figure 2. In vitro release of VCR over time from the VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs.

VCR-PBCA-NPs and F127-VCR-PBCA-NPs were 55.23 ± 0.96 %, 57.58 ± 0.77%, 7.87 ± 0.11% and 7.24 ± 0.15%, suggesting no obvious change after surface modification. In vitro VCR release In vitro VCR release behaviors from VCR-PBCA-NPs and F127-VCR-PBCA-NPs were evaluated and compared with VCR solution. As shown in Figure 2, the release of VCR from both kinds of nanoparticles was biphasic and much slower than VCR solution. The initial rapid increase phase in the release profiles of VCR-PBCA-NPs and F127-VCR-PBCANPs might be attributed to the fast release of surface absorbed and poorly entrapped VCR, and the following slow increase phase might be owing to the sustain release of VCR following the gradual disintegration of nanoparticles and/or slow diffusion of VCR through the polymer matrix [32]. The release of VCR from F127-VCR-PBCA-NPs was even slower than VCR-PBCA-NPs. The possible explanation was that Pluronic F127 further slowed down the diffusion of VCR to the outside medium. The release kinetics model fitting of VCR-loaded nanoparticles is listed in Table 1. The release behaviors of VCR from VCR-PBCA-NPs and F127-VCR-PBCA-NPs fitted

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Table 1. Release kinetics of free VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs.

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Formulation

Kinetic model

Regression equation

r

VCR-PBCA-NPs

First order Higuchi Weibull Ritger–Peppas Hixson–Crowell Baker–Lonsdale

ln(1  Q) ¼ 0.1278t  0.2338 Q ¼ 0.2347t1/2 + 0.0202 ln ln(1/(1  Q)) ¼ 0.9321 ln t  1.5157 ln Q ¼ 0.6791 ln t  1.6867 Q1/3 ¼ 0.0221t + 0.6131 1.5[1  (1  Q)2/3]  Q ¼ 0.0156t + 0.0140

0.9537 0.9426 0.9900 0.9626 0.7604 0.9505

F127-VCR-PBCA-NPs

First order Higuchi Weibull Ritger–Peppas Hixson–Crowell Baker–Lonsdale

ln(1  Q) ¼ 0.0695t  0.2386 Q ¼ 0.1999t1/2 + 0.0105 ln ln(1/(1  Q)) ¼ 0.0780 ln t  0.7447 ln Q ¼ 0.2519 ln t  0.6255 Q1/3 ¼ 0.0316t + 0.4296 1.5[1  (1  Q)2/3]  Q ¼ 0.0084t + 0.0130

0.8697 0.9282 0.4566 0.1608 0.6024 0.8934

Figure 3. VCR uptake into Raji cells after incubation with VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs (n ¼ 3).

well to the Weibull model and the Higuchi model, a diffusioncontrolled release model, respectively [33].

Figure 4. Mean VCR concentrations in rat plasma versus time profiles after a single dose (1 mg/kg) intravenous administration of VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs, respectively (n ¼ 5).

Cellular uptake of VCR from the VCR-PBCA-NPs and F127-VCR-PBCA-NPs After incubation with VCR-PBCA-NPs, F127-VCR-PBCANPs, or VCR solution for 4, 8, and 12 h, the VCR uptake amounts into Raji cells were measured and are shown in Figure 3. At 4 h, after the incubation, both kinds of VCRloaded nanoparticles showed significantly higher VCR delivery efficiencies (p50.05) than VCR solution, and the highest mean VCR uptake amount was observed for F127VCR-PBCA-NPs. However, at 8 and 12 h after the incubation, a drastic drop of VCR uptake amounts was seen for both kinds of VCR-loaded nanoparticles and VCR solution, which might be as a result of apoptosis and necrosis of the cells by uptaking VCR. Pharmacokinetics The mean VCR concentrations in rat plasma versus time profiles after the single dose intravenous administration of VCR-PBCA-NPs, F127-VCR-PBCA-NPs, and VCR solution are shown in Figure 4, and the corresponding pharmacokinetic parameters are listed in Table 2. As shown in Figure 4, the retention time of VCR in rat plasma was obviously prolonged for both kinds of VCR-loaded nanoparticles than VCR solution, and these changes in VCR pharmacokinetic properties were further confirmed by pharmacokinetic model fitting and comparison of pharmacokinetic parameters. The VCR concentration data in rat plasma following the

Table 2. Pharmacokinetic parameters of VCR in rat plasma after single dose (1 mg/kg) intravenous administration of VCR solution, VCRPBCA-NPs, and F127-VCR-PBCA-NPs, respectively (n ¼ 6).

Parameter

VCR solution

VCR-PBCANPs

F127-VCRPBCA-NPs

V (mg mL kg1 mg1) 0.14 ± 0.05 0.35 ± 0.06* 0.36 ± 0.13* AUC (h mg mL1) 0.72 ± 0.18 5.53 ± 1.32** 8.50 ± 1.79** 1 1 1 CL (mg mL kg h mg ) 1.65 ± 0.56 0.22 ± 0.03** 0.14 ± 0.01** * and ** represent p50.05 and p50.01, compared with the free VCR solution.

administration of both kinds of VCR-loaded nanoparticles fitted well to the three-compartment open pharmacokinetic model, whereas those data for VCR solution fitted well to the two-compartment model. The very significant lower (p50.01) CL of VCR were observed for both VCR-PBCANPs (0.22 ± 0.03 mgmL kg1 h1 mg1) and F127-VCRPBCA-NPs (0.14 ± 0.01 mgmL kg1 h1 mg1) than VCR solution (1.65 ± 0.56 mgmL kg1 h1 mg1), and the lowest CLs of VCR was seen for F127-VCR-PBCA-NPs, indicating prolonged retention time of VCR in rat plasma might be achieved by using nanoparticles as vehicles, especially after surface modification with hydrophilic polymers. These results were consistent with those reported previously [34]. The very significant higher AUC for both VCR-PBCA-NPs

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Table 3. Tissue distribution characteristics of VCR in rats after single dose (1 mg/kg) intravenous administration of VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs. TeC (%) VCR solution VCR-PBCA-NPs F127-VCR-PBCA-NPs

Heart

Liver

Spleen

Lung

Kidney

Lymph nodes

2.71 ± 0.21 2.39 ± 0.33 1.88 ± 0.17

8.66 ± 1.55 3.59 ± 0.52* 3.54 ± 0.47*

54.91 ± 8.23 58.21 ± 3.26 20.89 ± 5.40**

17.37 ± 1.84 9.22 ± 1.98 12.72 ± 2.65

9.99 ± 1.22 5.22 ± 1.59 7.90 ± 2.63

6.36 ± 1.45 21.38* ± 7.25 53.08** ± 11.07

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* and ** represent p50.05 and p50.01, compared with the free VCR solution.

(5.53 ± 1.32 (h mg mL1) and F127-VCR-PBCA-NPs (8.50 ± 1.79 h mg mL1) than VCR solution (0.72 ± 0.18 h mg mL1) were obtained, and the highest AUC was seen for F127-VCRPBCA-NPs. The partial explanation might be the obvious change of CLs. Similarly, significant higher apparent volumes of distribution (V) were seen for both VCR-PBCA-NPs (0.35 ± 0.06 mgmL kg1 mg1) and F127-VCR-PBCA-NPs (0.36 ± 0.13 mgmL kg1 mg1) than VCR solution (0.14 ± 0.05 mgmL kg1 mg1), suggesting the possible change in tissue distribution behavior following the administration of VCR-loaded nanoparticles. Biodistribution Targeting efficiency (TeC ) was used as an index for evaluating the tissue distribution properties of VCR, especially the lymphatic targeting efficiency, following the intravenous administration of VCR-loaded nanoparticles and VCR solution to rats. As seen in Table 3, significant (p50.05) and very significant (p50.01) improvement in lymphatic targeting efficiency of VCR was achieved by VCR-PBCA-NPs and F127-VCR-PBCA-NPs, respectively, compared with the VCR solution. These results were consistent with those reported previously, claiming higher lymphatic targeting efficiencies were achieved by the drug-loaded nanoparticles than the drug solution [35]. The highest lymphatic targeting efficiency was observed for F127-VCR-PBCA-NPs, and a decrease of VCR distribution into reticuloendothelial system (RES), especially the spleen, was seen for F127-VCR-PBCA-NPs than VCRPBCA-NPs. These results were in accordance with those reported previously. The possible explanation was the inverse targeting effect of RES-riched organs achieved when the nanocarrier containing the poloxamer 407(Pluronic F127) was employed as a vehicle for intravenous drug delivery [35]. The in vivo behaviors of the nanocarriers might be influenced obviously by the properties of the poloxamers. The length of the EO chain was shown to control the particle–macrophage interaction. The strong steric barrier of poloxamer 407, which arises from 98 EO units, suppressing particle opsonisation in lymph and/or interaction with macrophage receptors, while particles coated with poloxamer containing 4-15 EO units per chain (e.g. Poloxamer 401 and 402) are more liable to be captured by macrophages [36]. Therapeutic efficacy The antitumor efficacies of VCR-loaded nanoparticles to Human Burkitt’s lymphoma (Raji)-bearing nude mice were compared with the VCR solution, and the normal saline was used as a blank control. After multiple doses administration and an observation period, the tumor-bearing mice were sacrificed and the tumor was harvested. The mice and

Figure 5. Human Burkitt’s lymphoma (Raji) tumor bearing nude mice treated intravenously with VCR solution, VCR-PBCA-NPs, and F127VCR-PBCA-NPs (1 mg/kg) every 3 d in consecutive 9 d with normal saline as a control and sacrificed 21 d after the first treatment.

Figure 6. Human Burkitt’s lymphoma (Raji) tumors harvested from nude mice treated intravenously with VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs (1 mg/kg) every 3 d in consecutive 9 d with normal saline as a control and sacrificed 21 d after the first treatment.

harvested tumors were shown in Figures 5 and 6, respectively. As seen in Figures 5 and 6, tumors grew rapidly in mice given normal saline, while obvious tumor growth inhibition effect was observed in mice treated with VCR-loaded nanoparticles and VCR solution. The highest tumor growth inhibiting efficiency, indicated by lowest tumor volumes, was observed in mice treated with F127-VCR-PBCA-NPs. RTV, T/C, and TIR were employed as indices for further evaluating the antitumor efficacies of VCR-PBCA-NPs,

Preparation of VCR-PBCA-NPs modified with pluronic F127

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DOI: 10.3109/1061186X.2014.897708

F127-VCR-PBCA-NPs, and free VCR solution. As shown in Table 4, both kinds of VCR-loaded nanoparticles and VCR solution exhibited obvious tumor growth inhibition effect, indicated by significantly increased TIC, decreased RTV and T/C (p50.05), compared with the normal solution. The lowest RTV (229.61 ± 30.79%), T/C (12.15%), and highest TIC (88.79%) were observed for F127-VCR-PBCA-NPs, suggesting its highest antitumor efficiency. The possible explanation was the prolonged retention time of VCR in systemic circulation achieved by surface coating the nanoparticles with Pluronic F127, and the enhanced permeability and retention (EPR) effect of solid tumors to nanoscaled carriers. To provide further evidence for supporting the obviously improved therapeutic efficacy of VCR-loaded nanoparticles than VCR solution, the harvested tumors were sliced Table 4. Antitumor efficacy evaluation of VCR-PBCA-NPs, F127-VCRPBCA-NPs, and free VCR in comparison with normal saline after treating human Burkitt’s lymphoma (Raji) tumor bearing nude mice intravenously at a dose of 1 mg/kg every 3d in consecutive 9d with normal saline as a control and sacrificed 21d after the first treatment (n ¼ 5). Treatment/control VCR solution VCR-PBCA-NPs F127-VCR-PBCA-NPs Normal saline

RTV (%)

T/C (%)

TIC (%)

698.66 ± 120.75* 397.87 ± 70.20* 229.61 ± 30.79* 1889.97 ± 115.01

36.97* 21.05* 12.15* 100

67.26* 81.91* 88.79* 0

RTV, relative tumor volume; T/C, relative proliferation rate of the tumor; TIC, tumor inhibition rate; *p 50.05, compared with the normal saline.

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and stained with H & E. The representative slices are shown in Figure 7. In contrast to normal saline, the tumor cell death was observed after treating the tumor-bearing nude mice with VCR-loaded nanoparticles and VCR solution, and severe tumor cell necrosis was achieved by treating the tumor-bearing nude mice with F127VCR-PBCA-NPs. There are several reports about vincristine-loaded nanocarriers for tumor targeting, such as liposomes [37,38], liposome-templated calcium phosphate nanoshell [39], vesicular phospholipid gels [40], microemulsions [41], and nanoparticles [42–44]. VCR liposome injection has been approved by the US Food and Drug Administration for treatment of relapsed and clinically advanced Philadelphia chromosome-negative acute lymphoblastic leukemia. Our research indicated that the VCR-loaded poly (butylcyanoacrylate) nanoparticles modified with pluronic F127 is a potential delivery system for lymphatic targeting, and deserves further studies.

Conclusion In the present study, VCR-loaded nanoparticles exhibited obviously higher lymphatic targeting efficiencies than the VCR solution in experiments in vitro and in vivo, especially after surface modification with Pluronic F 127. Surfacemodified nanosacle carriers might be promising vehicles for improving the lymphatic targeting efficiencies and subsequent antitumor effect of chemotherapeutic agents to metastatic tumors and malignant lymphoma.

Figure 7. Representative hematoxylin and eosin (H & E) stained tumor slices from mice treated intravenously with VCR solution, VCR-PBCA-NPs, and F127-VCR-PBCA-NPs (1 mg/kg) every 3 d in consecutive 9 d with normal saline as a control and sacrificed 21 d after the first treatment.

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Acknowledgements We thank Mr. Qiangli Wang (Department of Histology and Embryology, Shanghai University of Traditional Chinese Medicine, Shanghai, China) for assistance with the cell culture experiments. Special thanks are due to Professor Kinam Park, Purdue University for the constructive suggestions.

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Declaration of interest

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The authors report no conflicts of interest in this work. This work was financially supported by program (10XD14303900) from the Science and Technology Commission of Shanghai Municipality, and projects (NCET08-0898) from the State Education Ministry of the PR China.

References 1. Nune SK, Gunda P, Majeti BK, et al. Advances in lymphatic imaging and drug delivery. Adv Drug Deliv Rev 2001;63:876–85. 2. Laakkonen P, Porkka K, Hoffman JA, Ruoslahti E. A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat Med 2002;8:751–5. 3. Apostolidou E, Swords R, Alvarado Y, Giles FG. Treatment of acute lymphoblastic leukaemia: a new era. Drugs 2007;67:2153–71. 4. Pulte D, Gondos A, Brenner H. Trends in survival after diagnosis with hematologic malignancy in adolescence or young adulthood in the United States, 1981–2005. Cancer 2009;115:4973–9. 5. Fielding AK, Richards SM, Chopra R, et al. Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood 2007;109:944–50. 6. Yang F, Fu DL, Long J, Ni QX. Magnetic lymphatic targeting drug delivery system using carbon nanotubes. Med Hypotheses 2008;70: 765–7. 7. Fujiwara K, Watanabe T. Effects of hyperthermia, radiotherapy and thermoradiotherapy on tumor microvascular permeability. Acta Pathol Jpn 1990;40:79–84. 8. Chen J, Wang L, Yao Q, et al. Drug concentrations in axillary lymph nodes after lymphatic chemotherapy on patients with breast cancer. Breast Cancer Res 2004;6:474–7. 9. Narvuzov SN, Zkbarov ET, Abduzhabborov SB, et al. Preliminary results of lymphatic chemotherapy in the treatment of rectal cancer. Klin Khir 2004;3:38–40. 10. Reddy GR, Bhojani MS, McConville P, et al. Vascular targeted nanoparticles for imaging and treatment of brain tumors. Clin Cancer Res 2006;12:6677–86. 11. Thomas PH, Joseph GA. Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature. Int J Pharm 2011;411:206–14. 12. Reddy ST, Rehor A, Schmoekel HG, et al. In vivo targeting of dendritic cells in lymph nodes with poly (propylene sulfide) nanoparticles. J Control Release 2006;112:26–34. 13. Rowinsky E, Donhower RC. Antimicrotubule agents. In: Chabner BA, Longo DL, eds. Cancer chemotherapy and biotherapy. Philadelphia: Lippincott-Raven Publishers; 1996:263–75. 14. Barthomeuf C, Grassi J, Demeule M, et al. Inhibition of P-glycoprotein transport function and reversion of MDR1 multidrug resistance by cnidiadin. Cancer Chemother Pharmacol 2005; 56:173–81. 15. Borst P, Zelcer N, van de Wetering K, Poolman B. On the putative co-transport of drugs by multidrug resistance proteins. FEBS Lett 2006;580:1085–93. 16. Marinina J, Shenderova A, Mallery SR, Schwendeman SP. Stabilization of vinca alkaloids encapsulated in poly(lactideco-glycolide) microspheres. Pharm Res 2000;17:677–83. 17. Ariasa JL, Linares-Molineroa F, Gallardoa V, Delgadob AV. Study of carbonyl iron/poly(butylcyanoacrylate) (core/shell) particles as anticancer drug delivery systems Loading and release properties. Eur J Pharm Sci 2008;33:252–61. 18. Soma CE, Dubernet C, Bentolila D, et al. Reversion of multidrug resistance by co-encapsulation of doxorubicin and

22. 23.

24.

25.

26.

27.

28. 29. 30. 31.

32.

33. 34.

35. 36.

37. 38.

cyclosporin A in polyalkylcyanoacrylate nanoparticles. Biomaterials 2000;21:1–7. Arias JL, Gallardo V, Ruiz MA, Delgado AV. Magnetite/poly (alkylcyanoacrylate) (core/shell) nanoparticles as 5-Fluorouracil delivery systems for active targeting. Eur J Pharm Biopharm 2008; 69:54–63. Arias JL, Gallardo V, Ruiz MA, Delgado AV. Ftorafur loading and controlled release from poly(ethyl-2-cyanoacrylate) and poly(butylcyanoacrylate) nanospheres. Int J Pharm 2007;337: 282–90. Vauthier C, Dubernet C, Chauvierre C, et al. Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles. J Control Release 2003;93:151–60. Nemati F, Dubernet C, Fessi H, et al. Reversion of multidrug resistance using nanoparticles in vitro: influence of the nature of the polymer. Int J Pharm 1996;138:237–46. Ambruosi A, Khalansky AS, Yamamoto H, et al. Biodistribution of polysorbate 80-coated doxorubicin-loaded [14C]-poly(butyl cyanoacrylate) nanoparticles after intravenous administration to glioblastoma-bearing rats. J Drug Target 2006;14:97–105. Friesea A, Seillera E, Quacka G, et al. Increase of the duration of the anticonvulsive activity of a novel NMDA receptor antagonist using poly(butylcyanoacrylate) nanoparticles as a parenteral controlled release system. Eur J Pharm Biopharm 2000;49: 103–9. Zhang W, Shi Y, Chen Y, et al. Enhanced antitumor efficacy by Paclitaxel-loaded Pluronic P123/F127 mixed micelles against nonsmall cell lung cancer based on passive tumor targeting and modulation of drug resistance. Eur J Pharm Biopharm 2010;75: 341–53. Tan R, Liu Y, Feng N, Zhao J. Preparation and in vitro release characteristics of vincristine sulphate loaded poly (butylcyanoacrylate) nanoparticles. Zhongguo Zhong Yao Za Zhi (in Chinese) 2011;36:1431–5. Ling G, Zhang P, Zhang W, et al. Development of novel selfassembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulfate by P-gp inhibition. J Control Release 2010;148:241–8. Na JH, Lee SY, Lee S, et al. Effect of the stability and deformability of self-assembled glycol chitosan nanoparticles on tumor-targeting efficiency. J Control Release 2012;163:2–9. Gu J, Song ZP, Gui DM, et al. Resveratrol attenuates doxorubicininduced cardiomyocyte apoptosis in lymphoma nude mice by heme oxygenase-1 induction. Cardiovasc Toxicol 2012;12:341–9. Salgado Oloris SC, Dagli NL, Guerra JL. Effect of b-carotene on the development of the solid Ehrlichtumor in mice. Life Sci 2002; 71:717–24. Kim WJ, Yockman JW, Jeong JH, et al. Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMVsFlt-1 complexes in tumor-bearing mice. J Control Release 2006; 114:381–8. Page ME, Pinto-Alphandary H, Chachaty E, et al. Entrapment of colistin into polyhexylcyanoacrylate nanoparticles: preparation, drug release and tissue distribution in mice. STP Pharm Sci 1996;6: 298–301. Costa P, Lobo JMS. Modeling and comparison of dissolution profiles. Eur J Pharm Sci 2001;13:123–33. Hee J Lee, Byung-N Ahn, Woo H Paik, et al. Inverse targeting of reticuloendothelial system-rich organs after intravenous administration of adriamycin-loaded neutral proliposomes containing poloxamer 407 to rats. Int J Pharm 1996;131:91–6. Shin SB, Cho HY, Kim DD, et al. Preparation and evaluation of tacrolimus-loaded nanoparticles for lymphatic delivery. Eur J Pharm Biopharm 2010;74:164–71. Moghimi SM. Modulation of lymphatic distribution of subcutaneously injected poloxamer 407-coated nanospheres: the effect of the ethylene oxide chain configuration. FEBS Lett 2003; 540:241–4. Zhang L, Gao H, Chen L, et al. Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells. Cancer Lett 2008;269:26–36. Davis T, Farag SS. Treating relapsed or refractory Philadelphia chromosome-negative acute lymphoblastic leukemia: liposome-encapsulated vincristine. Int J Nanomedicine 2013;8: 3479–88.

DOI: 10.3109/1061186X.2014.897708

Journal of Drug Targeting Downloaded from informahealthcare.com by University of Windsor on 07/14/14 For personal use only.

39. Thakkar HP, Baser AK, Parmar MP, et al. Vincristine-sulphateloaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system. J Liposome Res 2012;22: 139–47. 40. Gu¨thlein F, Burger AM, Brandl M, et al. Pharmacokinetics and antitumor activity of vincristine entrapped in vesicular phospholipid gels. Anticancer Drugs 2002;13:797–805. 41. Junping W, Takayama K, Nagai T, Maitani Y. Pharmacokinetics and antitumor effects of vincristine carried by microemulsions composed of PEG-lipid, oleic acid, vitamin E and cholesterol. Int J Pharm 2003;251:13–21.

Preparation of VCR-PBCA-NPs modified with pluronic F127

517

42. Wang C, Zhao M, Liu YR, et al. Suppression of colorectal cancer subcutaneous xenograft and experimental lung metastasis using nanoparticle-mediated drug delivery to tumor neovasculature. Biomaterials 2014;35:1215–26. 43. Chen J, Li S, Shen Q. Folic acid and cell-penetrating peptide conjugated PLGA-PEG bifunctional nanoparticles for vincristine sulfate delivery. Eur J Pharm Sci 2012;47:430–43. 44. Zhang P, Ling G, Sun J, et al. Multifunctional nanoassemblies for vincristine sulfate delivery to overcome multidrug resistance by escaping P-glycoprotein mediated efflux. Biomaterials 2011;32: 5524–33.

Preparation of vincristine sulfate-loaded poly (butylcyanoacrylate) nanoparticles modified with pluronic F127 and evaluation of their lymphatic tissue targeting.

In order to improve the lymphatic targeting efficiency of anti-cancer agent vincristine sulfate (VCR), the poly (butylcyanoacrylate) nanoparticles (VC...
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