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Redox-responsive targeted gelatin nanoparticles for delivery of combination wt-p53 expressing plasmid DNA and gemcitabine in the treatment of pancreatic cancer.

Xu J, Singh A, Amiji MM - BMC Cancer (2014)

Bottom Line: Efficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression.Gene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups.Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and significant decrease in the anti-apoptotic transcription factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA. m.amiji@neu.edu.

ABSTRACT

Background: Pancreatic adenocarcinoma is one of the most dreaded cancers with very low survival rate and poor prognosis to the existing frontline chemotherapeutic drugs. Gene therapy in combination with a cytotoxic agent could be a promising approach to circumvent the limitations of previously attempted therapeutic interventions.

Method: We have developed a redox-responsive thiolated gelatin based nanoparticle system that efficiently delivers its payload in the presence of glutathione-mediated reducing intra-cellular environment and could be successfully used for site-specific wt-p53 expressing plasmid DNA as well as gemcitabine delivery by targeting epidermal growth factor receptor (EGFR). Efficacy studies were performed in subcutaneous human adenocarcinoma bearing SCID beige mice along with molecular level p53 plasmid and apoptotic marker expression by PCR and western blot for all study groups.

Results: Efficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression. In all the treatment groups, the targeted nanoparticles showed better anti-tumor activity than their non-targeted as well as non-encapsulated, naked therapeutic agent counterparts (50.1, 61.7 and 77.3% tumor regression by p53 plasmid alone, gemcitabine alone and in combination respectively). Molecular analysis revealed a higher mRNA expression of transfected p53 gene, its corresponding protein and that the tumor cell death in all treatment groups was due to the induction of apoptotic pathways.

Conclusions: Gene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups. Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and significant decrease in the anti-apoptotic transcription factors.

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Assessment of apoptosis mediated cell death induction by gemcitabine conjugated gelatin nanoparticles. (a) mRNA expression profile for transcription factors in the apoptotic pathway (n = 6, *p < 0.05; **p < 0.01; ***p < 0.001). (b) Western blot analysis for cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (c) TUNEL stained images of treated tumors indicating DNA fragmentation (brown stain). All images were acquired at 20× with scale bar of 50 μm.
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Figure 4: Assessment of apoptosis mediated cell death induction by gemcitabine conjugated gelatin nanoparticles. (a) mRNA expression profile for transcription factors in the apoptotic pathway (n = 6, *p < 0.05; **p < 0.01; ***p < 0.001). (b) Western blot analysis for cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (c) TUNEL stained images of treated tumors indicating DNA fragmentation (brown stain). All images were acquired at 20× with scale bar of 50 μm.

Mentions: The mechanism for gemcitabine action is based on DNA damage that triggers the apoptotic pathway. Effect of gemcitabine treatment on apoptosis was evaluated quantitatively by measurement of mRNA levels of transcription factors (Bax, Bcl-2, DR5, Apaf-1, PUMA, caspase 3 and caspase 9) by qPCR and qualitatively by protein analysis. Targeted nanoparticles significantly increased mRNA expression of all the pro-apoptotic transcription factors (p < 0.001) while Gem-Gel-PEG and Gem-Gel nanoparticles were also able to increase the expression of these transcription factors, but to lesser extent (Figure 4a). Besides, anti-apoptotic transfection factor Bcl-2 mRNA level was slightly decreased with nanoparticle treatments. Protein analysis by western blot validated these results, where both cleaved caspase 3 and cleaved PARP show a moderate increase with nanoparticles treatment (Figure 4b) where the highest protein level was observed in Gem-Gel-PEG-peptide treated tumors. Gemcitabine induced DNA damage was also assessed by TUNEL staining, which showed maximum TUNEL-positive cells in targeted nanoparticle treatment group compared to non-targeted and non-PEG nanoparticle treatment (Figure 4c). These studies demonstrated that active targeted delivery of gemcitabine into tumors showed enhanced anti-tumor activity by successfully triggering the apoptotic pathway.


Redox-responsive targeted gelatin nanoparticles for delivery of combination wt-p53 expressing plasmid DNA and gemcitabine in the treatment of pancreatic cancer.

Xu J, Singh A, Amiji MM - BMC Cancer (2014)

Assessment of apoptosis mediated cell death induction by gemcitabine conjugated gelatin nanoparticles. (a) mRNA expression profile for transcription factors in the apoptotic pathway (n = 6, *p < 0.05; **p < 0.01; ***p < 0.001). (b) Western blot analysis for cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (c) TUNEL stained images of treated tumors indicating DNA fragmentation (brown stain). All images were acquired at 20× with scale bar of 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3927583&req=5

Figure 4: Assessment of apoptosis mediated cell death induction by gemcitabine conjugated gelatin nanoparticles. (a) mRNA expression profile for transcription factors in the apoptotic pathway (n = 6, *p < 0.05; **p < 0.01; ***p < 0.001). (b) Western blot analysis for cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (c) TUNEL stained images of treated tumors indicating DNA fragmentation (brown stain). All images were acquired at 20× with scale bar of 50 μm.
Mentions: The mechanism for gemcitabine action is based on DNA damage that triggers the apoptotic pathway. Effect of gemcitabine treatment on apoptosis was evaluated quantitatively by measurement of mRNA levels of transcription factors (Bax, Bcl-2, DR5, Apaf-1, PUMA, caspase 3 and caspase 9) by qPCR and qualitatively by protein analysis. Targeted nanoparticles significantly increased mRNA expression of all the pro-apoptotic transcription factors (p < 0.001) while Gem-Gel-PEG and Gem-Gel nanoparticles were also able to increase the expression of these transcription factors, but to lesser extent (Figure 4a). Besides, anti-apoptotic transfection factor Bcl-2 mRNA level was slightly decreased with nanoparticle treatments. Protein analysis by western blot validated these results, where both cleaved caspase 3 and cleaved PARP show a moderate increase with nanoparticles treatment (Figure 4b) where the highest protein level was observed in Gem-Gel-PEG-peptide treated tumors. Gemcitabine induced DNA damage was also assessed by TUNEL staining, which showed maximum TUNEL-positive cells in targeted nanoparticle treatment group compared to non-targeted and non-PEG nanoparticle treatment (Figure 4c). These studies demonstrated that active targeted delivery of gemcitabine into tumors showed enhanced anti-tumor activity by successfully triggering the apoptotic pathway.

Bottom Line: Efficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression.Gene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups.Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and significant decrease in the anti-apoptotic transcription factors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA. m.amiji@neu.edu.

ABSTRACT

Background: Pancreatic adenocarcinoma is one of the most dreaded cancers with very low survival rate and poor prognosis to the existing frontline chemotherapeutic drugs. Gene therapy in combination with a cytotoxic agent could be a promising approach to circumvent the limitations of previously attempted therapeutic interventions.

Method: We have developed a redox-responsive thiolated gelatin based nanoparticle system that efficiently delivers its payload in the presence of glutathione-mediated reducing intra-cellular environment and could be successfully used for site-specific wt-p53 expressing plasmid DNA as well as gemcitabine delivery by targeting epidermal growth factor receptor (EGFR). Efficacy studies were performed in subcutaneous human adenocarcinoma bearing SCID beige mice along with molecular level p53 plasmid and apoptotic marker expression by PCR and western blot for all study groups.

Results: Efficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression. In all the treatment groups, the targeted nanoparticles showed better anti-tumor activity than their non-targeted as well as non-encapsulated, naked therapeutic agent counterparts (50.1, 61.7 and 77.3% tumor regression by p53 plasmid alone, gemcitabine alone and in combination respectively). Molecular analysis revealed a higher mRNA expression of transfected p53 gene, its corresponding protein and that the tumor cell death in all treatment groups was due to the induction of apoptotic pathways.

Conclusions: Gene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups. Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and significant decrease in the anti-apoptotic transcription factors.

Show MeSH
Related in: MedlinePlus