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Solid tumor penetration by integrin-mediated pegylated poly(trimethylene carbonate) nanoparticles loaded with paclitaxel
Biomaterials 34 (2013) 1739e1746
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Solid tumor penetration by integrin-mediated pegylated poly(trimethylene carbonate) nanoparticles loaded with paclitaxel Xinyi Jiang a, b, Hongliang Xin a, c, Jijin Gu a, Ximing Xu b, Weiyi Xia a, Shuo Chen a, Yike Xie a, Liangcen Chen a, Yanzuo Chen a, Xianyi Sha a, Xiaoling Fang a, * a b c
Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, China Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang 212013, China Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 210029, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2012 Accepted 10 November 2012 Available online 2 December 2012
Limited penetration of antineoplastic agents is one of the contributing factors for chemotherapy failure of many solid tumors. In order to enhance drug penetration into solid cancer, especially, into the avascular regions inside tumors, we proposed cyclic RGD peptide functionalized PEGylated poly(trimethylene carbonate) nanoparticles (c(RGDyK)-NP). By integrin-mediated transcytosis and enhanced drug permeation, c(RGDyK)-NP could access the neoplastic cells distant from blood vessels, and consequently, avoiding the capability of cancer regeneration from these tumor cells. In the present study, the solid tumor penetration, homing specificity and anticancer efficacy were evaluated both on the ex vivo 3D tumor spheroids and on the subcutaneous xenograft mice model. In comparison with conventional nanoparticles (NP/PTX) and Taxol, c(RGDyK)-NP/PTX showed the strongest penetration and accumulation into 3D tumor spheroids, a marked tumor-homing specificity in vivo and the greatest tumor growth inhibitory effect in vitro and in vivo. Histochemistry analysis revealed that no obvious histopathological abnormalities or lesions were observed in major organs after intravenous administration with the treatment doses. In conclusion, cyclic RGD peptide-conjugated PEG-PTMC nanoparticle could facilitate drug penetration and accumulation in tumor tissues and may be a promising vehicle for enhancing the chemotherapy of solid cancers. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Solid cancer Tumor spheroids Integrin-mediated delivery Tumor penetration In vivo toxicity
1. Introduction Chemotherapeutic effect of solid tumors is often compromised due to several physiologic barriers of these cancers, including heterogeneous tumor perfusion and vascular permeability, high cell density, acidic pH, irregular blood flow, increased interstitial pressure and resultant hypoxia, which significantly limit the penetration of anticancer drugs into neoplastic cells distant from blood vessels [1e4]. Consequently, these anti-tumor agents are unable to access all of the cells within a tumor that are capable of regenerating it (that is, clonogenic cells or tumor stem cells), and then whatever their mode of action or potency, the effectiveness of chemotherapy will be compromised [1,5]. For a treatment to be effective, it should access the entire tumor, especially, the inside avascular regions [6e9]. Surface-engineered nanoplatform by cancer-specific targeting moieties (e.g., receptorbinding ligands or antibodies) is proffered as a promising strategy
* Corresponding author. Tel.: þ86 21 51980071; fax: þ86 21 51980072. E-mail address: [email protected] (X. Fang). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.11.016
for enhancing the drug delivery into solid tumors and lessening the non-special accumulation in MPS-related organs such as the liver and the spleen, and thus increasing the therapeutic index [10e13]. Previously, we have developed a cyclic RGD peptide-functionalized poly(trimethylene carbonate)-based micellar nanoparticulate system (c(RGDyK)-NP) which has been proved to be able to target integrin-rich malignant glioma cells [14]. In fact, extracellular matrixeintegrin receptors are also found to be overexpressed on most tumor cells and sprouting tumor vessels as compared to normal organs [15,16]. By integrin-mediated transcytosis and EPR effect, RGD sequence-based peptide functionalized nanosystem could actively accumulate to the neovascular region of the tumor after systemic administration [10,17,18]. However, for solid cancer, there are hypoxic and necrotic regions distant from the vascular bed, and these chemotherapy “blind areas” ineluctably lead to the relapse of cancer. It is postulated that enhancement in the ability of the delivery system to penetrate deeper into the avascular tumor tissues can significantly reduce the tumor regrowth and augment the therapeutic benefit of the treatment [2e4,19,20]. With this in mind, the solid tumor penetration capability of c(RGDyK)-NP was further investigated in the present study.
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It is critical for the targeting nanosystem to fulfill the anticipated functions without bringing any potential toxicity. Poly(trimethylene carbonate) (PTMC), one important type of aliphatic polycarbonates, has been widely used in biomedical field due to their tunable biodegradability and excellent biocompatibility [21e 23]. Some of the PTMC copolymers have been commercialized and used for clinical application approved by FDA. Our group and other researchers have described the synthesis and self-assembly behavior of di- or tri-block copolymer systems based on PEG and PTMC with a linear or star-shaped structure [24e26]. The reported copolymers have been demonstrated to be effective carriers for the sustained release of hydrophobic drugs [11,25,27,28]. Cyclic RGD peptide conjugation endowed nanostructures with tumor-homing ability. However, the changed biodistribution behavior of the nanosystem maybe induce some potential toxicities post systemic administration. Herein, the penetration, distribution, and accumulation of c(RGDyK)-NP into the avascular solid tumor were evaluated by ex vivo 3D tumor spheroids model. The in vivo tumorhoming capability and anticancer efficacy of c(RGDyK)-NP/PTX were investigated by subcutaneous xenograft mice model. In vivo toxicity was also assessed by histochemistry analysis after intravenous administration with the treatment doses.
2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Xi’an San jiang Bio-Engineering Co. Ltd. (Xi’an, China). Methoxyl poly (ethylene glycol)-co-poly (trimethylene carbonate) (MPEG3K-PTMC6K) and c(RGDyK) modified poly (ethylene glycol)-b-poly (trimethylene carbonate) (c(RGDyK)-PEG3.5K-PTMC6K) were synthesized as described previously [14]. Low melting-point agarose was obtained from Yixin Biotechnology Co., Ltd. (Shanghai, China). Coumarin 6 was purchased from Sigma (St Louis, MO, USA). 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindotricarbocyanine iodide (DiR) was obtained from Biotium (Invitrogen, USA). Cellulose ester membranes (dialysis bag) with a molecular weight cut off value (MWCO) of 3500 (Greenbird Inc. Shanghai, China) were used in dialysis experiments. Penicillinestreptomycin, DMEM, fetal bovine serum (FBS) and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). Purified deionized water was prepared by the Milli-Q plus system (Millipore Co., Billerica, MA, USA). All of other reagents and chemicals were analytical grade and were used without further purification. U87MG cells were obtained from Shanghai Institute of Cell Biology. It was cultured in special Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate. Female BALB/c mice (20 2 g), supplied by Department of Experimental Animals, Fudan University (Shanghai, China), were acclimated at 25 C and 55% of humidity under natural light/dark conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of the College of Pharmacy, Fudan University (Shanghai, China).
Fig. 1. Schematic representation of self-assembled micellar-like nanoparticles functioned with cyclic RGD peptide (c(RGDyK)-NP/PTX) (A); A TEM image of c(RGDyK) modified nanoparticles (B); Particle size and size distribution determined by DLS using a Malvern Nano ZS (Malvern Instruments, UK). Inset: a photo of a nanoparticle solution at the concentration of 20 mg/ml (C); Apparent zeta potential spectrum of c(RGDyK)-NP/PTX in buffered 1 mM NaCl solutions (D).
X. Jiang et al. / Biomaterials 34 (2013) 1739e1746 2.2. Preparation and characterization of drug-loaded c(RGDyK)-NP The c(RGDyK)-conjugated PTX-loaded NP was prepared according to the procedure described previously [14]. Particle mean size, size distribution and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) using a zeta plus analyzer (Zeta-sizer, Malvern Nano ZS, U.K.). The shape and morphology of the nanoparticles were observed using transmission electron microscopy (TEM) (JEOL JMPEG-PTMC-1230, Japan). In the DLS assay, the nanoparticles were diluted in physiological water, and DLS measurements were performed at a 90 scattering angle at 37 C. Z-average sizes of three sequential measurements were collected and analyzed. In TEM observation, the nanoparticle sample was negatively stained with sodium phosphotungstatic solution (2%, w/v).
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PTX and c(RGDyK)-NP/PTX, were applied to the wells where tumor spheroids had been incubated for 7 days. The final concentration of PTX was 0.5 mg/ml. And tumor spheroids incubated in DMEM medium without any formulation were used as blank controls. After treatments, tumor spheroids were observed under an inverted microscope (Chongqing Optical & Electrical Instrument Co. Ltd., Chongqing, China) on days 0, 1, 3, 5 and 7. Growth inhibition was calculated with the following formula: V ¼ (p dmax dmin)/6, where dmax is the maximum diameter and dmin is the minimum diameter of each spheroid. The change ratio of tumor spheroid volume was calculated with the following formula: ratio% ¼ (Vdayi/Vday0) 100, where Vdayi is the tumor spheroid volume on the ith day (day 1, 3, 5, 7) after applying drug, and Vday0 is the tumor spheroid volume prior to treatment. 2.6. Morphology of tumor spheroids
2.3. In vitro 3D tumor spheroids formation For evaluating the effects of the solid tumor penetration of peptidefunctionalized nanosystem in vitro, an ex vitro multicellular 3D tumor spheroids mimicking the solid tumors in vivo were developed using the lipid overlay system as reported previously [6,29,30]. Agarose was heated at 80 C for 30 min, and diluted to 2% (w/v) with the serum-free DMEM medium. Each well of 24-cell culture plates was coated with a thin layer (300 ml) of sterilized agarose-based DMEM. Tumor cells were seeded into each well at the density of 1000 cells/well (in complete medium), gently agitated for 5 min, and incubated at 37 C for 7 days, and the culture medium was changed every 3 days. The uniform and compact multicellular spheroids were selected for the follow-up studies. 2.4. Confocal microscopy of tumor spheroids To evaluate the penetration ability, 3D tumor spheroids were used for penetration experiments. Briefly, tumor spheroids were incubated with coumarin 6 labeled conventional nanoparticles or peptide-conjugated nanoparticles at a final concentration of 300 mg/ml, respectively. At 12 h after treatment, spheroids were rinsed with PBS three times, transferred to a chambered coverslips and analyzed using an Olympus FV1000 confocal microscope (Olympus, Center Valley, PA). Z-stack images were obtained by scanning the tumor spheroid step by step. The scanning began from the top of a spheroid. Each scanning layer was 15 mm in thickness, and the total scanning was 90 mm in depth in a spheroid.
For evaluating the intuitive effects on 3D tumor spheroids after similarly applying Taxol, NP/PTX or c(RGDyK)-NP/PTX, the treated spheroids on day 0 or on day 3 were rinsed 3 times by PBS, fixed by 2.5% glutaraldehyde for 2 h at 4 C, further washed 3 times by 0.1 M PBS, then dehydrated and embedded. These tumor spheroid specimens were viewed with a field emission scanning electron microscope (FESEM, Carl Zeiss Ultra 55, Germany) at instrumental magnification. 2.7. In vivo Near-Infrared (NIR) Imaging In vivo real-time fluorescence imaging analysis was used to evaluate the effect of tumor distribution and accumulation ability of c(RGDyK)-modified nanoparticles in solid tumor-bearing mice [31]. The subcutaneous tumor xenograft model was established by inoculation of 5 106 U87MG cells (in 200 ml cell culture medium) into the subcutaneous tissue of the right hind legs. Both of c(RGDyK)-NP and NP were labeled by Dir (Invitrogen, USA). In brief, Dir was co-dissolved with copolymer in DCM during nanoparticle preparation. Then, the free Dir was removed via CL-4B column (Hanbang Chemical Co. LTD, China). When the size of tumors reached 0.7e 0.9 cm in diameter, the tumor-bearing mice were injected with Dir-labeled NP and c(RGDyK)-NP via tail vein at a dose of 20 mg DiR/mouse, respectively. At the time points of 30 min, 1 h and 5 h, the mice were anesthetized and visualized using Cambridge Research & Instrumentation in vivo imaging system (CRi, MA, USA). After 5 h post-injection, the mice were humanely killed, following which the tumor and principle organs (including heart, liver, spleen, lung and kidney) were removed and also visualized using CRi in vivo imaging system.
2.5. Growth inhibition of 3D tumor spheroids 2.8. Frozen section analysis of tumor tissues The influence of various treatments on the growth of tumor spheroids was also investigated in this study. For evaluating the inhibition of tumor growth, the serumfree DMEM media but containing different PTX formulations, including Taxol, NP/
The in vivo solid tumor penetration and tumor-homing specificity of c(RGDyK)NP were studied qualitatively by fluorescence microscopic observation of equatorial
Fig. 2. Representative confocal images of tumor spheroids 12 h after treatment with fluorescein-labeled NP and c(RGDyK)-NP. Bright field (BF) is shown in white and black and coumarin 6 fluorescence is displayed in green. Z-stack images were obtained starting at the top of the spheroid in 15 mm intervals for a total of 90 mm into the spheroid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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plane sections of tumor tissues. The subcutaneous tumor bearing mice model was established as described above. When the size of tumors reached 0.7e0.9 cm in diameter, coumarin 6 labeled c(RGDyK)-NP or NP was injected into the tail vein of mice at a dose of 100 mg/kg, respectively. Approximately 45 min later, the animals were anesthetized with diethyl ether. Then, they underwent ventricular perfusion with saline and 4% paraformaldehyde for 30 min, respectively. After that, the tumor tissues were harvested, fixed in 4% paraformaldehyde for 24 h, placed in 15% sucrose PBS solution for 24 h until subsidence, and then in 30% sucrose for 24 h until subsidence. Afterwards, the tumor was frozen in OCT embedding medium (Sakura, Torrance, CA, USA) at 80 C. Frozen sections of 20 mm thickness were prepared and stained with 300 nM DAPI for 10 min at room temperature. After washed twice with PBS (pH 7.4), the sections were immediately examined under the fluorescence microscope (Leica DMI 4000B, Germany). 2.9. In vivo anti-tumor efficacy and safety evaluation In vivo anticancer activity against subcutaneous tumor was evaluated in mice. The dose schedule started when the tumor volume was about 40e80 mm3. The mice were randomized into four groups (n ¼ 5) and treated with 100 ml of Taxol, c(RGDyK)NP/PTX, NP/PTX, and physiological saline via tail vein injection on the day 0, 2, 4 and 6 (PTX dosage:10 mg/kg body weight, Taxol was diluted by physiological saline). Tumor size was monitored via serial caliper measurement for every 2 days and the tumor volume was estimated using the formula: Volume ¼ 0.5 length (width)2. On the day 14, the animals were sacrificed by cervical dislocation, and the tumor mass was harvested, photographed, and then fixed with paraformaldehyde for 48 h and embedded in paraffin. Each section was cut into 5 mm, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under fluorescent microscope (Leica DMI 4000B, Germany). The potential in vivo toxicity is always a great concern for nanoparticulate system used in biomedicine. To study the toxic effects of the nanocarriers, major organs such as heart, lung, liver, spleen, and kidney tissue were fixed with paraformaldehyde for 48 h and embedded in paraffin. Each section was cut into 5 mm, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under a Leica microscope.
surface. The PTX loading content and encapsulation ratio of c(RGDyK)-NP/PTX were about 6.4% and 93%, respectively. 3.2. In vitro tumor spheroids penetration In many solid tumors, there are hypoxic and avascular tumor regions. Due to the poor permeation of delivery systems, the amount of drug accessing inside the solid tumors is low. As a consequence, these chemotherapy “blind areas” eventually and ineluctably induced the recurrence of cancer, and the overall chemotherapeutic efficacy of anticancer agents is compromised. For a cancer treatment to be curative, the delivery system must efficiently penetrate tumor tissue to reach all of the viable cells. The ex vivo 3D tumor spheroids generated by liquid overlay technique are not only aggregates of cells in close contact but contain an organized extracellular matrix consisting of fibronectin, laminin, collagen, and GAG suggestive of the extracellular matrix of tumors in vivo [1,6,32]. Thereby, 3D multicellular model represents the
3. Results and discussion 3.1. Preparation and characterization of the nanoparticles The drug-loaded coreeshell type nanoparticle functionalized with cyclic RGD peptide was schematically illustrated in Fig. 1A. The c(RGDyK)-NP/PTX exhibited a spherical shape under the examination of TEM (Fig. 1B) with a Z-average diameter about 73 nm determined by the dynamic light scattering analysis (Fig. 1C). c(RGDyK)-NP/PTX solution at the concentration of 20 mg/ml was shown in Fig. 1C inset. The solution exhibited a slight whitish opalescence. The zeta potential for c(RGDyK)-NP/PTX was about 6.7 mV (Fig. 1D). As shown in the TEM photo, a clear peptide-engineered PEG corona could be seen on the nanoparticle
Fig. 3. Change ratios of tumor spheroid volume (%) after applying various PTX formulations and blank control. (a) P < 0.01, versus primary spheroid volume; (b) P < 0.01, versus Taxol; and (g) P < 0.01, versus conventional nanoparticles. Data represent mean SD (n ¼ 5).
Fig. 4. Morphology of tumor spheroids observed by scanning electronic microscope (SEM). Key: Spheroids at day 3 after applying serum-free DMEM culture medium (A), Taxol (B), NP/PTX (C) and c(RGDyK)-NP/PTX (D), respectively. Final PTX for all was at 0.5 mg/ml.
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Fig. 5. (A) In vivo fluorescence imaging of tumor-bearing nude mice after injection of DiR-labeled NP (A1) and DiR-labeled c(RGDyk)-NP (A2) was administered via a lateral tail vein. All NIR fluorescence images were acquired with a 230ms exposure time at 30 min and at 1 and 5 h (h). Arrow: the position of the tumor. Fluorescence signal from DiR was pseudocolored red. Representative images of dissected organs of a mouse bearing subcutaneous tumor sacrificed 5 h post intravenous injection of DiR-labeled NP (B) and DiR-labeled c(RGDyk)-NP (C). Key: t, solid tumor; li, liver; sp, spleen; lu, lung; h, heart; k, kidney. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
avascular regions found in many solid tumor tissues and can serve as an invaluable tool to evaluate the solid tumor penetration effect of drug delivery system. In this study, interstitial penetration and diffusion of c(RGDyK)-NP into avascular regions of the solid tumors were evaluated using the ex vivo 3D tumor spheroids as a model. Fig. 2 shows confocal laser scanning microscopic images of 3D tumor spheroids 12 h after applying coumarin 6 labeled nanoparticles. For conventional nanoparticles, fluorescence was observed primarily on the periphery of tumor spheroids. However, after applying nanoparticles conjugated with c(RGDyK), the fluorescence was able to be observed throughout the whole tumor spheroids and the depth of fluorescence that could be observed in
the spheroids reached to 90 mm, suggesting that solid tumor penetration was enhanced by the presence of RGD targeting ligand. 3.3. Growth inhibitory effect on tumor spheroids The influence of various treatments on the growth of tumor spheroids was also studied. Fig. 3 represents the in vitro tumor spheroid volume ratios after treatment with Taxol, NP/PTX and c(RGDyK)-NP/PTX at the final PTX concentration of 0.5 mg/ml, respectively. It was observed that tumor spheroids continued to grow in size and volume in the absence of any drug (128.7% of the primary volume after 7 days). The obvious reduction in volume of
Fig. 6. The qualitative valuation of in vivo tumor penetration and tumor-homing specificity of coumarin 6 labeled nanoparticles. Distribution of nanoparticles in tumor tissue of subcutaneous xenografts bearing mice treated with coumarin 6 labeled NP (AeC) and coumarin 6 labeled c(RGDyK)-NP (DeF) 45 min after i.v. administration. Image C is the combination of A and B; image F is the combination of D and E. Frozen sections (20 mm of thickness) of solid tumor were examined by fluorescent microscopy. Green: coumarin 6 labeled nanoparticles. Blue: cell nuclei. Images were obtained under Leica fluorescence microscope with the original magnification 100. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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tumor spheroids was observed for all PTX formulations after 7 days treatment, indicating that tumor spheroids were sensitive to PTX. The change ratios of tumor spheroid volumes (%) on day 7 were 87.2%, 74.3% and 31.7% for Taxol, NP/PTX and c(RGDyK)-NP/PTX, respectively. The result indicated that c(RGDyK)-NP/PTX significantly improved the inhibitory effects on the 3D tumor spheroids. For solid tumors, there are regions with high pressure and few vessels. Since the tumor spheroids could imitate the in vivo status because the tumor spheroids are free of blood vessels, the higher inhibitory effect suggests that c(RGDyK)-NP/PTX may improve therapeutic effect in vivo.
were tightly organized and the surfaces of these spheroids were covered with slices of agarose. After applying Taxol or NP/PTX, the surfaces of tumor spheroids were slightly disorganized and appeared concave-like holes (Fig. 4B and C). When the tumor spheroids were incubated with c(RGDyK)-NP/PTX, the margines of tumor cells became disintegrated and shrunken and the spheroids almost lost the three-dimensional structure (Fig. 4D). These observations were consistent with the results of the change ratios of tumor spheroid volumes. All of these results indicated that c(RGDyK) peptide could facilitate c(RGDyK)-NP/PTX penetration into the 3D tumor spheroids, and thus displayed much stronger inhibitory effects on tumor spheroids compare with Taxol and NP/PTX.
3.4. Morphology of tumor spheroids 3.5. Intravital Near-Infrared (NIR) Imaging Fig. 4 represents the SEM observations on tumor spheroids on day 3 after applying various PTX preparations including Taxol, NP/PTX and c(RGDyK)-NP/PTX. As a blank control, tumor spheroids (Fig. 4A)
The in vivo distribution and tumor accumulation ability of fluorescence-labeled c(RGDyK)-NP were determined non-
Fig. 7. Tumor growth curves of different groups after treatment indicated. Relative tumor volume ¼ tumor volume/primary tumor volume. Data are presented as mean SD (n ¼ 5) (A); Representative photos of tumors on mice (B) and Photographs of tumors harvested from each treatment group after various treatments (C); Images of H&E-stained sections tumor excised from subcutaneous tumor-bearing mice on 14th day after different treatment (D). Images were obtained under Leica microscope using a 20 and 40 objective.
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invasively in subcutaneous xenograft bearing nude mice. Compared with traditional NP group, the NIR fluorescence intensities in the tumor region of c(RGDyK)-NP group was much higher at any time post-injection ranged from 0.5 h to 5 h (Fig. 5A), suggesting that decoration of nanoparticles with c(RGDyK) could facilitate the accumulation of nanoparticles in tumor tissues. At 5 h after intravenous injection, the tumor-bearing mice were sacrificed by exsanguinations, and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were isolated and observed by the ex vivo images. The result also revealed that the tumor accumulation of c(RGDyK)-NP was much more than that of non-specific NP. Additionally, the fluorescence signal in liver and spleen of c(RGDyK)-NP group was lower than that of NP group (Fig. 5B and C), indicating that c(RGDyK)-NP localizing at tumor was denser than those in reticuloendothelial systems. These results implied that c(RGDyK)-NP could substantially home to solid cancer tissues and decrease non-specific accumulation in MPS-related organs. 3.6. Frozen section of tumor tissues The in vivo solid tumor permeation and targeting capability of coumarin 6 labeled c(RGDyK)-NP were studied qualitatively by fluorescence microscopic observation of the frozen sections of subcutaneous tumor. The result is presented in Fig. 6. For
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conventional nanoparticles, a slight green particles distributed in tumor tissue, indicated that normal nanoparticles could slightly accumulate in cancer tissue via the EPR effect (Fig. 6AeC). For c(RGDyK)-NP, an obviously higher and wider distribution of coumarin 6 labeled c(RGDyK)-NP than unmodified counterpart was observed (Fig. 6DeF). The result suggested that c(RGDyK) conjugation could facilitate permeation and enrichment of c(RGDyK)-NP in solid tumor tissues which was well consistent with the in vivo Near-Infrared Imaging experiment. 3.7. In vivo anti-tumor efficacy and safety evaluation The in vivo anti-tumor efficiency of Taxol, NP/PTX and c(RGDyk)-NP/PTX was validated in subcutaneous xenograft bearing mice. As shown in Fig. 7AeC, after treated with saline, Taxol and NP/ PTX c(RGDyk)-NP/PTX, the relative tumor volume (RTV) was 5.18, 3.22, 2.61 and 1.02 on day 14 respectively. Results showed that c(RGDyk)-NP/PTX exhibited the strongest inhibitory effect to the tumor volume. A combined effect of the passive targeting and enhanced cellular uptake could be the main reason for the significant suppression of tumor growth in c(RGDyk)-NP/PTX group. The tumor growth inhibitory effect in vivo for c(RGDyk)-NP/PTX was consistent well with the 3D tumor spheroids experiment in vitro. The tumor apoptosis was assessed by histopathological analysis. As
Fig. 8. Histopathological analysis of heart, lung, liver, spleen and kidney section stained with hematoxylin and eosin (H&E). Images were obtained under Leica microscope using a 40 objective.
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presented in Fig. 7D, it demonstrated that apoptosis occurred in tumor slices treated with various PTX formulations. It was clear that cell nuclei apoptosis of c(RGDyk)-NP/PTX group was more severe as compared to those of Taxol injection and NP/PTX. The result indicated that c(RGDyk)-NP could penetrate into tumor tissues and enhance the cellular uptake via integrin-mediated endocytosis and thus produced higher cytotoxicity than NP/PTX and Taxol. The toxicity is always a great concern for nanoparticulate system used in biomedicine. To further evaluate the safety of the delivery system, the toxicity of c(RGDyk)-NP/PTX was studied in vivo by histochemistry analysis. As shown in Fig. 8, no noticeable signal of organ damage was observed from hematoxylin and eosin (H&E) stained organ slices including heart, liver, spleen, lung and kidney. All of these results implied that c(RGDyk)-NP/PTX was a safe and an effective drug delivery system for solid tumor chemotherapy. 4. Conclusion Integrin-mediated poly(trimethylene carbonate)-based stealth nanoparticle was proposed as an efficient targeted vehicle for enhancing solid tumor penetration and chemotherapy. The penetration, distribution, and accumulation into 3D avascular tumor spheroid, and in vivo tumor accumulation of c(RGDyK)-NP were much higher than those of conventional nanoparticles. The antitumor efficacy of c(RGDyK)-NP/PTX was significantly enhanced in comparison with that of Taxol and NP/PTX. Histochemistry analysis revealed that no obvious histopathological abnormalities or lesions were observed in major organs after intravenous administration with the treatment doses. Our results indicate that c(RGDyK)decorated PEG-PTMC nanoparticle is a potential drug delivery system for enhancing drug penetration into tumor tissues and could be a promising vehicle for enhancing the chemotherapy of solid cancers. Acknowledgment This work was supported from the National Key Basic Research Program of China (2013CB932502), and National Science and Technology Major Project (2012ZX09304004). References [1] Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer 2006;6:583e92. [2] Cairns R, Papandreou I, Denko N. Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment. Mol Cancer Res 2006;4:61e70. [3] Jain RK. Delivery of molecular and cellular medicine to solid tumors. Adv Drug Deliv Rev 2012. http://dx.doi.org/10.1016/j.addr.2012.09.011. [4] Tannock IF, Lee CM, Tunggal JK, Cowan DSM, Egorin MJ. Limited penetration of anticancer drugs through tumor tissue a potential cause of resistance of solid tumors to chemotherapy. Clin Cancer Res 2002;8:878e84. [5] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Res 2009;69:7507e11. [6] Dhanikula RS, Argaw A, Bouchard JF, Hildgen P. Methotrexate loaded polyether-copolyester dendrimers for the treatment of gliomas: enhanced efficacy and intratumoral transport capability. Mol Pharm 2008;5:105e16. [7] Chilkoti A, Dreher MR, Meyer DE, Raucher D. Targeted drug delivery by thermally responsive polymers. Adv Drug Deliv Rev 2002;54:613e30.
[8] Jang SH, Wientjes MG, Lu D, Au JLS. Drug delivery and transport to solid tumors. Pharm Res 2003;20:1337e50. [9] Phillips R, Loadman P, Cronin B. Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours. Br J Cancer 1998;77:2112. [10] Li Z, Huang P, Zhang X, Lin J, Yang S, Liu B, et al. RGD-conjugated dendrimermodified gold nanorods for in vivo tumor targeting and photothermal therapy. Mol Pharm 2009;7:94e104. [11] Zhang W, Shi Y, Chen Y, Ye J, Sha X, Fang X. Multifunctional pluronic P123/ F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials 2011;32:2894e906. [12] Bagalkot V, Farokhzad OC, Langer R, Jon S. An aptameredoxorubicin physical conjugate as a novel targeted drug-delivery platform. Angew Chem Int Ed 2006;45:8149e52. [13] Xie J, Liu G, Eden HS, Ai H, Chen X. Surface-engineered magnetic nanoparticle platforms for cancer imaging and therapy. Acc Chem Res 2011;44:883e92. [14] Jiang X, Sha X, Xin H, Chen L, Gao X, Wang X, et al. Self-aggregated pegylated poly (trimethylene carbonate) nanoparticles decorated with c (RGDyK) peptide for targeted paclitaxel delivery to integrin-rich tumors. Biomaterials 2011;32:9457e69. [15] Cai W, Chen X. Anti-angiogenic cancer therapy based on integrin alphavbeta3 antagonism. Anti-cancer Agents Med Chem 2006;6:407. [16] Magnon C, Galaup A, Mullan B, Rouffiac V, Bidart JM, Griscelli F, et al. Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with avb3 and avb5 integrins. Cancer Res 2005;65:4353e61. [17] Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug delivery. J Med Chem 2005;48:1098e106. [18] Hu Z, Luo F, Pan Y, Hou C, Ren L, Chen J, et al. Arg-Gly-Asp (RGD) peptide conjugated poly (lactic acid)epoly (ethylene oxide) micelle for targeted drug delivery. J Biomed Mater Res A 2007;85:797e807. [19] Gwyther S. Modern techniques in radiological imaging related to oncology. Ann Oncol 1994;5:S3e7. [20] Yamashita T, Tomifuji M, Araki K, Kurioka T, Shiotani A. Endoscopic transoral oropharyngectomy using laparoscopic surgical instruments. Head & Neck 2011;33:1315e21. [21] Helou M, Miserque O, Brusson JM, Carpentier JF, Guillaume SM. Poly (trimethylene carbonate) from biometals-based initiators/catalysts: highly efficient immortal ring-opening polymerization processes. Adv Synth Catal 2009; 351:1312e24. [22] Liao L, Zhang C, Gong S. Preparation of poly (trimethylene carbonate)- blockpoly (ethylene glycol)-block-poly (trimethylene carbonate) triblock copolymers under microwave irradiation. React Funct Polym 2008;68:751e8. [23] Sabir MI, Xu X, Li L. A review on biodegradable polymeric materials for bone tissue engineering applications. J Mater Sci 2009;44:5713e24. [24] Buwalda SJ, Perez LB, Teixeira S, Calucci L, Forte C, Feijen J, et al. Self-assembly and photo-cross-linking of eight-armed PEG-PTMC star block copolymers. Biomacromolecules 2011;12:2746e54. [25] Jiang X, Xin H, Sha X, Gu J, Jiang Y, Law K, et al. PEGylated poly (trimethylene carbonate) nanoparticles loaded with paclitaxel for the treatment of advanced glioma: in vitro and in vivo evaluation. Int J Pharm 2011;420:385e94. [26] Odelius K, Albertsson AC. Precision synthesis of microstructures in star-shaped copolymers of 3-caprolactone, L-lactide, and 1,5-dioxepan-2-one. J Polym Sci Pol Chem 2007;46:1249e64. [27] Kluin OS, van der Mei HC, Busscher HJ, Neut D. A surface-eroding antibiotic delivery system based on poly-(trimethylene carbonate). Biomaterials 2009; 30:4738e42. [28] Zhang Y, Zhuo R. Synthesis and drug release behavior of poly (trimethylene carbonate)epoly (ethylene glycol)epoly (trimethylene carbonate) nanoparticles. Biomaterials 2005;26:2089e94. [29] Günther W, Pawlak E, Damasceno R, Arnold H, Terzis A. Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids. Br J Cancer 2003;88:463e9. [30] Ulrich TA, Jain A, Tanner K, MacKay JL, Kumar S. Probing cellular mechanobiology in three-dimensional culture with collageneagarose matrices. Biomaterials 2010;31:1875e84. [31] Huang P, Lin J, Wang X, Wang Z, Zhang C, He M, et al. Light-triggered theranostics based on photosensitizer-conjugated carbon dots for simultaneous enhanced-fluorescence imaging and photodynamic therapy. Adv Mater 2012;24:5104e10. [32] Davies C, Müller H, Hagen I, Gårseth M, Hjelstuen M. Comparison of extracellular matrix in human osteosarcomas and melanomas growing as xenografts, multicellular spheroids, and monolayer cultures. Anticancer Res 1997; 17:4317e26.
Contents lists available at SciVerse ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Solid tumor penetration by integrin-mediated pegylated poly(trimethylene carbonate) nanoparticles loaded with paclitaxel Xinyi Jiang a, b, Hongliang Xin a, c, Jijin Gu a, Ximing Xu b, Weiyi Xia a, Shuo Chen a, Yike Xie a, Liangcen Chen a, Yanzuo Chen a, Xianyi Sha a, Xiaoling Fang a, * a b c
Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, China Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang 212013, China Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 210029, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2012 Accepted 10 November 2012 Available online 2 December 2012
Limited penetration of antineoplastic agents is one of the contributing factors for chemotherapy failure of many solid tumors. In order to enhance drug penetration into solid cancer, especially, into the avascular regions inside tumors, we proposed cyclic RGD peptide functionalized PEGylated poly(trimethylene carbonate) nanoparticles (c(RGDyK)-NP). By integrin-mediated transcytosis and enhanced drug permeation, c(RGDyK)-NP could access the neoplastic cells distant from blood vessels, and consequently, avoiding the capability of cancer regeneration from these tumor cells. In the present study, the solid tumor penetration, homing specificity and anticancer efficacy were evaluated both on the ex vivo 3D tumor spheroids and on the subcutaneous xenograft mice model. In comparison with conventional nanoparticles (NP/PTX) and Taxol, c(RGDyK)-NP/PTX showed the strongest penetration and accumulation into 3D tumor spheroids, a marked tumor-homing specificity in vivo and the greatest tumor growth inhibitory effect in vitro and in vivo. Histochemistry analysis revealed that no obvious histopathological abnormalities or lesions were observed in major organs after intravenous administration with the treatment doses. In conclusion, cyclic RGD peptide-conjugated PEG-PTMC nanoparticle could facilitate drug penetration and accumulation in tumor tissues and may be a promising vehicle for enhancing the chemotherapy of solid cancers. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Solid cancer Tumor spheroids Integrin-mediated delivery Tumor penetration In vivo toxicity
1. Introduction Chemotherapeutic effect of solid tumors is often compromised due to several physiologic barriers of these cancers, including heterogeneous tumor perfusion and vascular permeability, high cell density, acidic pH, irregular blood flow, increased interstitial pressure and resultant hypoxia, which significantly limit the penetration of anticancer drugs into neoplastic cells distant from blood vessels [1e4]. Consequently, these anti-tumor agents are unable to access all of the cells within a tumor that are capable of regenerating it (that is, clonogenic cells or tumor stem cells), and then whatever their mode of action or potency, the effectiveness of chemotherapy will be compromised [1,5]. For a treatment to be effective, it should access the entire tumor, especially, the inside avascular regions [6e9]. Surface-engineered nanoplatform by cancer-specific targeting moieties (e.g., receptorbinding ligands or antibodies) is proffered as a promising strategy
* Corresponding author. Tel.: þ86 21 51980071; fax: þ86 21 51980072. E-mail address: [email protected] (X. Fang). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.11.016
for enhancing the drug delivery into solid tumors and lessening the non-special accumulation in MPS-related organs such as the liver and the spleen, and thus increasing the therapeutic index [10e13]. Previously, we have developed a cyclic RGD peptide-functionalized poly(trimethylene carbonate)-based micellar nanoparticulate system (c(RGDyK)-NP) which has been proved to be able to target integrin-rich malignant glioma cells [14]. In fact, extracellular matrixeintegrin receptors are also found to be overexpressed on most tumor cells and sprouting tumor vessels as compared to normal organs [15,16]. By integrin-mediated transcytosis and EPR effect, RGD sequence-based peptide functionalized nanosystem could actively accumulate to the neovascular region of the tumor after systemic administration [10,17,18]. However, for solid cancer, there are hypoxic and necrotic regions distant from the vascular bed, and these chemotherapy “blind areas” ineluctably lead to the relapse of cancer. It is postulated that enhancement in the ability of the delivery system to penetrate deeper into the avascular tumor tissues can significantly reduce the tumor regrowth and augment the therapeutic benefit of the treatment [2e4,19,20]. With this in mind, the solid tumor penetration capability of c(RGDyK)-NP was further investigated in the present study.
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It is critical for the targeting nanosystem to fulfill the anticipated functions without bringing any potential toxicity. Poly(trimethylene carbonate) (PTMC), one important type of aliphatic polycarbonates, has been widely used in biomedical field due to their tunable biodegradability and excellent biocompatibility [21e 23]. Some of the PTMC copolymers have been commercialized and used for clinical application approved by FDA. Our group and other researchers have described the synthesis and self-assembly behavior of di- or tri-block copolymer systems based on PEG and PTMC with a linear or star-shaped structure [24e26]. The reported copolymers have been demonstrated to be effective carriers for the sustained release of hydrophobic drugs [11,25,27,28]. Cyclic RGD peptide conjugation endowed nanostructures with tumor-homing ability. However, the changed biodistribution behavior of the nanosystem maybe induce some potential toxicities post systemic administration. Herein, the penetration, distribution, and accumulation of c(RGDyK)-NP into the avascular solid tumor were evaluated by ex vivo 3D tumor spheroids model. The in vivo tumorhoming capability and anticancer efficacy of c(RGDyK)-NP/PTX were investigated by subcutaneous xenograft mice model. In vivo toxicity was also assessed by histochemistry analysis after intravenous administration with the treatment doses.
2. Materials and methods 2.1. Materials Paclitaxel (PTX) was purchased from Xi’an San jiang Bio-Engineering Co. Ltd. (Xi’an, China). Methoxyl poly (ethylene glycol)-co-poly (trimethylene carbonate) (MPEG3K-PTMC6K) and c(RGDyK) modified poly (ethylene glycol)-b-poly (trimethylene carbonate) (c(RGDyK)-PEG3.5K-PTMC6K) were synthesized as described previously [14]. Low melting-point agarose was obtained from Yixin Biotechnology Co., Ltd. (Shanghai, China). Coumarin 6 was purchased from Sigma (St Louis, MO, USA). 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindotricarbocyanine iodide (DiR) was obtained from Biotium (Invitrogen, USA). Cellulose ester membranes (dialysis bag) with a molecular weight cut off value (MWCO) of 3500 (Greenbird Inc. Shanghai, China) were used in dialysis experiments. Penicillinestreptomycin, DMEM, fetal bovine serum (FBS) and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). Purified deionized water was prepared by the Milli-Q plus system (Millipore Co., Billerica, MA, USA). All of other reagents and chemicals were analytical grade and were used without further purification. U87MG cells were obtained from Shanghai Institute of Cell Biology. It was cultured in special Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate. Female BALB/c mice (20 2 g), supplied by Department of Experimental Animals, Fudan University (Shanghai, China), were acclimated at 25 C and 55% of humidity under natural light/dark conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of the College of Pharmacy, Fudan University (Shanghai, China).
Fig. 1. Schematic representation of self-assembled micellar-like nanoparticles functioned with cyclic RGD peptide (c(RGDyK)-NP/PTX) (A); A TEM image of c(RGDyK) modified nanoparticles (B); Particle size and size distribution determined by DLS using a Malvern Nano ZS (Malvern Instruments, UK). Inset: a photo of a nanoparticle solution at the concentration of 20 mg/ml (C); Apparent zeta potential spectrum of c(RGDyK)-NP/PTX in buffered 1 mM NaCl solutions (D).
X. Jiang et al. / Biomaterials 34 (2013) 1739e1746 2.2. Preparation and characterization of drug-loaded c(RGDyK)-NP The c(RGDyK)-conjugated PTX-loaded NP was prepared according to the procedure described previously [14]. Particle mean size, size distribution and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) using a zeta plus analyzer (Zeta-sizer, Malvern Nano ZS, U.K.). The shape and morphology of the nanoparticles were observed using transmission electron microscopy (TEM) (JEOL JMPEG-PTMC-1230, Japan). In the DLS assay, the nanoparticles were diluted in physiological water, and DLS measurements were performed at a 90 scattering angle at 37 C. Z-average sizes of three sequential measurements were collected and analyzed. In TEM observation, the nanoparticle sample was negatively stained with sodium phosphotungstatic solution (2%, w/v).
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PTX and c(RGDyK)-NP/PTX, were applied to the wells where tumor spheroids had been incubated for 7 days. The final concentration of PTX was 0.5 mg/ml. And tumor spheroids incubated in DMEM medium without any formulation were used as blank controls. After treatments, tumor spheroids were observed under an inverted microscope (Chongqing Optical & Electrical Instrument Co. Ltd., Chongqing, China) on days 0, 1, 3, 5 and 7. Growth inhibition was calculated with the following formula: V ¼ (p dmax dmin)/6, where dmax is the maximum diameter and dmin is the minimum diameter of each spheroid. The change ratio of tumor spheroid volume was calculated with the following formula: ratio% ¼ (Vdayi/Vday0) 100, where Vdayi is the tumor spheroid volume on the ith day (day 1, 3, 5, 7) after applying drug, and Vday0 is the tumor spheroid volume prior to treatment. 2.6. Morphology of tumor spheroids
2.3. In vitro 3D tumor spheroids formation For evaluating the effects of the solid tumor penetration of peptidefunctionalized nanosystem in vitro, an ex vitro multicellular 3D tumor spheroids mimicking the solid tumors in vivo were developed using the lipid overlay system as reported previously [6,29,30]. Agarose was heated at 80 C for 30 min, and diluted to 2% (w/v) with the serum-free DMEM medium. Each well of 24-cell culture plates was coated with a thin layer (300 ml) of sterilized agarose-based DMEM. Tumor cells were seeded into each well at the density of 1000 cells/well (in complete medium), gently agitated for 5 min, and incubated at 37 C for 7 days, and the culture medium was changed every 3 days. The uniform and compact multicellular spheroids were selected for the follow-up studies. 2.4. Confocal microscopy of tumor spheroids To evaluate the penetration ability, 3D tumor spheroids were used for penetration experiments. Briefly, tumor spheroids were incubated with coumarin 6 labeled conventional nanoparticles or peptide-conjugated nanoparticles at a final concentration of 300 mg/ml, respectively. At 12 h after treatment, spheroids were rinsed with PBS three times, transferred to a chambered coverslips and analyzed using an Olympus FV1000 confocal microscope (Olympus, Center Valley, PA). Z-stack images were obtained by scanning the tumor spheroid step by step. The scanning began from the top of a spheroid. Each scanning layer was 15 mm in thickness, and the total scanning was 90 mm in depth in a spheroid.
For evaluating the intuitive effects on 3D tumor spheroids after similarly applying Taxol, NP/PTX or c(RGDyK)-NP/PTX, the treated spheroids on day 0 or on day 3 were rinsed 3 times by PBS, fixed by 2.5% glutaraldehyde for 2 h at 4 C, further washed 3 times by 0.1 M PBS, then dehydrated and embedded. These tumor spheroid specimens were viewed with a field emission scanning electron microscope (FESEM, Carl Zeiss Ultra 55, Germany) at instrumental magnification. 2.7. In vivo Near-Infrared (NIR) Imaging In vivo real-time fluorescence imaging analysis was used to evaluate the effect of tumor distribution and accumulation ability of c(RGDyK)-modified nanoparticles in solid tumor-bearing mice [31]. The subcutaneous tumor xenograft model was established by inoculation of 5 106 U87MG cells (in 200 ml cell culture medium) into the subcutaneous tissue of the right hind legs. Both of c(RGDyK)-NP and NP were labeled by Dir (Invitrogen, USA). In brief, Dir was co-dissolved with copolymer in DCM during nanoparticle preparation. Then, the free Dir was removed via CL-4B column (Hanbang Chemical Co. LTD, China). When the size of tumors reached 0.7e 0.9 cm in diameter, the tumor-bearing mice were injected with Dir-labeled NP and c(RGDyK)-NP via tail vein at a dose of 20 mg DiR/mouse, respectively. At the time points of 30 min, 1 h and 5 h, the mice were anesthetized and visualized using Cambridge Research & Instrumentation in vivo imaging system (CRi, MA, USA). After 5 h post-injection, the mice were humanely killed, following which the tumor and principle organs (including heart, liver, spleen, lung and kidney) were removed and also visualized using CRi in vivo imaging system.
2.5. Growth inhibition of 3D tumor spheroids 2.8. Frozen section analysis of tumor tissues The influence of various treatments on the growth of tumor spheroids was also investigated in this study. For evaluating the inhibition of tumor growth, the serumfree DMEM media but containing different PTX formulations, including Taxol, NP/
The in vivo solid tumor penetration and tumor-homing specificity of c(RGDyK)NP were studied qualitatively by fluorescence microscopic observation of equatorial
Fig. 2. Representative confocal images of tumor spheroids 12 h after treatment with fluorescein-labeled NP and c(RGDyK)-NP. Bright field (BF) is shown in white and black and coumarin 6 fluorescence is displayed in green. Z-stack images were obtained starting at the top of the spheroid in 15 mm intervals for a total of 90 mm into the spheroid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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plane sections of tumor tissues. The subcutaneous tumor bearing mice model was established as described above. When the size of tumors reached 0.7e0.9 cm in diameter, coumarin 6 labeled c(RGDyK)-NP or NP was injected into the tail vein of mice at a dose of 100 mg/kg, respectively. Approximately 45 min later, the animals were anesthetized with diethyl ether. Then, they underwent ventricular perfusion with saline and 4% paraformaldehyde for 30 min, respectively. After that, the tumor tissues were harvested, fixed in 4% paraformaldehyde for 24 h, placed in 15% sucrose PBS solution for 24 h until subsidence, and then in 30% sucrose for 24 h until subsidence. Afterwards, the tumor was frozen in OCT embedding medium (Sakura, Torrance, CA, USA) at 80 C. Frozen sections of 20 mm thickness were prepared and stained with 300 nM DAPI for 10 min at room temperature. After washed twice with PBS (pH 7.4), the sections were immediately examined under the fluorescence microscope (Leica DMI 4000B, Germany). 2.9. In vivo anti-tumor efficacy and safety evaluation In vivo anticancer activity against subcutaneous tumor was evaluated in mice. The dose schedule started when the tumor volume was about 40e80 mm3. The mice were randomized into four groups (n ¼ 5) and treated with 100 ml of Taxol, c(RGDyK)NP/PTX, NP/PTX, and physiological saline via tail vein injection on the day 0, 2, 4 and 6 (PTX dosage:10 mg/kg body weight, Taxol was diluted by physiological saline). Tumor size was monitored via serial caliper measurement for every 2 days and the tumor volume was estimated using the formula: Volume ¼ 0.5 length (width)2. On the day 14, the animals were sacrificed by cervical dislocation, and the tumor mass was harvested, photographed, and then fixed with paraformaldehyde for 48 h and embedded in paraffin. Each section was cut into 5 mm, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under fluorescent microscope (Leica DMI 4000B, Germany). The potential in vivo toxicity is always a great concern for nanoparticulate system used in biomedicine. To study the toxic effects of the nanocarriers, major organs such as heart, lung, liver, spleen, and kidney tissue were fixed with paraformaldehyde for 48 h and embedded in paraffin. Each section was cut into 5 mm, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under a Leica microscope.
surface. The PTX loading content and encapsulation ratio of c(RGDyK)-NP/PTX were about 6.4% and 93%, respectively. 3.2. In vitro tumor spheroids penetration In many solid tumors, there are hypoxic and avascular tumor regions. Due to the poor permeation of delivery systems, the amount of drug accessing inside the solid tumors is low. As a consequence, these chemotherapy “blind areas” eventually and ineluctably induced the recurrence of cancer, and the overall chemotherapeutic efficacy of anticancer agents is compromised. For a cancer treatment to be curative, the delivery system must efficiently penetrate tumor tissue to reach all of the viable cells. The ex vivo 3D tumor spheroids generated by liquid overlay technique are not only aggregates of cells in close contact but contain an organized extracellular matrix consisting of fibronectin, laminin, collagen, and GAG suggestive of the extracellular matrix of tumors in vivo [1,6,32]. Thereby, 3D multicellular model represents the
3. Results and discussion 3.1. Preparation and characterization of the nanoparticles The drug-loaded coreeshell type nanoparticle functionalized with cyclic RGD peptide was schematically illustrated in Fig. 1A. The c(RGDyK)-NP/PTX exhibited a spherical shape under the examination of TEM (Fig. 1B) with a Z-average diameter about 73 nm determined by the dynamic light scattering analysis (Fig. 1C). c(RGDyK)-NP/PTX solution at the concentration of 20 mg/ml was shown in Fig. 1C inset. The solution exhibited a slight whitish opalescence. The zeta potential for c(RGDyK)-NP/PTX was about 6.7 mV (Fig. 1D). As shown in the TEM photo, a clear peptide-engineered PEG corona could be seen on the nanoparticle
Fig. 3. Change ratios of tumor spheroid volume (%) after applying various PTX formulations and blank control. (a) P < 0.01, versus primary spheroid volume; (b) P < 0.01, versus Taxol; and (g) P < 0.01, versus conventional nanoparticles. Data represent mean SD (n ¼ 5).
Fig. 4. Morphology of tumor spheroids observed by scanning electronic microscope (SEM). Key: Spheroids at day 3 after applying serum-free DMEM culture medium (A), Taxol (B), NP/PTX (C) and c(RGDyK)-NP/PTX (D), respectively. Final PTX for all was at 0.5 mg/ml.
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Fig. 5. (A) In vivo fluorescence imaging of tumor-bearing nude mice after injection of DiR-labeled NP (A1) and DiR-labeled c(RGDyk)-NP (A2) was administered via a lateral tail vein. All NIR fluorescence images were acquired with a 230ms exposure time at 30 min and at 1 and 5 h (h). Arrow: the position of the tumor. Fluorescence signal from DiR was pseudocolored red. Representative images of dissected organs of a mouse bearing subcutaneous tumor sacrificed 5 h post intravenous injection of DiR-labeled NP (B) and DiR-labeled c(RGDyk)-NP (C). Key: t, solid tumor; li, liver; sp, spleen; lu, lung; h, heart; k, kidney. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
avascular regions found in many solid tumor tissues and can serve as an invaluable tool to evaluate the solid tumor penetration effect of drug delivery system. In this study, interstitial penetration and diffusion of c(RGDyK)-NP into avascular regions of the solid tumors were evaluated using the ex vivo 3D tumor spheroids as a model. Fig. 2 shows confocal laser scanning microscopic images of 3D tumor spheroids 12 h after applying coumarin 6 labeled nanoparticles. For conventional nanoparticles, fluorescence was observed primarily on the periphery of tumor spheroids. However, after applying nanoparticles conjugated with c(RGDyK), the fluorescence was able to be observed throughout the whole tumor spheroids and the depth of fluorescence that could be observed in
the spheroids reached to 90 mm, suggesting that solid tumor penetration was enhanced by the presence of RGD targeting ligand. 3.3. Growth inhibitory effect on tumor spheroids The influence of various treatments on the growth of tumor spheroids was also studied. Fig. 3 represents the in vitro tumor spheroid volume ratios after treatment with Taxol, NP/PTX and c(RGDyK)-NP/PTX at the final PTX concentration of 0.5 mg/ml, respectively. It was observed that tumor spheroids continued to grow in size and volume in the absence of any drug (128.7% of the primary volume after 7 days). The obvious reduction in volume of
Fig. 6. The qualitative valuation of in vivo tumor penetration and tumor-homing specificity of coumarin 6 labeled nanoparticles. Distribution of nanoparticles in tumor tissue of subcutaneous xenografts bearing mice treated with coumarin 6 labeled NP (AeC) and coumarin 6 labeled c(RGDyK)-NP (DeF) 45 min after i.v. administration. Image C is the combination of A and B; image F is the combination of D and E. Frozen sections (20 mm of thickness) of solid tumor were examined by fluorescent microscopy. Green: coumarin 6 labeled nanoparticles. Blue: cell nuclei. Images were obtained under Leica fluorescence microscope with the original magnification 100. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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tumor spheroids was observed for all PTX formulations after 7 days treatment, indicating that tumor spheroids were sensitive to PTX. The change ratios of tumor spheroid volumes (%) on day 7 were 87.2%, 74.3% and 31.7% for Taxol, NP/PTX and c(RGDyK)-NP/PTX, respectively. The result indicated that c(RGDyK)-NP/PTX significantly improved the inhibitory effects on the 3D tumor spheroids. For solid tumors, there are regions with high pressure and few vessels. Since the tumor spheroids could imitate the in vivo status because the tumor spheroids are free of blood vessels, the higher inhibitory effect suggests that c(RGDyK)-NP/PTX may improve therapeutic effect in vivo.
were tightly organized and the surfaces of these spheroids were covered with slices of agarose. After applying Taxol or NP/PTX, the surfaces of tumor spheroids were slightly disorganized and appeared concave-like holes (Fig. 4B and C). When the tumor spheroids were incubated with c(RGDyK)-NP/PTX, the margines of tumor cells became disintegrated and shrunken and the spheroids almost lost the three-dimensional structure (Fig. 4D). These observations were consistent with the results of the change ratios of tumor spheroid volumes. All of these results indicated that c(RGDyK) peptide could facilitate c(RGDyK)-NP/PTX penetration into the 3D tumor spheroids, and thus displayed much stronger inhibitory effects on tumor spheroids compare with Taxol and NP/PTX.
3.4. Morphology of tumor spheroids 3.5. Intravital Near-Infrared (NIR) Imaging Fig. 4 represents the SEM observations on tumor spheroids on day 3 after applying various PTX preparations including Taxol, NP/PTX and c(RGDyK)-NP/PTX. As a blank control, tumor spheroids (Fig. 4A)
The in vivo distribution and tumor accumulation ability of fluorescence-labeled c(RGDyK)-NP were determined non-
Fig. 7. Tumor growth curves of different groups after treatment indicated. Relative tumor volume ¼ tumor volume/primary tumor volume. Data are presented as mean SD (n ¼ 5) (A); Representative photos of tumors on mice (B) and Photographs of tumors harvested from each treatment group after various treatments (C); Images of H&E-stained sections tumor excised from subcutaneous tumor-bearing mice on 14th day after different treatment (D). Images were obtained under Leica microscope using a 20 and 40 objective.
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invasively in subcutaneous xenograft bearing nude mice. Compared with traditional NP group, the NIR fluorescence intensities in the tumor region of c(RGDyK)-NP group was much higher at any time post-injection ranged from 0.5 h to 5 h (Fig. 5A), suggesting that decoration of nanoparticles with c(RGDyK) could facilitate the accumulation of nanoparticles in tumor tissues. At 5 h after intravenous injection, the tumor-bearing mice were sacrificed by exsanguinations, and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were isolated and observed by the ex vivo images. The result also revealed that the tumor accumulation of c(RGDyK)-NP was much more than that of non-specific NP. Additionally, the fluorescence signal in liver and spleen of c(RGDyK)-NP group was lower than that of NP group (Fig. 5B and C), indicating that c(RGDyK)-NP localizing at tumor was denser than those in reticuloendothelial systems. These results implied that c(RGDyK)-NP could substantially home to solid cancer tissues and decrease non-specific accumulation in MPS-related organs. 3.6. Frozen section of tumor tissues The in vivo solid tumor permeation and targeting capability of coumarin 6 labeled c(RGDyK)-NP were studied qualitatively by fluorescence microscopic observation of the frozen sections of subcutaneous tumor. The result is presented in Fig. 6. For
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conventional nanoparticles, a slight green particles distributed in tumor tissue, indicated that normal nanoparticles could slightly accumulate in cancer tissue via the EPR effect (Fig. 6AeC). For c(RGDyK)-NP, an obviously higher and wider distribution of coumarin 6 labeled c(RGDyK)-NP than unmodified counterpart was observed (Fig. 6DeF). The result suggested that c(RGDyK) conjugation could facilitate permeation and enrichment of c(RGDyK)-NP in solid tumor tissues which was well consistent with the in vivo Near-Infrared Imaging experiment. 3.7. In vivo anti-tumor efficacy and safety evaluation The in vivo anti-tumor efficiency of Taxol, NP/PTX and c(RGDyk)-NP/PTX was validated in subcutaneous xenograft bearing mice. As shown in Fig. 7AeC, after treated with saline, Taxol and NP/ PTX c(RGDyk)-NP/PTX, the relative tumor volume (RTV) was 5.18, 3.22, 2.61 and 1.02 on day 14 respectively. Results showed that c(RGDyk)-NP/PTX exhibited the strongest inhibitory effect to the tumor volume. A combined effect of the passive targeting and enhanced cellular uptake could be the main reason for the significant suppression of tumor growth in c(RGDyk)-NP/PTX group. The tumor growth inhibitory effect in vivo for c(RGDyk)-NP/PTX was consistent well with the 3D tumor spheroids experiment in vitro. The tumor apoptosis was assessed by histopathological analysis. As
Fig. 8. Histopathological analysis of heart, lung, liver, spleen and kidney section stained with hematoxylin and eosin (H&E). Images were obtained under Leica microscope using a 40 objective.
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presented in Fig. 7D, it demonstrated that apoptosis occurred in tumor slices treated with various PTX formulations. It was clear that cell nuclei apoptosis of c(RGDyk)-NP/PTX group was more severe as compared to those of Taxol injection and NP/PTX. The result indicated that c(RGDyk)-NP could penetrate into tumor tissues and enhance the cellular uptake via integrin-mediated endocytosis and thus produced higher cytotoxicity than NP/PTX and Taxol. The toxicity is always a great concern for nanoparticulate system used in biomedicine. To further evaluate the safety of the delivery system, the toxicity of c(RGDyk)-NP/PTX was studied in vivo by histochemistry analysis. As shown in Fig. 8, no noticeable signal of organ damage was observed from hematoxylin and eosin (H&E) stained organ slices including heart, liver, spleen, lung and kidney. All of these results implied that c(RGDyk)-NP/PTX was a safe and an effective drug delivery system for solid tumor chemotherapy. 4. Conclusion Integrin-mediated poly(trimethylene carbonate)-based stealth nanoparticle was proposed as an efficient targeted vehicle for enhancing solid tumor penetration and chemotherapy. The penetration, distribution, and accumulation into 3D avascular tumor spheroid, and in vivo tumor accumulation of c(RGDyK)-NP were much higher than those of conventional nanoparticles. The antitumor efficacy of c(RGDyK)-NP/PTX was significantly enhanced in comparison with that of Taxol and NP/PTX. Histochemistry analysis revealed that no obvious histopathological abnormalities or lesions were observed in major organs after intravenous administration with the treatment doses. Our results indicate that c(RGDyK)decorated PEG-PTMC nanoparticle is a potential drug delivery system for enhancing drug penetration into tumor tissues and could be a promising vehicle for enhancing the chemotherapy of solid cancers. Acknowledgment This work was supported from the National Key Basic Research Program of China (2013CB932502), and National Science and Technology Major Project (2012ZX09304004). References [1] Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer 2006;6:583e92. [2] Cairns R, Papandreou I, Denko N. Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment. Mol Cancer Res 2006;4:61e70. [3] Jain RK. Delivery of molecular and cellular medicine to solid tumors. Adv Drug Deliv Rev 2012. http://dx.doi.org/10.1016/j.addr.2012.09.011. [4] Tannock IF, Lee CM, Tunggal JK, Cowan DSM, Egorin MJ. Limited penetration of anticancer drugs through tumor tissue a potential cause of resistance of solid tumors to chemotherapy. Clin Cancer Res 2002;8:878e84. [5] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Res 2009;69:7507e11. [6] Dhanikula RS, Argaw A, Bouchard JF, Hildgen P. Methotrexate loaded polyether-copolyester dendrimers for the treatment of gliomas: enhanced efficacy and intratumoral transport capability. Mol Pharm 2008;5:105e16. [7] Chilkoti A, Dreher MR, Meyer DE, Raucher D. Targeted drug delivery by thermally responsive polymers. Adv Drug Deliv Rev 2002;54:613e30.
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