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Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells.

Nair MS, Mony U, Menon D, Koyakutty M, Sidharthan N, Pavithran K, Nair SV, Menon KN - Int J Nanomedicine (2015)

Bottom Line: Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems.Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest.Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo.

View Article: PubMed Central - PubMed

Affiliation: Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kerala, India.

ABSTRACT
Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

No MeSH data available.


Related in: MedlinePlus

Scanning electron microscopy (SEM) images of 3-D micro-nanofibrous polyurethane (PU)/poly-l-lactic acid (PLLA) scaffolds phase separated using thermally induced phase separation in tetrahydrofuran at −80°C.Notes: (A–C) 100% PU; (D–F) 80:20 PU/PLLA; (G–I) 60:40 PU/PLLA; (J–L) 50:50 PU/PLLA; (M–O) 40:60 PU/PLLA; (P–R) 20:80 PU/PLLA; (S–U) 100% PLLA; (V) SEM image of bone marrow extracellular matrix (ECM) showing the micro-nanofibrous structure. Note that the micro-nanofibrous nature of PU/PLLA 60:40 (I) is similar to that of bone marrow (V).
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f1-ijn-10-3603: Scanning electron microscopy (SEM) images of 3-D micro-nanofibrous polyurethane (PU)/poly-l-lactic acid (PLLA) scaffolds phase separated using thermally induced phase separation in tetrahydrofuran at −80°C.Notes: (A–C) 100% PU; (D–F) 80:20 PU/PLLA; (G–I) 60:40 PU/PLLA; (J–L) 50:50 PU/PLLA; (M–O) 40:60 PU/PLLA; (P–R) 20:80 PU/PLLA; (S–U) 100% PLLA; (V) SEM image of bone marrow extracellular matrix (ECM) showing the micro-nanofibrous structure. Note that the micro-nanofibrous nature of PU/PLLA 60:40 (I) is similar to that of bone marrow (V).

Mentions: In order to generate a scaffold with micro-nanofibrous morphology similar to that of decellularized bone marrow, we decided to exploit the properties of PU and PLLA using TIPS technology, as PLLA has been successfully used for the creation of nanofibrous morphology using TIPS technology.24,26 Unlike previous studies using PU/PLLA blends, as far as we are aware, no study has been conducted before that has analyzed a composite of PU/PLLA generated using TIPS strategy providing unique micro-nanofibrous morphology similar to decellularized bone marrow niche, as shown in this study.21 Thus, we generated different composites of PU/PLLA using TIPS. While 100% PU scaffolds exhibited a featureless film-like morphology devoid of any fibers (Figure 1A–C), 100% PLLA had a distinct, homogenous, complete nanofibrous structure (Figure 1S–U) throughout. The mixing of PU with varying concentrations of PLLA (20%, 40%, 50%, 60%, 80%, and 100%) resulted in PU/PLLA compositions of varying morphology. At ≤50% PU, the morphology of PU/PLLA composites showed nanofibrous structures with differently sized spherical structures of PU (Figure 1J–R). With the increasing concentration of PU (60% and 80%), the spherical structures got transformed into thick, fibrous structures at −80°C (Figure 1M–R). The morphology of the different composites of scaffolds was compared with an SEM image of human decellularized bone marrow extra cellular matrix (BM-ECM) (Figure 1V). Among the different composites, the PU/PLLA 60:40 scaffold showed indistinguishable similarity with the micro-nanofibrous nature of BM-ECM (Figure 1I vs V).


Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells.

Nair MS, Mony U, Menon D, Koyakutty M, Sidharthan N, Pavithran K, Nair SV, Menon KN - Int J Nanomedicine (2015)

Scanning electron microscopy (SEM) images of 3-D micro-nanofibrous polyurethane (PU)/poly-l-lactic acid (PLLA) scaffolds phase separated using thermally induced phase separation in tetrahydrofuran at −80°C.Notes: (A–C) 100% PU; (D–F) 80:20 PU/PLLA; (G–I) 60:40 PU/PLLA; (J–L) 50:50 PU/PLLA; (M–O) 40:60 PU/PLLA; (P–R) 20:80 PU/PLLA; (S–U) 100% PLLA; (V) SEM image of bone marrow extracellular matrix (ECM) showing the micro-nanofibrous structure. Note that the micro-nanofibrous nature of PU/PLLA 60:40 (I) is similar to that of bone marrow (V).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440427&req=5

f1-ijn-10-3603: Scanning electron microscopy (SEM) images of 3-D micro-nanofibrous polyurethane (PU)/poly-l-lactic acid (PLLA) scaffolds phase separated using thermally induced phase separation in tetrahydrofuran at −80°C.Notes: (A–C) 100% PU; (D–F) 80:20 PU/PLLA; (G–I) 60:40 PU/PLLA; (J–L) 50:50 PU/PLLA; (M–O) 40:60 PU/PLLA; (P–R) 20:80 PU/PLLA; (S–U) 100% PLLA; (V) SEM image of bone marrow extracellular matrix (ECM) showing the micro-nanofibrous structure. Note that the micro-nanofibrous nature of PU/PLLA 60:40 (I) is similar to that of bone marrow (V).
Mentions: In order to generate a scaffold with micro-nanofibrous morphology similar to that of decellularized bone marrow, we decided to exploit the properties of PU and PLLA using TIPS technology, as PLLA has been successfully used for the creation of nanofibrous morphology using TIPS technology.24,26 Unlike previous studies using PU/PLLA blends, as far as we are aware, no study has been conducted before that has analyzed a composite of PU/PLLA generated using TIPS strategy providing unique micro-nanofibrous morphology similar to decellularized bone marrow niche, as shown in this study.21 Thus, we generated different composites of PU/PLLA using TIPS. While 100% PU scaffolds exhibited a featureless film-like morphology devoid of any fibers (Figure 1A–C), 100% PLLA had a distinct, homogenous, complete nanofibrous structure (Figure 1S–U) throughout. The mixing of PU with varying concentrations of PLLA (20%, 40%, 50%, 60%, 80%, and 100%) resulted in PU/PLLA compositions of varying morphology. At ≤50% PU, the morphology of PU/PLLA composites showed nanofibrous structures with differently sized spherical structures of PU (Figure 1J–R). With the increasing concentration of PU (60% and 80%), the spherical structures got transformed into thick, fibrous structures at −80°C (Figure 1M–R). The morphology of the different composites of scaffolds was compared with an SEM image of human decellularized bone marrow extra cellular matrix (BM-ECM) (Figure 1V). Among the different composites, the PU/PLLA 60:40 scaffold showed indistinguishable similarity with the micro-nanofibrous nature of BM-ECM (Figure 1I vs V).

Bottom Line: Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems.Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest.Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo.

View Article: PubMed Central - PubMed

Affiliation: Amrita Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham University, Kerala, India.

ABSTRACT
Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-L-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34(+)/CD38(-)/CD33(-) phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27(Kip1) leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27(Kip1) in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

No MeSH data available.


Related in: MedlinePlus