Correlation between Protonation of Tailor-Made Polypiperazines and Endosomal Escape for Cytosolic Protein Delivery

Abstract

Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or pK a value within the physiologically relevant range (pH 6–7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with pK a values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased pK a values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters

“…[168,178,179] Primarily, live cell microscopy is used, also allowing kinetic studies. [172,183] Less commonly, cells are studied after fixation. [178,190] Fixation with subsequent permeabilization may complicate the interpretation of the calcein assay due to false positive results as a result of fixation artifacts.…”

Stimuli-Responsive Thiomorpholine Oxide-Derived Polymers with Tailored Hydrophilicity and Hemocompatible Properties

“…However, this value is lower than that of polymers presenting a tertiary amine function, such as poly(2-(dimethyl amino)ethyl methacrylate PDMAEMA (pK a = 7.5) [22] and could be due to the steric hindrance of the heterocycle which renders the tertiary amine less accessible to protonation by the acid [23]. Hausig et al [13] reported low pK a values (between 6 and 7.1) for polymers comporting N-alkyl-piperazine units. Low pK a values were also observed for poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methylamino]-ethyl ester), [24] a polymer used in cellular transfection.…”

“…Changes in this physiological pH are often symptomatic of the presence of infections or cancer [12]. Morpholine-derived polymers contain tertiary amino groups which are protonated in acidic pH (pK a of morpholine = 8.3) [13,14]. This property can be used to tailor the behaviour of the polymer in different biological environments [13,14].…”

“…Morpholine-derived polymers contain tertiary amino groups which are protonated in acidic pH (pK a of morpholine = 8.3) [13,14]. This property can be used to tailor the behaviour of the polymer in different biological environments [13,14]. Moreover, poly(N-acryloylmorpholine) (PNAM) and copolymers of NAM present a high water solubility and biocompatibility and are thus particularly interesting for biological applications [15].…”

“…Moreover, poly(N-acryloylmorpholine) (PNAM) and copolymers of NAM present a high water solubility and biocompatibility and are thus particularly interesting for biological applications [15]. Furthermore, pH-responsive materials with pK a values ranging between 5 and 7.4 are further interesting candidates for entering the cells and facilitating a pHinduced endosomal escape [13,16].…”