Since the development of simple polymer conjugates, many new polymers for drug delivery have been developed that employ a huge array of functions that control biodistribution, bioavailability, biodegradability, cell targeting, and subcellular trafficking. Polymers of different types are incorporated into these nanocarriers that include micelles, polymersomes, dendrimers, nanohydrogels, nanoparticles and nanotubules (129). However, these innovations are still in their infancy and basic knowledge of how these materials interact with living organisms, and how to control the various biological functions of these materials remains lacking. The motivation of the work described in Chapter 4 was to gain a systematic understanding of how cells can interact with a variety of synthetic materials.
A review of contemporary literature revealed that there are large gaps in our understanding of how synthetic macromolecules interact with living cells. Experiments designed to directly transduce HPMA copolymers into the cytoplasm of living cells was difficult to evaluate since little is known about how basic physio-chemical characteristics of synthetic materials affect the distribution and transport of nanoparticles within cells. Basic information, such as the diffusivity of synthetic polymers through cytoplasm, was unknown or incomplete. Other general characterizations were also incomplete. Little was
known regarding how charge, molecular weight, and relative hydrophilicity affect binding to cell membranes, cell uptake, intracellular trafficking, membrane transduction, cell toxicity, intracellular distribution and fate.
1.7.1 An HPMA Copolymer Array
HPMA copolymers are extremely versatile for incorporating monomers containing various other chemical moieties and functional groups. In contrast with PEG, the incorporation of methacryloylated comonomers is straightforward and the contents of the resulting copolymers is predictable based on the ratios of the feed monomers. Given this flexibility, a large array of HPMA copolymers was created that possessed a very wide range of different charges, charge densities and hydrophilicities (Figure 1.8.). The comonomers used included weak and strong acids, weak and strong bases, and methacryoylated (MA) peptide monomers with varying numbers of hydrophobic amino acids. The general reaction scheme is shown in Figure 1.9. Each polymer was fractionated based on molecular weight using identical size-exclusion chromatography protocols to create parallel “ladders” of copolymer molecular weight standards.
This work represents a systematic approach to characterizing how the physio- chemical features of water soluble polymers interact with the cell. In one experimental series, each of the polymers was microinjected in live cultured cells and the distribution of the cells over time was monitored in detail using time-lapse confocal microscopy (130). In the other experimental series, live cells were incubated in media containing each copolymer and the differences in cell uptake and trafficking were observed by flow cytometry and by confocal microscopy. Details of the differences in the endocytic pathways taken by each
copolymer in this array were studied by Liu et al., and are summarized in Chapter 5 (131).
1.7.2 Nuclear Entry
Special interest was devoted to the rates of entry into the nucleus. Initial microinjection experiments using array copolymers revealed significant differences in the rates of polymer diffusion into the nuclei. Cytoplasmic macromolecules are typically excluded from entering membrane-limited organelles, such as mitochondria, lysosomes, the ER, in a nonspecific manner. The exception to this is the nucleus whose membrane possesses channels that allow the passive uptake of intermediate-sized macromolecules. The NPC (nuclear pore complex) of the nuclear envelope is composed of about 30 different nucleoporin proteins and is the conduit for both nuclear import and export of macromolecules, such as proteins and nucleic acids. In active transport, cargos as large as 40 nm possessing NLS (nuclear localization sequence) or NES (nuclear export sequence) signaling peptides are guided through the channel after binding to NTR (nuclear transport receptor) proteins (132). For smaller macromolecules below 10 nm, however, NPCs have been shown to act as nonspecific pores that allow exchange between the nucleus and cytoplasm by diffusion (133). As a conduit for nonbiological macromolecules, the NPCs have been shown to transmit PEG-coated gold colloid particles 4-7 nm in diameter (134). In contrast, 27 nm and 39 nm PEG-gold particles and 25 nm quantum dot particles conjugated with NLS peptide sequences have been shown to require the active nuclear import mechanism for uptake by the nucleus (132, 134, 135).
Dynamic structural modeling of several of the nucleoporins in the core of the NPC has been used to explain the dual size selection of active vs. passive nuclear transport
selection. The structure of several nucleoporins notably contains natively unfolded domains containing hydrophobic FG (phenylalanine-glycine) domains, consisting of FG, GFLG, and FXFG peptide repeats. Several different models are currently under debate to account for the dynamics of NPC transport. Ribbeck et al. (136, 137) proposed that the FG domains form a polymer brush conformation in the NPC channel that excludes larger solutes by entropic hydrophobic repulsion. Frey et al. suggested that nucleoporins form an unstructured hydrogel mesh in the core of the NPC channel through reversible crosslinks of FG peptide repeats that they possess (138). Nuclear transport receptors have been shown to possess a number of hydrophobic regions that specifically bind to nucleoporins in their FG repeat regions and have been proposed to disassociate interchain crosslinks. The pore proteins, thereby, form a 3-dimensional mesh with a simple molecular weight permeability cut-off when crosslinked. In this model of active transport, NTRs are able to open the nucleoporin mesh by “melting” the hydrogel structure and guide large macromolecules in or out of the nuclear compartment (139). Alternately, Melčák et al. proposed an interface between the α-helical regions of nucleoporins that selectively slide circumferentially to dilate the NPC pore opening after NTR binding (140).
Since this work was done, many models have been proposed for the structure and function of the NPC. However, the subject currently remains an active area of contention (141-145). Models developed since the time of this research will be discussed in detail in Chapter 5.