June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Carboplatin Loaded Small Extracellular Vesicles Reverse the Chemoresistance of RetinoblastomaY79 cell lines-Invitro
Author Affiliations & Notes
  • Attem Jyothi
    School of Medical Sciences, University of Hyderabad, Hyderabad, Telangana, India
  • Narayana RVL
    School of Medical Sciences, University of Hyderabad, Hyderabad, Telangana, India
  • Radhika Manukonda
    Operation Eyesight Universal Institute, LV Prasad Eye Institute, Hyderabad, Telangana, India
    Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad, Telangana, India
  • Ram Mukka Raju Jogula
    School of Medical Sciences, University of Hyderabad, Hyderabad, Telangana, India
  • Swathi Kaliki
    Operation Eyesight Universal Institute, LV Prasad Eye Institute, Hyderabad, Telangana, India
    Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad, Telangana, India
  • *Geeta K Vemuganti
    School of Medical Sciences, University of Hyderabad, Hyderabad, Telangana, India
  • Footnotes
    Commercial Relationships   Attem Jyothi None; Narayana RVL None; Radhika Manukonda None; Ram Mukka Raju Jogula None; Swathi Kaliki None; *Geeta K Vemuganti None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3278. doi:
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      Attem Jyothi, Narayana RVL, Radhika Manukonda, Ram Mukka Raju Jogula, Swathi Kaliki, *Geeta K Vemuganti; Carboplatin Loaded Small Extracellular Vesicles Reverse the Chemoresistance of RetinoblastomaY79 cell lines-Invitro. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3278.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Chemoresistance is an emerging challenge and is attributed to the small extracellular vesicles (sEVs) in the tumor through various mechanisms. Interestingly, small EVs are also being explored as a drug-delivery carrier system. Therefore, in the study, we evaluated the efficacy of sEVs derived from Rb Y79 cells, loaded with carboplatin (CPT) as a drug delivery vehicle to reverse the chemoresistance in drug- resistance clones derived from the same cell line.

Methods : Drug resistance (DR) RbY79 cells were derived by serial and increasing concentration of CPT. The sEVs were isolated from conditioned media of RbY79 cells by ultracentrifugation and then loaded with CPT by incubation method. The sEVs before and after drug uptake were characterized based on morphology (TEM, FE-SEM), size distribution (DLS), and phenotypic markers (WB). The encapsulation efficiency of CPT in sEV, release, and internalization of CPT-loaded sEVs by RbY79, pH 6.0, and pH 7.4 were measured by HPLC, confocal microscopy, and dialysis. The encapsulated sEVs were cryopreserved for 1 month to ensure stability and then evaluated for release and internalization. The cytotoxicity of CPT-loaded sEVs was assessed by MTT assay using the standard drug (CPT) as control

Results : DR clones derived from RbY79 cells showed six times greater inhibitory concentration compared to untreated RbY79 (IC50 41.38µg/mL and 6.17 µg/mL). The morphology of sEVs before and after drug uptake demonstrated typical cup shape structures with an average particle size distribution of 170 nm and 219 nm, a zeta potential of -12.8 mV. The sEVs were immunoreactive for CD63, CD9, TSG 101, and HSP 70. Zeta potential after cryopreservation at 4 °C was -15.06 mV, suggesting stable CPT-sEV. The entrapment efficiency of sEVs-CPT was (22.24%). The CPT release from the sEVs was increased at pH 6.0 compared to pH 7.4 (53.99±1.4%, and 50.74±2.44%), after 48 h. The cytotoxicity of CPT-sEVs was significantly higher in DR resistance cells as compared to CPT (IC50 28.18 µg/mL vs 41.21µg/mL) (P<0.05)

Conclusions : The study demonstrates that sequential exposure to CPT generates drug-resistant DR clones from the Rb Y 79 cell line which served as an appropriate model to evaluate the efficacy of CPT-loaded sEVs in reversing the chemoresistance, Further preclinical studies are required to validate these preliminary results of in-vitro experiments

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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