Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle–Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage

In this study, interactions of polyethylene glycol (PEG)-coated gold nanoparticles (AuNPs) with cells were investigated with particular focus on the relationship between the PEG layer properties (conformation, grafting density, and hydrodynamic volume) and cell cycle arrest, apoptosis, and DNA damage. Steric hindrance and PEG hydrodynamic volume controlled the protein adsorption, whereas the AuNP core size and PEG hydrodynamic volume were primary factors for cell uptake and viability. At all PEG grafting densities, the particles caused significant cell cycle arrest and DNA damage against CaCo2 and PC3 cells without apoptosis. However, at a particular PEG grafting density (∼0.65 chains/nm²), none of these severe damages were observed on 3T3 cells indicating discriminating behavior of the healthy (3T3) and cancer (PC3 and CaCo2) cells. It was concluded that the PEG grafting density and hydrodynamic volume, tuned with the PEG concentration and AuNP size, played an important role in particle–cell interactions

Well-Defined Cholesterol Polymers with pH-Controlled Membrane Switching Activity

Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly­(methacrylic acid-<i>co</i>-cholesteryl methacrylate) P­(MAA-<i>co</i>-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5–7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV–visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer–lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter–Blue assay showed that the copolymers did not affect the cell viability up to 30 μM over a period of 72 h

Effect of Molecular Architecture on Cell Interactions and Stealth Properties of PEG

PEGylation, covalent attachment of PEG to therapeutic biomolecules, in which suboptimal pharmacokinetic profiles limiting their therapeutic utility are of concern, is a widely applied technology. However, this technology has been challenged by reduced bioactivity of biomolecules upon PEGylation and immunogenicity of PEG triggering immune response and abrogating clinical efficacy, which collectively necessitate development of stealth polymer alternatives. Here we demonstrate that comb-shape poly­[oligo­(ethylene glycol) methyl ether methacrylate] (POEGMA), a stealth polymer alternative, has a more compact structure than PEG and self-organize into nanoparticles in a molecular weight dependent manner. Most notably, we show that comb-shape POEGMA promotes significantly higher cellular uptake and exhibits less steric hindrance imposed on the conjugated biomolecule than PEG. Collectively, comb-shape POEGMA offers a versatile alternative to PEG for stealth polymer–biomolecule conjugation applications

Assessment of Cholesterol-Derived <i>Ionic</i> Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived <i>ionic</i> copolymers were previously synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization as ‘smart’ gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly­(methacrylic acid-co-cholesteryl methacrylate) (P­(MAA-<i>co</i>-CMA)) and cationic poly­(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P­(DMAEMA-<i>co</i>-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P­(DMAEMA-<i>co</i>-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P­(MAA-<i>co</i>-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P­(MAA-<i>co</i>-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P­(MAA-<i>co</i>-CMA)-DNA) and 20:1 (Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P­(MAA-<i>co</i>-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P­(DMAEMA-<i>co</i>-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA particles are extremely promising candidates for <i>in vivo</i> applications of siRNA therapy

Insight into Serum Protein Interactions with Functionalized Magnetic Nanoparticles in Biological Media

Surface modification with linear polymethacrylic acid (20 kDa), linear and branched polyethylenimine (25 kDa), and branched oligoethylenimine (800 Da) is commonly used to improve the function of magnetite nanoparticles (MNPs) in many biomedical applications. These polymers were shown herein to have different adsorption capacity and anticipated conformations on the surface of MNPs due to differences in their functional groups, architectures, and molecular weight. This in turn affects the interaction of MNPs surfaces with biological serum proteins (fetal bovine serum). MNPs coated with 25 kDa branched polyethylenimine were found to attract the highest amount of serum protein while MNPs coated with 20 kDa linear polymethacrylic acid adsorbed the least. The type and amount of protein adsorbed, and the surface conformation of the polymer was shown to affect the size stability of the MNPs in a model biological media (RPMI-1640). A moderate reduction in <i>r</i>₂ relaxivity was also observed for MNPs suspended in RPMI-1640 containing serum protein compared to the same particles suspended in water. However, the relaxivities following protein adsorption are still relatively high making the use of these polymer-coated MNPs as Magnetic Resonance Imaging (MRI) contrast agents feasible. This work shows that through judicious selection of functionalization polymers and elucidation of the factors governing the stabilization mechanism, the design of nanoparticles for applications in biologically relevant conditions can be improved

Keto-Functionalized Polymer Scaffolds as Versatile Precursors to Polymer Side-Chain Conjugates

A new methacrylate monomer with a reactive ketone side chain, 2-(4-oxopentanoate)­ethyl methacrylate (PAEMA), was synthesized and subsequently polymerized by reversible addition–fragmentation chain transfer (RAFT) polymerization to give a polymer with a narrow molecular weight distribution (PDI = 1.25). The polymer was chain extended with poly­(ethylene glycol methyl ether methacrylate) (PEGMA) to yield a block copolymer. Aminooxy-containing small molecules and oligoethylene glycol were conjugated to the ketone functionality of the side chains in high yields. Cytotoxicity of the oxime-linked tetra­(ethylene glycol) polymer to mouse fibroblast cells was investigated; the polymer was found to be noncytotoxic up to 1 mg/mL. The ease with which this polymer is functionalized suggests that it may be useful in forming tailored polymeric medicines

Dicer-Labile PEG Conjugates for siRNA Delivery

Poly­(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3′-sense or 5′-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3′-sense or 5′-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3′-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells