Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

Abstract

Endocytic uptake and subcellular trafficking of a large array of HPMA (N-(2-hydroxypropyl)methacrylamide) based copolymers possessing positively or negatively charged residues, or hydrophobic groups were evaluated by flow cytometry and living cell confocal microscopy in cultured prostate cancer cells. The degrees of cellular uptake of various copolymer fractions with narrow polydispersities were quantified. The copolymer charge was the predominant physicochemical feature in terms of cellular uptake. Fast and efficient uptake occurred in positively charged copolymers due to non-specific adsorptive endocytosis, whereas slow uptake of negatively charged copolymers was observed. The uptake of copolymers was also molecular weight dependent. The copolymers were internalized into the cells through multiple endocytic pathways: positively charged copolymers robustly engaged clathrin-mediated endocytosis, macropinocytosis and dynamin-dependent endocytosis, while weakly negatively charged copolymers weakly employed these pathways; strongly negatively charged copolymers only mobilized macropinocytosis. HPMA copolymer possessing 4 mol% of moderately hydrophobic functional groups did not show preferential uptake. All copolymers ultimately localized in late endosomes/lysosomes via early endosomes; with varying kinetics among the copolymers. This study indicates that cell entry and subsequent intracellular trafficking of polymeric drug carriers are strongly dependent on the physicochemical characteristics of the nanocarrier, such as charge and molecular weight.

Introduction

Employment of macromolecular drug carriers for the delivery of anticancer therapeutics has resulted in significant improvements in treatment efficacy [1]. A variety of macromolecular drug carriers are currently under investigation. An ideal macromolecular drug carrier should possess preferable pharmacokinetics, highly efficient cellular uptake and preferential subcelluar trafficking. These biological merits are determined to a large extent by physicochemical characteristics of the macromolecular drug carriers. For instance, molecular weight has an impact on the intravascular half-life and renal clearance [2]. Positively charged macromolecular drug carriers are internalized by cells more efficiently than negatively charged carriers, however their uptake occurs in the majority of cells [3]. Nevertheless, how the physicochemical characteristics of drug carriers determine their uptake and subcellular trafficking remains largely unknown.

Understanding the uptake and subcellular trafficking of macromolecular drug carriers by cells relies on knowledge of the complexity and diversity of endocytic pathways. Many endocytic pathways have been identified and a variety of classification schemes have been proposed. Based on the cargo, endocytosis is classified as phagocytosis or pinocytosis (fluid-phase endocytosis) [4]. Based on receptors, endocytosis can be designated as receptor mediated endocytosis specified by involvement of specific high-affinity receptors, adsorptive endocytosis denoted by nonspecific binding of solutes to the cell membrane, and regular endocytosis taking up surrounding fluid unspecifically [5]. Most common classification schemes of endocytosis are based on protein machinery that facilitates the process, such as clathrin-mediated endocytosis, and clathrin independent endocytosis [5], [6], [7], [8]. Clathrin independent endocytosis is further categorized as caveolae-mediated endocytosis and clathrin- and caveolin-independent endocytosis [5], [7] or dynamin-dependent and dynamin-dependent endocytosis [7], [8]. Dynamin is a GTPase protein that surrounds the neck of vesicle pits and facilitates the scission of many, but not all vesicles, such as clathrin-coated, caveolae-mediated and clathrin- and caveolin-independent vesicles [9]. Macropinocytosis is a distinct pathway of pinocytosis [5], [10], [11]; traditionally, macropinocytosis was designated as bulk non-selective and constitutive uptake of extracellular fluid through plasma membrane protrusion or ruffling. It has been recently recognized as an elaborately coordinated process including actin-mediated membrane reorganization and regulation of signaling, such as phosphoinositide (PI) 3-kinases [10], [11].

Hydrophilic and neutral copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) have been broadly used as linear water soluble polymeric drug carriers for therapeutic applications [12], [13]. Several drug-polymer conjugates based on HPMA copolymers have been studied clinically. A doxorubicin–HPMA copolymer conjugate PK1 was the first HPMA copolymer drug conjugate to enter clinical trials [14]. Phase II trial of PK1 in breast, non-small cell lung and colorectal cancers suggested that polymer-bound therapeutics are capable of improving anticancer activities [15]. PK2 [16], a compound related to PK1, and several HPMA copolymer drug conjugates containing taxol, camptothecin, and platinum entered Phase I clinical trials [17], [18], [19], [20]. HPMA copolymers are internalized into cells through endocytosis as shown in an early study on HPMA copolymers containing different degradable peptidyl side chains using rat visceral yolk sacs cultured in vitro [21]. Incorporation of hydrophobic tyrosinamide residues, bound either directly to the polymer main chain or through a glycylglycine linker, enhanced the uptake of HPMA copolymers [22]. More recently, endocytic uptake and subcellular trafficking of a HPMA antibody conjugate have been studied [23].

In the present study, the relationship between the mechanisms of endocytic uptake and subcellular trafficking of a large array of fluorescently-labeled, HPMA based copolymers and their physicochemical characteristics, such as charge, molecular weight and hydrophobicity, was studied. The array was composed of 9 HPMA copolymers with 5 comonomers possessing functional groups with positive or negative charges or containing a short hydrophobic peptide. FITC-labeled polyHPMA (P-FITC) was used as control. These copolymers were fractionated using size exclusion chromatography to create parallel Mw “ladders” consisting of 10 narrowly polydisperse fractions with molecular weights ranging from 10 to 200 kDa. Cellular uptake of the HPMA copolymer fractions and involvement of different endocytic pathways were assessed by flow cytometry in cultured prostate cancer cells. Subcellular trafficking of membrane vesicles carrying copolymers was characterized by living cell confocal microscopy.