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A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

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Related in: MedlinePlus

This figure shows the scanning electron microscope picture of a Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticle containing 1.25% chitosan and 5% gelatin (100 mg PLGA, 1.25 mg chitosan, and 5 mg gelatin). Surface morphology of microparticles was characterized by scanning electron microscopy (SEM). The average diameter of formulated microparticle in this study ranged from 145 μm to 162 μm.
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f2: This figure shows the scanning electron microscope picture of a Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticle containing 1.25% chitosan and 5% gelatin (100 mg PLGA, 1.25 mg chitosan, and 5 mg gelatin). Surface morphology of microparticles was characterized by scanning electron microscopy (SEM). The average diameter of formulated microparticle in this study ranged from 145 μm to 162 μm.

Mentions: In the current study, we prepared a 3-D tumor model with PLGA–gelatin microparticles for in vitro evaluation of different anticancer drugs (native and loaded in nanoparticles). It was previously well documented that gelatin is a suitable candidate for scaffold materials because of its biocompatibility, low immunogenicity, and biodegradability. Gelatin contains Arg–Gly–Asp (RGD)-like sequences that promote cell adhesion and migration by forming a polyelectrolyte complex. Furthermore, the anionic cell surface provided by the gelatin–chitosan-blended microparticles provide better cell adhesion and cellular bioactivity for proliferating cells. A successful microparticle formulation was achieved with a highly porous matrix to facilitate the infiltration of proliferating cells. As evidenced from the SEM image, the formulated microparticle showed a porous infrastructure with interconnected void structures and had a spherical shape covered with a thin film of polymer (Figure 2). This rough surface could be due to deposition of cationic chitosan on the anionic microparticle surface, which is apparent from the surface characteristics [7]. Similarly, particle size is an important parameter that could affect the degradation of the polymer matrix [10]. To achieve a microparticle of a desired diameter, the conditions for emulsification and formulation composition were optimized. The average diameter of the formulated microparticle in this study ranged from 145 μm to 162 μm.


A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

This figure shows the scanning electron microscope picture of a Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticle containing 1.25% chitosan and 5% gelatin (100 mg PLGA, 1.25 mg chitosan, and 5 mg gelatin). Surface morphology of microparticles was characterized by scanning electron microscopy (SEM). The average diameter of formulated microparticle in this study ranged from 145 μm to 162 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369889&req=5

f2: This figure shows the scanning electron microscope picture of a Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticle containing 1.25% chitosan and 5% gelatin (100 mg PLGA, 1.25 mg chitosan, and 5 mg gelatin). Surface morphology of microparticles was characterized by scanning electron microscopy (SEM). The average diameter of formulated microparticle in this study ranged from 145 μm to 162 μm.
Mentions: In the current study, we prepared a 3-D tumor model with PLGA–gelatin microparticles for in vitro evaluation of different anticancer drugs (native and loaded in nanoparticles). It was previously well documented that gelatin is a suitable candidate for scaffold materials because of its biocompatibility, low immunogenicity, and biodegradability. Gelatin contains Arg–Gly–Asp (RGD)-like sequences that promote cell adhesion and migration by forming a polyelectrolyte complex. Furthermore, the anionic cell surface provided by the gelatin–chitosan-blended microparticles provide better cell adhesion and cellular bioactivity for proliferating cells. A successful microparticle formulation was achieved with a highly porous matrix to facilitate the infiltration of proliferating cells. As evidenced from the SEM image, the formulated microparticle showed a porous infrastructure with interconnected void structures and had a spherical shape covered with a thin film of polymer (Figure 2). This rough surface could be due to deposition of cationic chitosan on the anionic microparticle surface, which is apparent from the surface characteristics [7]. Similarly, particle size is an important parameter that could affect the degradation of the polymer matrix [10]. To achieve a microparticle of a desired diameter, the conditions for emulsification and formulation composition were optimized. The average diameter of the formulated microparticle in this study ranged from 145 μm to 162 μm.

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

Show MeSH
Related in: MedlinePlus