Hydrophobicity Regulates the Cellular Interaction of Cyanine5-Labeled Poly(3-hydroxypropionate)-Based Comb Polymers.

An in-depth understanding of the effect of physicochemical properties of nanocarriers on their cellular uptake and fate is crucial for the development of novel delivery systems. In this study, well-defined hydrophobic carboxylated poly(3-hydroxypropionate)-based comb polymers were synthesized. Two oligo(3-hydroxypropionate) (HPn) of different degrees of polymerization (DP; 5 and 9) bearing α-vinyl end-groups were obtained by an hydrogen transfer polymerization (HTP)-liquid/liquid extraction strategy. 2-Carboxyethyl acrylate (CEA), representing the DP 1 analogue of HPn, was also included in the study. (Macro)monomers were polymerized via reversible addition-fragmentation chain-transfer (RAFT) polymerization and fully characterized by 1H NMR spectroscopy and size exclusion chromatography. All polymers were non-hemolytic and non-cytotoxic against NIH/3T3 cells. Detailed cellular association and uptake studies of Cy5-labeled polymers by flow cytometry and confocal laser scanning microscopy (CLSM) revealed that the carboxylated water-soluble PCEA, the polymer with the shortest side chain, efficiently targets mitochondria. However, increasing the side-chain DP led to a change in the intracellular fate. P(HP5) was trafficked to both mitochondria and lysosomes, while P(HP9) was exclusively found in lysosomes. Importantly, FLIM-FRET investigation of P(HP5) provided initial insight into the mitochondria subcompartment location of Cy5-labeled carboxylated polymers. Moreover, intracellular uptake mechanism studies were performed. Blocking scavenger receptors by dextran sulfate or cooling cells to 4 °C significantly affected the cell association of hydrophobic carboxylated polymers with an insignificant response to membrane-potential inhibitors. In contrast, water-soluble carboxylated polymers' cellular association was substantially inhibited in cells treated with compounds depleting the mitochondrial potential (ΔΨ). Overall, this study highlights hydrophobicity as a valuable means to tune the cellular interaction of carboxylated polymers and thus will inform the design of future drug carriers based on Cy5-modified carboxylated polymers.

Polymeric Nanotubes as Drug Delivery Vectors─Comparison of Covalently and Supramolecularly Assembled Constructs.

Rod-shaped nanoparticles have been identified as promising drug delivery candidates. In this report, the in vitro cell uptake and in vivo pharmacokinetic/bio-distribution behavior of molecular bottle-brush (BB) and cyclic peptide self-assembled nanotubes were studied in the size range of 36-41 nm in length. It was found that BB possessed the longest plasma circulation time (t1\2 > 35 h), with the cyclic peptide system displaying an intermediate half-life (14.6 h), although still substantially elevated over a non-assembling linear control (2.7 h). The covalently bound BB underwent substantial distribution into the liver, whereas the cyclic peptide nanotube was able to mostly circumvent organ accumulation, highlighting the advantage of the inherent degradability of the cyclic peptide systems through their reversible aggregation of hydrogen bonding core units.

An Evaluation of Optimal PEGylation Strategies for Maximizing the Lymphatic Exposure and Antiviral Activity of Interferon after Subcutaneous Administration.

Interferon α2 is an antiviral/antiproliferative protein that is currently used to treat hepatitis C infections and several forms of cancer. Two PEGylated variants of interferon α2 (containing 12 and 40 kDa PEGs) are currently marketed and display longer plasma circulation times than that of unmodified interferon. With increasing realization that the lymphatic system plays an important role in the extrahepatic replication of the hepatitis C virus and in the metastatic dissemination of cancers, this study sought to evaluate PEGylation strategies to optimally enhance the antiviral activity and plasma and lymphatic exposure of interferon after subcutaneous administration in rats. The results showed that conjugation with a linear 20 kDa PEG provided the most ideal balance between activity and plasma and lymph exposure. A linear 5 kDa PEG variant also exhibited excellent plasma and lymph exposure to interferon activity when compared to those of unmodified interferon and the clinically available linear 12 kDa PEGylated construct.

Depolymerization of hyaluronan using PEGylated human recombinant hyaluronidase promotes nanoparticle tumor penetration.

Aim: Delivery of nanoparticles (NPs) to tumors can be impeded by high levels of hyaluronan (HA) in the stroma. Enzymatic depolymerization of HA with PEGylated hyaluronidase (PEGPH20) improves the delivery of antibodies to tumors. However, it is unknown whether NP delivery is enhanced by this strategy. Methods: The impact of PEGPH20 pretreatment on the uptake and tumor penetration of model PEGylated polystyrene NPs was studied in mice with orthotopic breast cancers. Results: Tumor oxygenation and NP penetration, but not overall tumor uptake, of 50 nm NPs, was significantly enhanced by PEGPH20 pre-administration. Conclusion: PEGPH20 has the potential to improve intratumoral penetration of NP-based drug delivery systems and enhance access to cancer cells in poorly vascularized regions of the tumor.

Local inflammation alters the lung disposition of a drug loaded pegylated liposome after pulmonary dosing to rats.

The development of inhalable 'nanomedicines' based on biocompatible lipids and polymers is attracting increasing interest worldwide. Our understanding of how pulmonary inflammation impacts on lung distribution and clearance kinetics however, is limited. Similarly, there is limited information on how the inhaled delivery of biocompatible nanomaterials affects existing respiratory disease. We have addressed these knowledge gaps by describing and comparing the pulmonary pharmacokinetic behaviour of a 3H-labelled PEGylated liposome loaded with a model drug (ciprofloxacin) after intratracheal administration to healthy rats and rats with bleomycin-induced lung inflammation by following both 3H label and drug. Cell- and cytokine-based markers of lung inflammation were used to evaluate the response of healthy and inflamed lungs to the liposome. Liposomes were initially cleared more rapidly from inflamed lungs than from healthy lungs, but exhibited similar rates of lung clearance after 3 days. This was interesting given that mucociliary clearance was more efficient from healthy lungs, despite evidence of higher mucus retention in inflamed lungs and reduced association of the liposome with lung tissue. Although the plasma pharmacokinetics of ciprofloxacin did not differ between rats with healthy or inflamed lungs after pulmonary administration, the plasma pharmacokinetics of 3H-phosphatidylcholine suggested higher liposome bioavailability and more prolonged absorption from inflamed lungs. Concentrations of the pro-inflammatory cytokine IL-1ß were increased in bronchoalveolar lavage fluid after a single pulmonary dose of liposomes to rats with inflamed lungs, but no other significant changes in lung inflammatory markers were identified in healthy or bleomycin-challenged rats.

A 30 kDa polyethylene glycol-enfuvirtide complex enhances the exposure of enfuvirtide in lymphatic viral reservoirs in rats.

HIV therapy with anti-retroviral drugs is limited by the poor exposure of viral reservoirs, such as lymphoid tissue, to these small molecule drugs. We therefore investigated the effect of PEGylation on the anti-retroviral activity and subcutaneous lymphatic pharmacokinetics of the peptide-based fusion inhibitor enfuvirtide in thoracic lymph duct cannulated rats. Both the peptide and the PEG were quantified in plasma and lymph via ELISA. Conjugation to a single 5 kDa linear PEG decreased anti-HIV activity three-fold compared to enfuvirtide. Whilst plasma and lymphatic exposure to peptide mass was moderately increased, the loss of anti-viral activity led to an overall decrease in exposure to enfuvirtide activity. A 20 kDa 4-arm branched PEG conjugated with an average of two enfuvirtide peptides decreased peptide activity by six-fold. Plasma and lymph exposure to enfuvirtide, however, increased significantly such that anti-viral activity was increased two- and six-fold respectively. The results suggest that a multi-enfuvirtide-PEG complex may optimally enhance the anti-retroviral activity of the peptide in plasma and lymph.

'Stealth' lipid-based formulations: poly(ethylene glycol)-mediated digestion inhibition improves oral bioavailability of a model poorly water soluble drug.

For over 20years, stealth drug delivery has been synonymous with nanoparticulate formulations and intravenous dosing. The putative determinants of stealth in these applications are the molecular weight and packing density of a hydrophilic polymer (commonly poly(ethylene glycol) (PEG)) that forms a steric barrier at the surface of the nanoparticle. The current study examined the potential translation of the concepts learned from stealth technology after intravenous administration to oral drug delivery and specifically, to enhance drug exposure after administration of oral lipid-based formulations (LBFs) containing medium-chain triglycerides (MCT). MCT LBFs are rapidly digested in the gastrointestinal tract, typically resulting in losses in solubilisation capacity, supersaturation and drug precipitation. Here, non-ionic surfactants containing stealth PEG headgroups were incorporated into MCT LBFs in an attempt to attenuate digestion, reduce precipitation risk and enhance drug exposure. Stealth capabilities were assessed by measuring the degree of digestion inhibition that resulted from steric hindrance of enzyme access to the oil-water interface. Drug-loaded LBFs were assessed for maintenance of solubilising capacity during in vitro digestion and evaluated in vivo in rats. The data suggest that the structural determinants of stealth LBFs mirror those of parenteral formulations, i.e., the key factors are the molecular weight of the PEG in the surfactant headgroup and the packing density of the PEG chains at the interface. Interestingly, the data also show that the presence of labile ester bonds within a PEGylated surfactant also impact on the stealth properties of LBFs, with digestible surfactants requiring a PEG Mw of ~1800g/mol and non-digestible ether-based surfactants ~800g/mol to shield the lipidic cargo. In vitro evaluation of drug solubilisation during digestion showed stealth LBFs maintained drug solubilisation at or above 80% of drug load and reduced supersaturation in comparison to digestible counterparts. This trend was also reflected in vivo, where the relative bioavailability of drug after administration in two stealth LBFs increased to 120% and 182% in comparison to analogous digestible (non-stealth) formulations. The results of the current study indicate that self-assembled "stealth" LBFs have potential as a novel means of improving LBF performance.