Abstract

AbstractEndocytosis is an essential function of cells, with key roles in the internalisation of nutrients, signal molecules and also drugs. Endocytic processes are therefore widely investigated in the context of drug delivery, and inhibitors of endocytic pathways have been used to provide information regarding uptake mechanisms of drug carrier materials. Here we describe studies in which two established inhibitors of clathrin dependent and independent endocytosis, chlorpromazine and methyl‐β‐cyclodextrin respectively, were employed to probe endocytic pathways of three cell lines chosen to represent tumour‐relevant or associated phenotypes: 3 T3 (fibroblasts), HCT 116 (colon cancer) and MGLVA‐1 (gastric cancer). For clathrin mediated endocytosis the data highlight that chlorpromazine inhibition of transferrin internalization, via clathrin dependent endocytosis, is cell and time dependent. We also show that inhibition of uptake is transient with a resumption of transferrin internalization after a maximal inhibition period. The same endocytosis inhibitors were used to probe the internalization of 50 and 100 nm carboxylated polystyrene nanoparticles (C‐PS‐NPs) as model drug delivery carriers. Flow cytometry data indicated that internalisation of C‐PS‐NPs varied considerably with the incubation time of cells with chlorpromazine or methyl‐β‐cyclodextrin, and that the effects were also markedly cell‐line dependent. These data highlight that the effects of endocytosis inhibitors on the internalisation pathways even of relatively simple nanoparticles are complex and interdependent. We suggest that mechanistic investigations of the endocytic processes which govern practical applications of nanoparticles for diagnostic and therapeutic applications should be considered on a cell, time and concentration basis.

Highlights

  • In the specific context of nanoparticle delivery, we evaluate the use of chlorpromazine (CPZ), an established inhibitor of clathrin mediated endocytosis (CME), and methyl-β-cyclodextrin (MβCD), which extracts cholesterol from the membrane of cells and inhibits clathrin independent pathways (CIE), including those which take place via caveolae

  • The gastric cancer cells were derived from the MGLVA-1 line –as ascites of a variant of MKN45 human gastric adenocarcinoma cells and were cultured according to procedures developed in our laboratories.(Watson et al, 1990) Other cell lines including HCT 116 human colon cancer cells (Brattain et al, 1981), and 3 T3-Swiss albino mouse embryo fibroblasts were purchased from the American Type Culture Collection (ATCC) and LGC Standards, Teddington, UK

  • CPZ is a dopamine antagonist and has an amphiphilic structure enabling intercalation in the inner leaflet of cell membranes.(Ferrell Jr. et al, 1988) Its cationic portion interacts with the negative charge of phospholipids and, in particular, phosphoinositides.(Chen et al, 2003) CPZ has been reported to inhibit endocytosis by binding to calmodulin, which regulates the recruitment of the myristoylated alanine-rich C-kinase substrate protein (MARCKS), which in turn acts to sequester the phospholipid phosphatidylinositol 4,5bisphosphate (PI(4,5)P2).(Marshak et al, 1985) The process of CME requires the phospholipid in the binding of the AP-2 adaptor protein with the plasma membrane.(Eisenberg et al, 2008; Levin and Weiss, 1976) when CPZ binds to calmodulin, the cascade of protein and lipid recognition is disrupted and clathrin-mediated uptake is reduced

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Summary

Introduction

In the specific context of nanoparticle delivery, we evaluate the use of chlorpromazine (CPZ), an established inhibitor of clathrin mediated endocytosis (CME), and methyl-β-cyclodextrin (MβCD), which extracts cholesterol from the membrane of cells and inhibits clathrin independent pathways (CIE), including those which take place via caveolae. Carboxylated polystyrene nanoparticles (C-PSNPs) of 50 and 100 nm were chosen as simple, readily available models of drug delivery carriers and their internalization was probed in these three cell lines treated with the inhibitors at different incubation times. The data further indicate that the actions of CPZ and MβCD, which are widely used in drug delivery and cell biology experiments, are more elaborate than commonly reported and that experimental protocols using these inhibitors need to be adapted for the particular cell and carrier systems under investigation. Given the reported concerns on the low success rates for translating lab-based nanomedicines to the clinic, we believe the experiments reported here can help in the search for standardised and readily transferable studies of drug delivery nanoparticles in vitro

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