Liposomal Drug Delivery System for Treatment of Drug Resistant Ovarian Cancer

Samantha Gardner, Kharimat L. Alatise, Angela A. Alexander-Bryant, PhD
Department of Bioengineering, Clemson University, Clemson, SC

Introduction

Ovarian cancer is the fifth leading cause of cancer mortality in women, with four out of five patients diagnosed in advanced stages, III and IV [1]. Late detection significantly reduces the survival rate from 90% in women with localized, early-stage diagnosis, to 30% when diagnosed in the late stages [2]. A debulking surgery prior to platinum-based chemotherapy is the current standard of care for treatment of ovarian cancer [2]. However, recurrence is a common problem, with nearly 75% of women who respond to initial platinum-based treatment experiencing relapse due to drug resistance [3]. Liposomes are a promising solution to overcoming drug resistance due to their biocompatibility and capacity to encapsulate both hydrophilic and hydrophobic drugs as well as complex small interfering RNA (siRNA). siRNAs are double-stranded RNA molecules that have the potential to downregulate the expression of genes related to drug resistance [4]. However, a drug delivery vehicle is necessary for the transport of siRNAs due to degradation of the molecules in a biological environment [4]. We aim to synthesize and characterize a liposomal system to deliver siRNA and paclitaxel to ovarian cancer cells.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754410/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754410/

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Methods

Cholesterol (CHOL) and cholesteryl hemisuccinate (CHEMS) liposomes were synthesized.

https://www.sigmaaldrich.com/technical-documents/articles/biology/liposome-preparation.html

https://www.sigmaaldrich.com/technical-documents/articles/biology/liposome-preparation.html

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Uptake of liposomes into OVCAR3 and OVCAR3-T40, a wild-type and a paclitaxel-resistant human adenocarcinoma cell line, was examined using fluorescence microscopy. Basal expression of ABCB1 was identified using immunoblotting in OVCAR3 and OVCAR3-T40 cells.

Results

Figure 1. Characterization of CHOL and CHEMS Liposomes. Size, PDI, and Zeta potential were measured using DLS. siRNA encapsulation efficiency was determined using fluorescent siRNA. HPLC was used to ascertain paclitaxel loading efficiency.

Figure 2. Cytotoxicity of liposomes on sensitive ovarian cancer cells. OVCAR-3 cells were treated at various concentrations of blank CHOL and CHEMS liposomes for 48 hrs. Viability was assessed via MTS assay.

Figure 3. Cellular uptake of coumarin 6 loaded CHOL liposomes on sensitive and resistant ovarian cancer cells. Coumarin 6 loaded CHOL liposomes were imaged using fluorescent microscopy. Nuclei are stained with DAPI (blue). Coumarin 6 is green.

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Figure 4. Basal expression of target genes. Protein expression of ABCB1 was assessed via immunoblotting on sensitive, OVCAR3, and resistant, OVCAR3-T40, ovarian cancer cell lines.

Conclusions

  • Liposomes were successfully formed with a monodisperse size, exhibited effective drug and siRNA loading, and were internalized in OVCAR3 and OVCAR3-T40 cells
  • Unloaded liposomes were not cytotoxic to both the wild-type and drug-resistant cell lines
  • Cytotoxicity of drug-loaded liposomes can be attributed to paclitaxel, siRNA or combination treatment
  • CHOL liposomes were successfully internalized in OVCAR3 and OVCAR3-T40 cells

Future Work

  • Examining expression of JAK2 and CFLAR in sensitive and resistant cell lines
  • Examine knockdown of ABCB1, JAK2, and CFLAR after treatment with liposomal system
  • Viability with paclitaxel and ABCB1, paclitaxel and JAK2, and paclitaxel and CFLAR

Acknowledgements

Advisor: Dr. Angela Alexander-Bryant, PhD

Liposome Group: Lora Alatise, Emily Grant, and Emily Miller, MS

Lab Members: Jessie Boulos, Serena Gilmore, Anthony Hazelton, Alex Nukovic, Megan Pitz, Timmy Samec

Thank you to George Duran from Stanford University for the donation of the resistant OVCAR3 cells.

Funding: This work was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Any Opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect those of the National Science Foundation

References

[1] Torre, L. A., CA Cancer J Clin. 2018;68(4):284-296

[2] Cortez, A. J., Cancer Chemother Pharmacol. 2018;81(1):17-38

[3], Norouzi-Barough L., J Cell Physiol. 2018;233(6):4546–4562

[4] Farra R., Pharmaceutics. 2019;11(10):547

[5] Miller, Emily Margaret, “Liposomal Combination Drug and siRNA Delivery to Combat Drug-Resistant Ovarian Cancer” (2018). All Theses. 2997.

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