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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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Transfection efficiency and apoptotic activity of wt-p53 loaded gelatin nanoparticles. (a) p53 mRNA expression in tumors. Results are presented as mean ± SD (n = 3 for day 7 and 18; n = 6 for day 33). mRNA expression level for downstream apoptotic markers after (b) day 7, (c) day 18 and (d) day 33. (n = 3 for day 7 and 18; n = 6 for day 33) (*p < 0.05; **p < 0.01; ***p < 0.001). (e) Western blot analysis for p53, cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (f) TUNEL analysis of apoptotic activity in the wt-p53 treated tumors. Sections of tumor tissues imaged after treatment for 7, 18 and 33 days for TUNEL positive (brown) cells. All images were acquired at 20× with scale bar of 50 μm.
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Figure 2: Transfection efficiency and apoptotic activity of wt-p53 loaded gelatin nanoparticles. (a) p53 mRNA expression in tumors. Results are presented as mean ± SD (n = 3 for day 7 and 18; n = 6 for day 33). mRNA expression level for downstream apoptotic markers after (b) day 7, (c) day 18 and (d) day 33. (n = 3 for day 7 and 18; n = 6 for day 33) (*p < 0.05; **p < 0.01; ***p < 0.001). (e) Western blot analysis for p53, cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (f) TUNEL analysis of apoptotic activity in the wt-p53 treated tumors. Sections of tumor tissues imaged after treatment for 7, 18 and 33 days for TUNEL positive (brown) cells. All images were acquired at 20× with scale bar of 50 μm.

Mentions: The expression of wt-p53 and downstream apoptotic markers in the tumors after day 7, 18 and 33 were confirmed quantitatively by qPCR (Figure 2a-d) and qualitatively by western blot analysis (Figure 2e). SH-Gel-PEG-peptide treated tumors show remarkably higher levels of p53 mRNA (p < 0.001) as well as protein after 7 and 18 days of treatment while SH-Gel-PEG treated tumors also show higher p53 mRNA expression compared to control. Naked plasmid on the other hand did not show any change in mRNA level, confirming that a delivery system is essential for successful transfection and gene activity in vivo. Further, no significant p53 mRNA or protein expression was observed in any of the treatment groups (Figure 2a,e).


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)

Transfection efficiency and apoptotic activity of wt-p53 loaded gelatin nanoparticles. (a) p53 mRNA expression in tumors. Results are presented as mean ± SD (n = 3 for day 7 and 18; n = 6 for day 33). mRNA expression level for downstream apoptotic markers after (b) day 7, (c) day 18 and (d) day 33. (n = 3 for day 7 and 18; n = 6 for day 33) (*p < 0.05; **p < 0.01; ***p < 0.001). (e) Western blot analysis for p53, cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (f) TUNEL analysis of apoptotic activity in the wt-p53 treated tumors. Sections of tumor tissues imaged after treatment for 7, 18 and 33 days for TUNEL positive (brown) cells. 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 2: Transfection efficiency and apoptotic activity of wt-p53 loaded gelatin nanoparticles. (a) p53 mRNA expression in tumors. Results are presented as mean ± SD (n = 3 for day 7 and 18; n = 6 for day 33). mRNA expression level for downstream apoptotic markers after (b) day 7, (c) day 18 and (d) day 33. (n = 3 for day 7 and 18; n = 6 for day 33) (*p < 0.05; **p < 0.01; ***p < 0.001). (e) Western blot analysis for p53, cleaved PARP, cleaved caspase 3 and β-actin protein expression in treated tumors. (f) TUNEL analysis of apoptotic activity in the wt-p53 treated tumors. Sections of tumor tissues imaged after treatment for 7, 18 and 33 days for TUNEL positive (brown) cells. All images were acquired at 20× with scale bar of 50 μm.
Mentions: The expression of wt-p53 and downstream apoptotic markers in the tumors after day 7, 18 and 33 were confirmed quantitatively by qPCR (Figure 2a-d) and qualitatively by western blot analysis (Figure 2e). SH-Gel-PEG-peptide treated tumors show remarkably higher levels of p53 mRNA (p < 0.001) as well as protein after 7 and 18 days of treatment while SH-Gel-PEG treated tumors also show higher p53 mRNA expression compared to control. Naked plasmid on the other hand did not show any change in mRNA level, confirming that a delivery system is essential for successful transfection and gene activity in vivo. Further, no significant p53 mRNA or protein expression was observed in any of the treatment groups (Figure 2a,e).

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