Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer-doxorubicin

Seib, Friedrich Philipp, Jones, Arwyn Tomos ORCID: https://orcid.org/0000-0003-2781-8905 and Duncan, Ruth 2006. Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer-doxorubicin. Journal of drug targeting 14 (6) , pp. 375-390. 10.1080/10611860600833955

Full text not available from this repository.

Abstract

Polymer therapeutics are being designed for lysosomotropic, endosomotropic and transcellular drug delivery. Their appropriate intracellular routing is thus crucial for successful use. For example, polymer–anticancer drug conjugates susceptible to lysosomal enzyme degradation will never deliver their drug payload unless they encounter the appropriate activating enzymes. Many studies use confocal microscopy to monitor intracellular fate, but there is a pressing need for more quantitative methods able to define intracellular compartmentation over time. Only then will it be possible to optimise the next generation of polymer therapeutics for specific applications. The aim of this study was to establish a subcellular fractionation method for B16F10 murine melanoma cells and subsequently to use it to define the intracellular trafficking of N-(2-hydroxyproplylmethacrylamide) (HPMA) copolymer-bound doxorubicin (PK1). Free doxorubicin was used as a reference. The cell cracker method was used to achieve cell breakage and optimised to reproducibly achieve 90% breakage efficiency. This ensured that subsequent subcellular fractionation experiments were representative for the whole cell population. To characterise the subcellular fractions obtained by differential centrifugation, DNA (nuclei), succinate dehydrogenase (mitochondria), N-acetyl-β-glucosaminidase (lysosomes), alkaline phosphatase (plasma membrane) and lactate dehydrogenase (cytosol) were selected as markers and their assay was carefully validated. The relative specific activity (RSA) of the fractions obtained from B16F10 cells were: nuclei (2.2), mitochondria (4.1), lysosomes (3.7) and cytosol (2.5). When used to study the intracellular distribution at non-toxic concentrations of PK1 and doxorubicin, time-dependent accumulation of PK1 in lysosomes was evident and the expected nuclear localisation of free doxorubicin was seen. Live cell fluorescence microscopy and confocal co-localisation studies gave qualitative corroboration of these results, but by using this method, we were unable to accurately define organelle localisation. In conclusion, the B16F10 subcellular fractionation method developed here provides a useful tool to allow comparison of the intracellular trafficking of other polymer conjugates.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Pharmacy
Subjects: R Medicine > RM Therapeutics. Pharmacology
R Medicine > RS Pharmacy and materia medica
Uncontrolled Keywords: Subcellular fractionation, differential centrifugation, marker enzymes, endocytosis, PK1, polymer therapeutics
Publisher: Taylor & Francis
ISSN: 1061-186X
Last Modified: 19 Oct 2022 10:45
URI: https://orca.cardiff.ac.uk/id/eprint/25410

Citation Data

Cited 39 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item Edit Item