Synthesis and Antitumor Activity of Doxycycline Polymeric Nanoparticles: Effect on Tumor Apoptosis in Solid Ehrlich Carcinoma
Abstract
:1. Introduction
2. Results
2.1. Estimation of Encapsulation Efficiency and Loading Capacity of DOXY-PNPs
2.2. The Particle Size, ZP and PDI of DOXY-PNPs
2.3. The In-Vitro Release of DOXY from the Prepared PNPs
2.4. The Surface Morphology of the Prepared DOXY-PNPs
2.5. Selecting the Best Formulation
2.6. In-Vivo Pharmacological Activity for DOXY-PNP3
2.6.1. Survival Data
2.6.2. Mass of the Solid Tumors
2.6.3. Quantitative Polymerase Chain Reaction for Tumoral mRNA Expression of BAX and Caspase 3
2.6.4. Assessment of Histologic Features and Tumor Necrosis in Sections Stained with H&E
2.6.5. BAX and Caspase 3 Immunostaining in the Solid Tumors
2.6.6. Simple Linear Regression Analysis for the Tumor Data
3. Discussion
3.1. Characterization of DOXY-PNPs
3.2. In-Vivo Pharmacological Activity of DOXY-PNPs
4. Materials and Methods
4.1. Materials
4.2. Synthesis of Doxycycline Polymeric Nanoparticles
4.2.1. Doxycycline Polymeric Nanoparticle Preparation
4.2.2. Estimation of DOXY-PNPs Encapsulation Efficiency and Loading Capacity
4.2.3. Determination of Particle Size, Zeta Potential and Polydispersity Index of the Prepared DOXY-PNPs
4.2.4. The Surface Morphology Determination Using Transmission Electron Microscopy
4.2.5. The In-vitro Release Study of DOXY from Prepared PNPs
4.2.6. The Selection of the Best Formulation of DOXY-PNPs
4.3. The Pharmacological Activity of DOXY-PNPs
4.3.1. Animals
4.3.2. Inoculation of Ehrlich Solid Tumors in Female Mice
4.3.3. Experimental Design
4.3.4. Sacrification and Separation of Solid Tumors
4.3.5. Quantitative polymerase chain reaction (PCR) of caspase 3 and BAX genes
- RNA Extraction: Homogenized tumor samples from the study groups were lysed and RNAeasy Mini Kit (Qiagen) Kit was used for isolation of total RNA which was examined for both quality and quantity with a dual spectrophotometer (Beckman, Waltham, MA, USA).
- Real Time PCR: For quantitative determination of mRNAs expression of caspase 3 and BAX, the following procedure was performed. A quantity of 10 ng of the total RNA was directed for the synthesis of cDNA employing the Applied Biosystems high capacity cDNA reverse transcriptase kit (USA). Amplification of the cDNA was then achieved in a 48-well plate by Syber Green I PCR Master Kit (Fermentas) using the Step One instrument (Applied Biosystems, Waltham, MA, USA). Alterations in gene expression were normalized to the mean critical threshold values obtained with GAPDH by the ΔΔCt method [82]. 1 μM of the gene primers was used in the assay and are demonstrated in Table 3.
4.3.6. Histologic Investigation of the Solid Ehrlich Carcinomas
4.3.7. Immunohistochemical Determination of BAX and Caspase 3
4.3.8. Data Analysis
5. Conclusion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
Formula Code | Drug: Polymer Mass Ratio | HPMC Concentration | Tween 80 Concentration | Organic: Aqueous Phase |
---|---|---|---|---|
PNP1 | 1:2 | 0.8 g% | 1% | 1:8 |
PNP2 | 1:1 | |||
PNP3 | 2:1 |
Formula Code | EE % | Drug Loading % | Particle Size (nm) | ZP (mv) | PDI |
---|---|---|---|---|---|
PNP1 | 42.15 ± 0.84 | 30.29 ± 0.15 | 203.6 ± 1.4 | −21.8 ± 4.89 | 0.431 ± 0.02 |
PNP2 | 56.78 ± 0.52 £ | 49.57 ± 0.11 £ | 489.7 ± 6.7 £ | −23 ± 5.68 | 0.851 ± 0.05 £ |
PNP3 | 84.65 ± 0.93 £,& | 66.13 ± 0.36 £,& | 615.3 ± 8.3 £,& | −15.1 ± 4.84 | 1.00 ± 0.04 £,& |
Target Gene | Primer Sequence: 5′-3′ |
---|---|
Caspase-3 | F: ATGTCAGCTCGCAATGG R: AAGAAATTATGGAATTG |
BAX | F: CAGATCATGAAGACAGG R: GTGGATACAGACTCCCC |
GAPDH | F: TAGGTATATGTTAAATTT R: GCTGACATTTAGGTAGAA |
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Gardouh, A.R.; Attia, M.A.; Enan, E.T.; Elbahaie, A.M.; Fouad, R.A.; El-Shafey, M.; Youssef, A.M.; Alomar, S.Y.; Ali, Z.A.-E.; Zaitone, S.A.; et al. Synthesis and Antitumor Activity of Doxycycline Polymeric Nanoparticles: Effect on Tumor Apoptosis in Solid Ehrlich Carcinoma. Molecules 2020, 25, 3230. https://doi.org/10.3390/molecules25143230
Gardouh AR, Attia MA, Enan ET, Elbahaie AM, Fouad RA, El-Shafey M, Youssef AM, Alomar SY, Ali ZA-E, Zaitone SA, et al. Synthesis and Antitumor Activity of Doxycycline Polymeric Nanoparticles: Effect on Tumor Apoptosis in Solid Ehrlich Carcinoma. Molecules. 2020; 25(14):3230. https://doi.org/10.3390/molecules25143230
Chicago/Turabian StyleGardouh, Ahmed R., Mohammed A. Attia, Eman T. Enan, Alaaeldeen M. Elbahaie, Rania A. Fouad, Mohamed El-Shafey, Amal M. Youssef, Suliman Y. Alomar, Zinab Abd-Elhady Ali, Sawsan A. Zaitone, and et al. 2020. "Synthesis and Antitumor Activity of Doxycycline Polymeric Nanoparticles: Effect on Tumor Apoptosis in Solid Ehrlich Carcinoma" Molecules 25, no. 14: 3230. https://doi.org/10.3390/molecules25143230