Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–5 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.968820
A collagen-based multicellular tumor spheroid model for evaluation of the efficiency of nanoparticle drug delivery Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Dalhousie University on 03/05/15 For personal use only.
Van-Minh Le1, Mei-Dong Lang2, Wei-Bin Shi3 & Jian-Wen Liu1 1Department of Molecular & Cellular Pharmacology, Biomedical Nanotechnology Center, State Key Laboratory of Bioreactor
Engineering & Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai , P.R. China, 2Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, China, and 3Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, P.R. China
radation)(Danhier et al. 2012, Kadam et al. 2012), and/or reducing their side effects by targeted delivery, for example in the case of cancer therapy (De Jong and Borm 2008), and finally, enhancing the therapeutic value of the drug (Thakor and Gambhir 2013, Vrignaud et al. 2011). While in vivo models can be used to assess the actual therapeutic values of these delivery systems better, the experimental procedure could be complex, with low throughput, and time-consuming. On the other hand, in traditional two-dimensional (2-D) cell cultures, the cells are directly exposed to drugs and therefore do not reflect the effect of restriction by many extracellular barriers in the body that could otherwise lead to significant reductions in the infiltration capacity and in the therapeutic effect of the drug-conjugated nanocarriers on the targeted tissue. It has been suggested that tumor cells respond very differently to therapeutic factors in vitro compared to in vivo, due to the lack of complex interactions within their specialized microenvironments (Kuo et al. 2012, Mikhail et al. 2013, He et al. 2014). There were also significant distinctions in terms of morphology, gene expression, drug resistance, migration and remodel matrices between 2-D and 3-D cultures, which was thought to closely mimic the cell-matrix interactions of in vivo conditions (Kuo et al. 2012, Cen et al. 2008, Aszodi et al. 2006, Even-Ram and Yamada 2005). Rat tail-type I collagen, extracted from rat tail tendons(Rajan et al. 2006), is one of the most widely used biomaterials in tissue engineering (Cen et al. 2008, Aszodi et al. 2006), due to its excellent biocompatibility and low antigenicity (Techatanawat et al. 2011). In this study, the tumor cells were embedded and cultured in a collagen scaffold to resemble the dimensional effects of the in vivo microenvironment of the tumor. Collagenbased tumor spheroids were developed and optimized using 95-D, U87 and HCT116 cells respectively. The delivery and
Abstract Targeted drug delivery systems, especially those that use nanoparticles, have been the focus of research into cancer therapy during the last decade, to improve the bioavailability and delivery of anticancer drugs to specific tumor sites, thereby reducing the toxicity and side effects to normal tissues. However, the positive antitumor effects of these nanocarriers observed in conventional monolayer cultures frequently fail in vivo, due to the lack of physical and biological barriers resembling those seen in the actual body. Therefore, the collagen-based 3-D multicellular culture system, to screen new nanocarriers for drug delivery and to obtain more adequate and better prediction of therapeutic outcomes in preclinical experiments, was developed. This 3-D culture model was successfully established using optimized density of cells. Our result showed that 3-D cell colonies were successfully developed from 95-D, U87 and HCT116 cell lines respectively, after a seven-day culture in the collagen matrix. The coumarin-conjugated nanoparticles were able to penetrate the matrix gel to reach the tumor cells. The model is supposedly more accurate in reflecting/predicting the dynamics and therapeutic outcomes of candidates for drug transport in vivo, and/or investigation of tumor biology, thus speeding up the pace of discovery of novel drug delivery systems for cancer therapy. Keywords: cancer, drug delivery system, nanoparticles, three dimensional model, tissue engineering
Introduction In recent years, nanoparticles have been studied extensively to develop delivery systems for chemotherapeutic agents. Nanoparticles can be engineered for multiple therapeutic purposes, including increasing the drug’s water solubility and stability (protecting it from denaturation and/or deg-
Correspondence: Jian-Wen Liu, Ph.D., East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237, People’s Republic of China. Tel/Fax: 021-64252044, 64252262. E-mail: [email protected] (Received 4 August 2014; revised 3 September 2014; accepted 12 September 2014)
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antitumor efficiencies of drug-conjugated nanoparticles in this tumor spheroid model were assessed by uptake and cytotoxicity studies.
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Materials The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 5-fluorouracil (5-FU) were purchased from Sigma (Sigma-Aldrich China, Inc., Shanghai, China). The 5-FU-loaded polymicelles and the coumarin-loaded polymicelles were synthesized and kindly provided by Professor Lang’s Lab (East China University of Science and Technology, Shanghai, China). These micelles had a mean diameter of 151.9 nm, with an encapsulation efficiency of 37.46%,, a polydispersity index of 0.155, and a zeta potential of ⫺ 0.29 mV.
Methods Cells and cell culture The human lung cancer cell line (95-D), which is highly metastatic, and the human colon cancer cell line (HCT116), were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The glioblastoma cell line (U87) was obtained from the American Type Culture Collection (ATCC, USA). The 95-D and HCT116 cells were cultured in RPMI 1640 medium (Hyclone Laboratories Inc., Australia) and the U87 cells were maintained in DMEM (Hyclone). The culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone), 100 U/ mL penicillin, and 100 μg/mL streptomycin (Gibco, Paisley, UK). The cells were maintained at 37°C in a humidified atmosphere of 95% O2 and 5% CO2, and were routinely passaged using trypsin/EDTA solution (Gibco) when they reached confluence. The cell suspension was harvested by trypsin digestion and used for the spheroid formation.
Multicellular spheroid culture To culture multicellular 3-D tumor spheres in the collagen gel, the cells were embedded in rat tail-type I collagen (Shengyou Biotechnology Company Ltd., Hangzhou, China) at the desired concentrations, after the cell culture surfaces were pre-chilled and pre-coated with a thin layer of collagen. The collagen gel was formed according to the manufacturer’s protocol and modified from reference (Lee et al. 2007). The volume medium containing a density of cells was obtained as follows: VMedium ⫽ VFresh medium ⫹ Vcollagen (5 mg/mL) ⫹ VNaOH (0.1 M) ⫹ V10⫻ (RPMI 1640 medium), where VNaOH ⫽ 0.06 ⫻ Vcollagen,V10⫻ ⫽ 0.01 ⫻ Vmedium. The appropriate volume of fresh medium was added after collagen gel formulation. The cell culture medium was changed every two days. Photographs were taken with a Nikon Eclipse Ti-S fluorescence microscope (MA, U.S.A.).
Cytotoxicity assay For a spheroid culture in collagen gel, monolayer tumor cells were trypsinized and centrifuged, and a final density of 3 ⫻ 104 cells/mL was resuspended in 1 mg/mL collagen gel and then immediately seeded onto a 96-well plate to
generate 3-D tumor spheroids. A proper volume of medium was added on top of the cell scaffold. The cells were adapted for five days. To test drug responses, cells in 2-D or 3-D cultures were incubated with a concentration of drug equivalent to 50 μg/mL, for 2 days. Prior to the finished culture in 3-D models, the matrix gel was digested by type I collagenase, centrifuged and re-incubated in a medium containing 10% MTT for 4 h. The absorbance was monitored by the EnSpire Multimode Plate Reader (PerkinElmer, Waltham, Massachusetts) at 492 and 630 nm. The rate of inhibition was calculated using the formula: Inhibition rate (%) ⫽ (1 – OD treatment/ OD control) ⫻ 100%. For the 2-D monolayer, 95-D cells (4 ⫻ 104 cells/mL) were seeded in a 96-well plate and were incubated for 24 h. Then, solutions of the drug were added to cells and further incubated for 48 h. Thereafter, the viability of cells was determined using the MTT method.
Confocal microscopy and estimation of nanoparticle penetration The spheroids obtained from collagen gel-embedded 3-D culture methods were fixed in a fixative (3:1 ratio of methanol and acetic acid glacial) for 10 min after the digestion of type I collagenase. They were washed three times with cold phosphate buffer saline (PBS) of pH 7.4, and then treated with propidium iodide (PI, 5 μg/mL, Sigma-Aldrich China, Inc., Shanghai, China) for staining of the nuclei. Thereafter, the spheroids were rinsed with PBS again and were transferred to microscope slides. To detect fluorescence, a Nikon A1 plus confocal laser scanning microscope system (Nikon Instech Co., Ltd., Tokyo, Japan) with a laser excitation line at 488 nm and an emission bandpass filter of 610‒630 nm was used. As described above, HCT116 cells were embedded in collagen gel and were cultured for 3 days. After that, coumarin-loaded nanoparticles (100 μg/mL) were added to the cell culture wells and incubated for 4 h at 37°C. After washing with PBS to remove unbound and excess components, the spheroids were visualized with fluorescent microscopy (Nikon Ti–S).
Results and discussion Morphology of tumor cells in 2-D vs 3-D To develop 3-D culture models, the 95-D, U87 and HCT116 cell lines were employed. The U87 cells had slow growth activity, compared to the HCT116 and 95-D which showed rapid growth and high malignancy (Figure 1). In the 2-D culture models, U87 cells exhibited the classic tear-drop shape and HCT116 exhibited an oval shape, while the 95-D cells showed spread-out, sheet-like trigonal or polygonal morphologies (Figure 1, left). By contrast, the morphology of cells cultured in the 3-D conditions appears dramatically different from that of cells cultured in the monolayer. The phase-contrast images revealed that the U87 (Figure 1B) and HCT116 (Figure 1C) cells formed tightly packed and rounded spheroids, but the 95-D (Figure 1A) cells were of irregular shapes, and loosely clustered. All three tumor cells were successfully entrapped, expanded, and propagated in 3-D collagen scaffolds, which
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Figure 1. Tumor cell morphology in different culture models. Typical morphology of cells grown in 2-D substrata (left) at day-2 vs. cells grown in the 3-D culture model (middle, 20⫻; right, 40⫻). The 95-D (A), U87 (B) and HCT116 (C) grown for 9 days in the collagen scaffold in the same conditions. Phase-contrast images were obtained at day-7 using an inverted microscope. Scale bar ⫽ 100 μm.
indicated that our collagen-based 3-D culture models could be used for these three cell line cultures.
Optimization of the density of cell seeding The density of cell seeding can affect cell growth, nutrition supply and reliability of experimental results. For optimization studies of the density of cell seeding and the culture time, 95-D cells were obtained from a monolayer culture and cell numbers were counted by Trypan blue exclusion methods. Then, the cells were transferred onto collagen gel matrices in replicates of 24 wells, at densities of 1 ⫻ 103, 1 ⫻ 104, 5 ⫻ 104 and 1 ⫻ 105 cells/mL for 3-D culture (Figure 2). The first three days were the adaptation period during which the
cells exhibited slow growth rate, and only small cell populations were generated. After that, the cell density increased and formed defined colonies. At a seeding density of 1 ⫻ 103 cells/mL, cell growth was slow and clusters were also formed with irregular shapes and with longer culture time. At the highest planting density of 1 ⫻ 105 cells/mL, the adaptation time was shorter than that for experiments with low cell seeding density. However, they formed smaller colonies compared to those at the densities of 1 ⫻ 104 or 5 ⫻ 104 cells/ mL. Furthermore, most of these cells died at day-7. The 95-D cell line formed middle clusters with a wide range of different sizes from day- 3. Seeding cells at 1 ⫻ 104 or 5 ⫻ 104 cells/ mL showed that cells had formed the multicellular spheroids
Figure 2. Morphological appearance of 95-D tumor cells cultured on the collagen gel with different seeding densities. Cultures maintained for 9 days and images captured every 3 days using light microscopy. Scale bar ⫽ 100 μm.
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Figure 3. Image of HCT116 cell spheroids stained with Propidium Iodide nuclear labeling dye. (A) Monolayer cells; (B) The organization of granule cell layer in multicellular spheroids; (C) The complete 3-D cells (nuclei) in 3-D image. All pictures were taken using confocal laser scanning microscopy.
after day-3, with a uniform size and shape. Little cell death was observed when seeding at the density of 5 ⫻ 104 cells/ mL during the last culture period. Based on these results, the seeding densities from 1 ⫻ 104 to 5 ⫻ 104 cells/mL was chosen for the subsequent research according to experimental design. The treatment time point was selected at day-5 after culture for 48 h of subsequent drug incubation.
Validation of established 3-D tumor models In the above experiments, the entire spheroid morphology was just illustrated in bright field microscopy. In order to gain further understanding of spheroid architecture, the fluorescent agent, propidium iodide (PI), was employed to visualize cells and extracellular matrix structures after fixation. PI diffused into cells and intercalated with nuclear and cytoplasmic nucleic acids of the fixative cells. As shown in Figure 3, cells cultured in the 2-D monolayer and in the multicellular spheroid culture model were stained with the fluorescent dye, thereby enabling visualization of the spheroids, which are composed of individual cells. Here, the difference in cell morphology and cell assembly in the 2-D monolayer vs. the 3-D model could present a different response by cells to nanoparticles. For the assessment of the interactions between nanoparticles and cancer cells, 95-D cells were individually cultured in 2-D or 3-D scaffolds for two or three days before treatment. Following that, 50 μg/mL of free 5-FU or 5-FU loaded polymer micelles (5-FU/PM) were added to the two
different culture models for 48 h, respectively. Then, the inhibition of the 5-FU formulation was determined by an MTT assay. Figure 4A shows that the tumor inhibition effect of the drugs in the 3-D culture model was lower than that in the 2-D monolayer culture. In the concentration used in this study, no significant difference in the effect of free 5-FU and 5-FU/PM was found using the 2-D model; however in the 3-D model, 5-FU/PM exhibited strikingly lower antitumor activity than free 5-FU . The inhibition rate of free 5-FU and 5-FU/PM were respectively 61.09 ⫾ 2.06 and 47.03 ⫾ 3.93% after 2 days, and 40.02 ⫾ 5.28 and 36.48 ⫾ 1.89% after 3 days of cultivation in the 3-D model. It could be due to small drug molecules that are nonpolar being able to diffuse across the membrane more readily than the large polymer micelles. The cytotoxicity assay in the 3-D cultures reveals that 3-D cell cultures in collagen gel showed significantly higher drug resistance compared to 2-D cell cultures, after treatments with 5-FU during the same period of time . Apart from the effect of growth inhibition, the morphological differences of cells between the control and treatment groups were also substantial. The size of the sphere, the reduction in cell density, irregular shape, and cellular shrinkage, were observed (Figure 4B). On the other hand, these results also showed that the spheres grew larger with time, suggesting a continuous reduction in drug sensitivity of the 3-D tumor spheroid to the drug (Figure 4A).
Figure 4. Cytotoxicity of 95-D cells induced by 5-FU formulations. (A) Growth inhibition of 5-FU or 5-FU/PM of tumor cells in different models was measured by MTT assay. Cells were incubated in 2-D model for 2 days or in 3-D for 5 days and then treated with 50 μg/mL 5-FU equivalent concentration of 5-FU/PM for 48 h. (B) Morphology of cells after exposure to 5-FU-loaded micelles for 48 h. Bright field images were captured using an inverted microscope to monitor morphology of cells in the control group (left) and treated group (right). Data from three independent experiments were presented as mean ⫾ SD, and the Student’s t-test was used to compare the means of two samples, ns: no significance; **p ⬍ 0.01; ***p ⬍ 0.001. Scale bar ⫽ 100 μm.
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This work was supported by the Shanghai Committee of Science and Technology (No. 13140902300), Nano Science and Technology Special Funding of the Shanghai Committee of Science and Technology (No. 11nm0503700), the Shanghai Committee of Science and Technology [grant 11DZ2260600] and the Fundamental Research Funds for the Central Universities.
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Figure 5. Fluorescence microscopy images of multicellular spheroids HCT116 cells incubated with coumarin-polymer micelles for 4 h. Left image is bright field and right is the fluorescence image.
To observe the ability of the nanoscale polymicelles to penetrate the collagen matrix, and whether death of cells was induced by 5-FU/MC, the HCT116 cells were seeded in the collagen scaffold for 3 days and subsequently incubated with coumarin-loaded polymicelles for 4 h. As shown in Figure 5, the penetration of micelles into the tumor spheroids was very limited. There was only a minor portion of the micelles infiltrating the matrix and translocating into the cells after 4 h of incubation (green fluorescence). This suggests that in 3-D tumor spheroids, the uptake of nanoparticles (large molecules) could be less efficient than that of the free drug (small molecules). Similarly, in vivo tumors have also been shown to exhibit poor nanoparticle penetration due to the presence of the extracellular matrix (Grantab et al. 2006, Kuppen et al. 2001, Goodman et al. 2008) and the formation of the tight junctions in the spheroid cell (Goodman et al. 2008, Thorne and Nicholson 2006). Hence, our 3-D culture models can closely mimic not only the dimensional obstacles as found in vivo, but can also reproduce the biological responses of the tumor to antitumor drugs, which have been associated with tumor initiation, progression and propensity to malignancy (invasion and metastasis)(Godugu et al. 2013). This result again demonstrated the rationality of the 3-D culture model established to evaluate the efficiency of the drug delivery system for cancer therapy.
Conclusion In this study, a befitting model based on type I collagen to evaluate the efficacy of drug carriers was established and optimized. The behavior of cells grown in the 3-D model, including their shape, density, interactions, drug sensitivity and drug resistance, reveal marked differences from those of cells cultured on the traditional monolayer. These results showed that the size of multicellular spheroids and agglomeration of cells can be controlled by optimizing the densities of the seeding cells. This 3-D culture model is applicable for in vitro screening of the drug, because it is cheaper than in vivo experiments and can recreate numerous features of living tissues such as the structure, function, microenvironment, and so on.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References Aszodi A , Legate KR, Nakchbandi I, Fässler R. What mouse mutants teach us about extracellular matrix function. Annu Rev Cell Dev Biol. 2006;22:591–621. Cen L, Liu W, Cui L, Zhang W, Cao Y. Collagen tissue engineering: development of novel biomaterials and applications. Pediatr Res. 2008;63:492–496. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A , Preat V. PLGAbased nanoparticles: an overview of biomedical applications. J Control Release. 2012;161:505–522. De Jong WH, Borm PJ. Drug delivery and nanoparticles:applications and hazards. Int J Nanomedicine. 2008;3:133–149. Even-Ram S, Yamada KM. Cell migration in 3D matrix. Curr Opin Cell Biol. 2005;17:524–532. Godugu C, Patel AR, Desai U, Andey T, Sams A , Singh M. AlgiMatrix based 3D cell culture system as an in-vitro tumor model for anticancer studies. PloS One 2013;8:e53708. Goodman TT, Ng CP, Pun SH. 3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. Bioconjug Chem. 2008;19:1951–1959. Grantab R, Sivananthan S, Tannock IF. The penetration of anticancer drugs through tumor tissue as a function of cellular adhesion and packing density of tumor cells. Cancer Res. 2006; 66:1033–1039. He W, Kuang Y, Xing X, Simpson RJ, Huang H, Yang T, et al. Proteomic comparison of 3D and 2D glioma models reveals increased HLA-E expression in 3D models is associated with resistance to NK cell-mediated cytotoxicity. J Proteome Res. 2014; 13:2272–2281. Kadam RS, Bourne DW, Kompella UB. Nano-advantage in enhanced drug delivery with biodegradable nanoparticles: contribution of reduced clearance. Drug Metab Dispos. 2012;40:1380–1388. Kuo C-T, Chiang C-L, Yun-Ju Huang R, Lee H, Wo AM. Configurable 2D and 3D spheroid tissue cultures on bioengineered surfaces with acquisition of epithelial–mesenchymal transition characteristics. NPG Asia Mater. 2012;4:1–8. Kuppen PJK, van der Eb MM, Jonges LE, Hagenaars M, Hokland ME, Nannmlark U, et al. Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer. Histochem Cell Biol. 2001;115:67–72. Lee GY, Kenny PA , Lee EH, Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods. 2007;4:359–365. Mikhail AS, Eetezadi S, Allen C . Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomedicines in vitro: a comparison of docetaxelloaded block copolymer micelles and Taxotere(R). PloS One 2013;8:e62630. Rajan N, Habermehl J, Coté MF, Doillon CJ, Mantovani D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc. 2006;1:2753–2758. Techatanawat S, Surarit R, Suddhasthira T, Khovidhunkit SOP. Type I collagen extracted from rat-tail and bovine Achilles tendon for dental application: a comparative study. Asian Biomed. 2011; 5:787–798. Thakor AS, Gambhir SS. Nanooncology: the future of cancer diagnosis and therapy. CACancer J Clin. 2013;63:395–418. Thorne RG, Nicholson C. In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space. Proc Natl Acad Sci U S A. 2006;103:5567–5572. Vrignaud S, Benoit JP, Saulnier P. Strategies for the nanoencapsulation of hydrophilic molecules in polymer-based nanoparticles. Biomaterials. 2011;32:8593–8604.
A collagen-based multicellular tumor spheroid model for evaluation of the efficiency of nanoparticle drug delivery Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Dalhousie University on 03/05/15 For personal use only.
Van-Minh Le1, Mei-Dong Lang2, Wei-Bin Shi3 & Jian-Wen Liu1 1Department of Molecular & Cellular Pharmacology, Biomedical Nanotechnology Center, State Key Laboratory of Bioreactor
Engineering & Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai , P.R. China, 2Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, China, and 3Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, P.R. China
radation)(Danhier et al. 2012, Kadam et al. 2012), and/or reducing their side effects by targeted delivery, for example in the case of cancer therapy (De Jong and Borm 2008), and finally, enhancing the therapeutic value of the drug (Thakor and Gambhir 2013, Vrignaud et al. 2011). While in vivo models can be used to assess the actual therapeutic values of these delivery systems better, the experimental procedure could be complex, with low throughput, and time-consuming. On the other hand, in traditional two-dimensional (2-D) cell cultures, the cells are directly exposed to drugs and therefore do not reflect the effect of restriction by many extracellular barriers in the body that could otherwise lead to significant reductions in the infiltration capacity and in the therapeutic effect of the drug-conjugated nanocarriers on the targeted tissue. It has been suggested that tumor cells respond very differently to therapeutic factors in vitro compared to in vivo, due to the lack of complex interactions within their specialized microenvironments (Kuo et al. 2012, Mikhail et al. 2013, He et al. 2014). There were also significant distinctions in terms of morphology, gene expression, drug resistance, migration and remodel matrices between 2-D and 3-D cultures, which was thought to closely mimic the cell-matrix interactions of in vivo conditions (Kuo et al. 2012, Cen et al. 2008, Aszodi et al. 2006, Even-Ram and Yamada 2005). Rat tail-type I collagen, extracted from rat tail tendons(Rajan et al. 2006), is one of the most widely used biomaterials in tissue engineering (Cen et al. 2008, Aszodi et al. 2006), due to its excellent biocompatibility and low antigenicity (Techatanawat et al. 2011). In this study, the tumor cells were embedded and cultured in a collagen scaffold to resemble the dimensional effects of the in vivo microenvironment of the tumor. Collagenbased tumor spheroids were developed and optimized using 95-D, U87 and HCT116 cells respectively. The delivery and
Abstract Targeted drug delivery systems, especially those that use nanoparticles, have been the focus of research into cancer therapy during the last decade, to improve the bioavailability and delivery of anticancer drugs to specific tumor sites, thereby reducing the toxicity and side effects to normal tissues. However, the positive antitumor effects of these nanocarriers observed in conventional monolayer cultures frequently fail in vivo, due to the lack of physical and biological barriers resembling those seen in the actual body. Therefore, the collagen-based 3-D multicellular culture system, to screen new nanocarriers for drug delivery and to obtain more adequate and better prediction of therapeutic outcomes in preclinical experiments, was developed. This 3-D culture model was successfully established using optimized density of cells. Our result showed that 3-D cell colonies were successfully developed from 95-D, U87 and HCT116 cell lines respectively, after a seven-day culture in the collagen matrix. The coumarin-conjugated nanoparticles were able to penetrate the matrix gel to reach the tumor cells. The model is supposedly more accurate in reflecting/predicting the dynamics and therapeutic outcomes of candidates for drug transport in vivo, and/or investigation of tumor biology, thus speeding up the pace of discovery of novel drug delivery systems for cancer therapy. Keywords: cancer, drug delivery system, nanoparticles, three dimensional model, tissue engineering
Introduction In recent years, nanoparticles have been studied extensively to develop delivery systems for chemotherapeutic agents. Nanoparticles can be engineered for multiple therapeutic purposes, including increasing the drug’s water solubility and stability (protecting it from denaturation and/or deg-
Correspondence: Jian-Wen Liu, Ph.D., East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237, People’s Republic of China. Tel/Fax: 021-64252044, 64252262. E-mail: [email protected] (Received 4 August 2014; revised 3 September 2014; accepted 12 September 2014)
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antitumor efficiencies of drug-conjugated nanoparticles in this tumor spheroid model were assessed by uptake and cytotoxicity studies.
Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by Dalhousie University on 03/05/15 For personal use only.
Materials The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 5-fluorouracil (5-FU) were purchased from Sigma (Sigma-Aldrich China, Inc., Shanghai, China). The 5-FU-loaded polymicelles and the coumarin-loaded polymicelles were synthesized and kindly provided by Professor Lang’s Lab (East China University of Science and Technology, Shanghai, China). These micelles had a mean diameter of 151.9 nm, with an encapsulation efficiency of 37.46%,, a polydispersity index of 0.155, and a zeta potential of ⫺ 0.29 mV.
Methods Cells and cell culture The human lung cancer cell line (95-D), which is highly metastatic, and the human colon cancer cell line (HCT116), were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The glioblastoma cell line (U87) was obtained from the American Type Culture Collection (ATCC, USA). The 95-D and HCT116 cells were cultured in RPMI 1640 medium (Hyclone Laboratories Inc., Australia) and the U87 cells were maintained in DMEM (Hyclone). The culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone), 100 U/ mL penicillin, and 100 μg/mL streptomycin (Gibco, Paisley, UK). The cells were maintained at 37°C in a humidified atmosphere of 95% O2 and 5% CO2, and were routinely passaged using trypsin/EDTA solution (Gibco) when they reached confluence. The cell suspension was harvested by trypsin digestion and used for the spheroid formation.
Multicellular spheroid culture To culture multicellular 3-D tumor spheres in the collagen gel, the cells were embedded in rat tail-type I collagen (Shengyou Biotechnology Company Ltd., Hangzhou, China) at the desired concentrations, after the cell culture surfaces were pre-chilled and pre-coated with a thin layer of collagen. The collagen gel was formed according to the manufacturer’s protocol and modified from reference (Lee et al. 2007). The volume medium containing a density of cells was obtained as follows: VMedium ⫽ VFresh medium ⫹ Vcollagen (5 mg/mL) ⫹ VNaOH (0.1 M) ⫹ V10⫻ (RPMI 1640 medium), where VNaOH ⫽ 0.06 ⫻ Vcollagen,V10⫻ ⫽ 0.01 ⫻ Vmedium. The appropriate volume of fresh medium was added after collagen gel formulation. The cell culture medium was changed every two days. Photographs were taken with a Nikon Eclipse Ti-S fluorescence microscope (MA, U.S.A.).
Cytotoxicity assay For a spheroid culture in collagen gel, monolayer tumor cells were trypsinized and centrifuged, and a final density of 3 ⫻ 104 cells/mL was resuspended in 1 mg/mL collagen gel and then immediately seeded onto a 96-well plate to
generate 3-D tumor spheroids. A proper volume of medium was added on top of the cell scaffold. The cells were adapted for five days. To test drug responses, cells in 2-D or 3-D cultures were incubated with a concentration of drug equivalent to 50 μg/mL, for 2 days. Prior to the finished culture in 3-D models, the matrix gel was digested by type I collagenase, centrifuged and re-incubated in a medium containing 10% MTT for 4 h. The absorbance was monitored by the EnSpire Multimode Plate Reader (PerkinElmer, Waltham, Massachusetts) at 492 and 630 nm. The rate of inhibition was calculated using the formula: Inhibition rate (%) ⫽ (1 – OD treatment/ OD control) ⫻ 100%. For the 2-D monolayer, 95-D cells (4 ⫻ 104 cells/mL) were seeded in a 96-well plate and were incubated for 24 h. Then, solutions of the drug were added to cells and further incubated for 48 h. Thereafter, the viability of cells was determined using the MTT method.
Confocal microscopy and estimation of nanoparticle penetration The spheroids obtained from collagen gel-embedded 3-D culture methods were fixed in a fixative (3:1 ratio of methanol and acetic acid glacial) for 10 min after the digestion of type I collagenase. They were washed three times with cold phosphate buffer saline (PBS) of pH 7.4, and then treated with propidium iodide (PI, 5 μg/mL, Sigma-Aldrich China, Inc., Shanghai, China) for staining of the nuclei. Thereafter, the spheroids were rinsed with PBS again and were transferred to microscope slides. To detect fluorescence, a Nikon A1 plus confocal laser scanning microscope system (Nikon Instech Co., Ltd., Tokyo, Japan) with a laser excitation line at 488 nm and an emission bandpass filter of 610‒630 nm was used. As described above, HCT116 cells were embedded in collagen gel and were cultured for 3 days. After that, coumarin-loaded nanoparticles (100 μg/mL) were added to the cell culture wells and incubated for 4 h at 37°C. After washing with PBS to remove unbound and excess components, the spheroids were visualized with fluorescent microscopy (Nikon Ti–S).
Results and discussion Morphology of tumor cells in 2-D vs 3-D To develop 3-D culture models, the 95-D, U87 and HCT116 cell lines were employed. The U87 cells had slow growth activity, compared to the HCT116 and 95-D which showed rapid growth and high malignancy (Figure 1). In the 2-D culture models, U87 cells exhibited the classic tear-drop shape and HCT116 exhibited an oval shape, while the 95-D cells showed spread-out, sheet-like trigonal or polygonal morphologies (Figure 1, left). By contrast, the morphology of cells cultured in the 3-D conditions appears dramatically different from that of cells cultured in the monolayer. The phase-contrast images revealed that the U87 (Figure 1B) and HCT116 (Figure 1C) cells formed tightly packed and rounded spheroids, but the 95-D (Figure 1A) cells were of irregular shapes, and loosely clustered. All three tumor cells were successfully entrapped, expanded, and propagated in 3-D collagen scaffolds, which
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Figure 1. Tumor cell morphology in different culture models. Typical morphology of cells grown in 2-D substrata (left) at day-2 vs. cells grown in the 3-D culture model (middle, 20⫻; right, 40⫻). The 95-D (A), U87 (B) and HCT116 (C) grown for 9 days in the collagen scaffold in the same conditions. Phase-contrast images were obtained at day-7 using an inverted microscope. Scale bar ⫽ 100 μm.
indicated that our collagen-based 3-D culture models could be used for these three cell line cultures.
Optimization of the density of cell seeding The density of cell seeding can affect cell growth, nutrition supply and reliability of experimental results. For optimization studies of the density of cell seeding and the culture time, 95-D cells were obtained from a monolayer culture and cell numbers were counted by Trypan blue exclusion methods. Then, the cells were transferred onto collagen gel matrices in replicates of 24 wells, at densities of 1 ⫻ 103, 1 ⫻ 104, 5 ⫻ 104 and 1 ⫻ 105 cells/mL for 3-D culture (Figure 2). The first three days were the adaptation period during which the
cells exhibited slow growth rate, and only small cell populations were generated. After that, the cell density increased and formed defined colonies. At a seeding density of 1 ⫻ 103 cells/mL, cell growth was slow and clusters were also formed with irregular shapes and with longer culture time. At the highest planting density of 1 ⫻ 105 cells/mL, the adaptation time was shorter than that for experiments with low cell seeding density. However, they formed smaller colonies compared to those at the densities of 1 ⫻ 104 or 5 ⫻ 104 cells/ mL. Furthermore, most of these cells died at day-7. The 95-D cell line formed middle clusters with a wide range of different sizes from day- 3. Seeding cells at 1 ⫻ 104 or 5 ⫻ 104 cells/ mL showed that cells had formed the multicellular spheroids
Figure 2. Morphological appearance of 95-D tumor cells cultured on the collagen gel with different seeding densities. Cultures maintained for 9 days and images captured every 3 days using light microscopy. Scale bar ⫽ 100 μm.
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V. M. Le et al.
Figure 3. Image of HCT116 cell spheroids stained with Propidium Iodide nuclear labeling dye. (A) Monolayer cells; (B) The organization of granule cell layer in multicellular spheroids; (C) The complete 3-D cells (nuclei) in 3-D image. All pictures were taken using confocal laser scanning microscopy.
after day-3, with a uniform size and shape. Little cell death was observed when seeding at the density of 5 ⫻ 104 cells/ mL during the last culture period. Based on these results, the seeding densities from 1 ⫻ 104 to 5 ⫻ 104 cells/mL was chosen for the subsequent research according to experimental design. The treatment time point was selected at day-5 after culture for 48 h of subsequent drug incubation.
Validation of established 3-D tumor models In the above experiments, the entire spheroid morphology was just illustrated in bright field microscopy. In order to gain further understanding of spheroid architecture, the fluorescent agent, propidium iodide (PI), was employed to visualize cells and extracellular matrix structures after fixation. PI diffused into cells and intercalated with nuclear and cytoplasmic nucleic acids of the fixative cells. As shown in Figure 3, cells cultured in the 2-D monolayer and in the multicellular spheroid culture model were stained with the fluorescent dye, thereby enabling visualization of the spheroids, which are composed of individual cells. Here, the difference in cell morphology and cell assembly in the 2-D monolayer vs. the 3-D model could present a different response by cells to nanoparticles. For the assessment of the interactions between nanoparticles and cancer cells, 95-D cells were individually cultured in 2-D or 3-D scaffolds for two or three days before treatment. Following that, 50 μg/mL of free 5-FU or 5-FU loaded polymer micelles (5-FU/PM) were added to the two
different culture models for 48 h, respectively. Then, the inhibition of the 5-FU formulation was determined by an MTT assay. Figure 4A shows that the tumor inhibition effect of the drugs in the 3-D culture model was lower than that in the 2-D monolayer culture. In the concentration used in this study, no significant difference in the effect of free 5-FU and 5-FU/PM was found using the 2-D model; however in the 3-D model, 5-FU/PM exhibited strikingly lower antitumor activity than free 5-FU . The inhibition rate of free 5-FU and 5-FU/PM were respectively 61.09 ⫾ 2.06 and 47.03 ⫾ 3.93% after 2 days, and 40.02 ⫾ 5.28 and 36.48 ⫾ 1.89% after 3 days of cultivation in the 3-D model. It could be due to small drug molecules that are nonpolar being able to diffuse across the membrane more readily than the large polymer micelles. The cytotoxicity assay in the 3-D cultures reveals that 3-D cell cultures in collagen gel showed significantly higher drug resistance compared to 2-D cell cultures, after treatments with 5-FU during the same period of time . Apart from the effect of growth inhibition, the morphological differences of cells between the control and treatment groups were also substantial. The size of the sphere, the reduction in cell density, irregular shape, and cellular shrinkage, were observed (Figure 4B). On the other hand, these results also showed that the spheres grew larger with time, suggesting a continuous reduction in drug sensitivity of the 3-D tumor spheroid to the drug (Figure 4A).
Figure 4. Cytotoxicity of 95-D cells induced by 5-FU formulations. (A) Growth inhibition of 5-FU or 5-FU/PM of tumor cells in different models was measured by MTT assay. Cells were incubated in 2-D model for 2 days or in 3-D for 5 days and then treated with 50 μg/mL 5-FU equivalent concentration of 5-FU/PM for 48 h. (B) Morphology of cells after exposure to 5-FU-loaded micelles for 48 h. Bright field images were captured using an inverted microscope to monitor morphology of cells in the control group (left) and treated group (right). Data from three independent experiments were presented as mean ⫾ SD, and the Student’s t-test was used to compare the means of two samples, ns: no significance; **p ⬍ 0.01; ***p ⬍ 0.001. Scale bar ⫽ 100 μm.
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This work was supported by the Shanghai Committee of Science and Technology (No. 13140902300), Nano Science and Technology Special Funding of the Shanghai Committee of Science and Technology (No. 11nm0503700), the Shanghai Committee of Science and Technology [grant 11DZ2260600] and the Fundamental Research Funds for the Central Universities.
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Figure 5. Fluorescence microscopy images of multicellular spheroids HCT116 cells incubated with coumarin-polymer micelles for 4 h. Left image is bright field and right is the fluorescence image.
To observe the ability of the nanoscale polymicelles to penetrate the collagen matrix, and whether death of cells was induced by 5-FU/MC, the HCT116 cells were seeded in the collagen scaffold for 3 days and subsequently incubated with coumarin-loaded polymicelles for 4 h. As shown in Figure 5, the penetration of micelles into the tumor spheroids was very limited. There was only a minor portion of the micelles infiltrating the matrix and translocating into the cells after 4 h of incubation (green fluorescence). This suggests that in 3-D tumor spheroids, the uptake of nanoparticles (large molecules) could be less efficient than that of the free drug (small molecules). Similarly, in vivo tumors have also been shown to exhibit poor nanoparticle penetration due to the presence of the extracellular matrix (Grantab et al. 2006, Kuppen et al. 2001, Goodman et al. 2008) and the formation of the tight junctions in the spheroid cell (Goodman et al. 2008, Thorne and Nicholson 2006). Hence, our 3-D culture models can closely mimic not only the dimensional obstacles as found in vivo, but can also reproduce the biological responses of the tumor to antitumor drugs, which have been associated with tumor initiation, progression and propensity to malignancy (invasion and metastasis)(Godugu et al. 2013). This result again demonstrated the rationality of the 3-D culture model established to evaluate the efficiency of the drug delivery system for cancer therapy.
Conclusion In this study, a befitting model based on type I collagen to evaluate the efficacy of drug carriers was established and optimized. The behavior of cells grown in the 3-D model, including their shape, density, interactions, drug sensitivity and drug resistance, reveal marked differences from those of cells cultured on the traditional monolayer. These results showed that the size of multicellular spheroids and agglomeration of cells can be controlled by optimizing the densities of the seeding cells. This 3-D culture model is applicable for in vitro screening of the drug, because it is cheaper than in vivo experiments and can recreate numerous features of living tissues such as the structure, function, microenvironment, and so on.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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