Hervé Hillaireau

Nanocarriers’ entry into the cell: relevance to drug delivery

Nanocarriers offer unique possibilities to overcome cellular barriers in order to improve the delivery of various drugs and drug candidates, including the promising therapeutic biomacromolecules (i.e., nucleic acids, proteins). There are various mechanisms of nanocarrier cell internalization that are dramatically influenced by nanoparticles’ physicochemical properties. Depending on the cellular uptake and intracellular trafficking, different pharmacological applications may be considered. This review will discuss these opportunities, starting with the phagocytosis pathway, which, being increasingly well characterized and understood, has allowed several successes in the treatment of certain cancers and infectious diseases. On the other hand, the non-phagocytic pathways encompass various complicated mechanisms, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis, which are more challenging to control for pharmaceutical drug delivery applications. Nevertheless, various strategies are being actively investigated in order to tailor nanocarriers able to deliver anticancer agents, nucleic acids, proteins and peptides for therapeutic applications by these non-phagocytic routes.

Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells

Background Because of the described hazards related to inhalation of manufactured nanoparticles, we investigated the lung toxicity of biodegradable poly (lactide-co-glycolide) (PLGA) nanoparticles displaying various surface properties on human bronchial Calu-3 cells. Methods Positively and negatively charged as well as neutral nanoparticles were tailored by coating their surface with chitosan, Poloxamer, or poly (vinyl alcohol), respectively. Nanoparticles were characterized in terms of size, zeta potential, and surface chemical composition, confirming modifications provided by hydrophilic polymers. Results Although nanoparticle internalization by lung cells was clearly demonstrated, the cytotoxicity of the nanoparticles was very limited, with an absence of inflammatory response, regardless of the surface properties of the PLGA nanoparticles. Conclusion These in vitro results highlight the safety of biodegradable PLGA nanoparticles in the bronchial epithelium and provide initial data on their potential effects and the risks associated with their use as nanomedicines.

Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma.

The EWS–Fli1 fusion gene encodes for a chimeric oncogenic transcription factor considered to be the cause of the Ewing sarcoma. The efficiency of small interfering RNAs (siRNAs) targeted toward the EWS–Fli1 transcript (at the junction point type 1) was studied, free or encapsulated into recently developed polyisobutylcyanoacrylate aqueous core nanocapsules. Because this mRNA sequence is only present in cancer cells, it therefore constituted a relevant target. Studies of the intracellular penetration by confocal microscopy in NIH/3T3 EWS–Fli1 cells showed that nanocapsules improved the intracellular penetration of siRNA with mainly a cytoplasmic localization. These biodegradable siRNA-loaded nanocapsules were then tested in vivo on a mice xenografted EWS–Fli1-expressing tumor; they were found to trigger a dose-dependant inhibition of tumor growth after intratumoral injection. A specific inhibition of EWS–Fli1 was observed, too. These findings now open new prospects for the treatment of experimental cancers with junction oncogenes.

Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor.

The mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand.

Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells

In vitro cytotoxicity and inflammatory response following exposure to nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) have been investigated on A549 human lung epithelial cells. Three different PLGA NPs (230 nm) were obtained using different stabilizers (polyvinyl alcohol, chitosan, or Pluronic(®) F68) to form respectively neutral, positively or negatively charged NPs. Polystyrene NPs were used as polymeric but non-biodegradable NPs, and titanium dioxide (anatase and rutile) as inorganic NPs, for comparison. Cytotoxicity was evaluated through mitochondrial activity as well as membrane integrity (lactate dehydrogenase release, trypan blue exclusion, propidium iodide staining). The cytotoxicity of PLGA-based and polystyrene NPs was lower or equivalent to the one observed after exposure to titanium dioxide NPs. The inflammatory response, evaluated through the release of the IL-6, IL-8, MCP-1, TNF-α cytokines, was low for all NPs. However, some differences were observed, especially for negative PLGA NPs that led to a higher inflammatory response, which can be correlated to a higher uptake of these NPs. Taken together, these results show that both coating of PLGA NPs and the nature of the core play a key role in cell response.

Biodegradable Nanoparticles Meet the Bronchial Airway Barrier: How Surface Properties Affect Their Interaction with Mucus and Epithelial Cells

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.

Physicochemical characteristics and preliminary in vivo biological evaluation of nanocapsules loaded with siRNA targeting estrogen receptor alpha.

Specific siRNAs that target estrogen receptor alpha (ERalpha) were encapsulated in nanocapsules (NCs). We produced small (approximately 100-200 nm) ERalpha-siRNA NCs with a water core by incorporating two mixed duplexes of specific ERalpha-siRNAs (ERalpha-mix-siRNA) into NCs. The encapsulation yield that was obtained with poly(iso-butylcyanoacrylate) (PIBCA) NCs was low, whereas no release of trapped siRNA was observed for poly(ethylene)glycol-poly(D,L-lactide-co-glycolide) (PEG-PLGA) NCs. High levels of ERalpha-siRNA incorporation into PEG-epsilon-caprolactone-malic acid (PEG-PCL/MA) NCs (3.3 microM in a polymer solution at 16 mg/mL) were observed (72% yield). No difference in size or zeta potential was observed between siRNA NCs that were based on PEG-PCL/MA and empty NCs. Fluorescence quenching assays confirmed the incorporation of siRNA into the NC core. A persistent loss of ERalpha (90% over 5 days) was observed in MCF-7 human breast cancer cells that were exposed to PEG-PCL/MA NCs that were loaded with ERalpha-siRNA. The intravenous injection of these NCs into estradiol-stimulated MCF-7 cell xenografts led to a significant decrease in tumor growth and a decrease in ERalpha expression in tumor cells. These data indicate that a novel strategy, based on ERalpha-siRNA delivery, could be developed for the treatment of hormone-dependent breast cancers.

Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages

The purpose of this study was to investigate the toxicity of a series of poly(lactide-co-glycolic) (PLGA) nanoparticles on human-like THP-1 macrophages. Positively-, negatively-charged and neutral nanoparticles (200 nm) were prepared using chitosan (CS), poloxamer 188 (PF68) and poly(vinyl alcohol) (PVA) as stabilizer. Stabilizer-free PLGA nanoparticles were obtained as well. When used at therapeutically relevant concentrations (up to 0.1 mg/mL in vitro), all tested nanoparticles showed no or scarce signs of toxicity, as assessed by cell mitochondrial activity, induction of apoptosis and necrosis, production of intracellular reactive oxygen species (ROS) and secretion of pro-inflammatory cytokines. At high concentrations (above 1mg/mL), cytotoxicity was found to be induced by the presence of stabilizers, whatever the toxicological pattern of the stabilizer itself. While stabilizer-free PLGA nanoparticles exerted no cytotoxicity, the slightly cytotoxic CS polymer conferred PLGA nanoparticles significant cytotoxicity when used as nanoparticle stabilizer; more surprisingly, the otherwise innocuous PVA and PF68 polymers also conferred a significant cytotoxicity to PLGA nanoparticles. These results unveiled the critical toxicological contribution played by stabilizers used for the formulation of PLGA nanoparticles when used at high concentrations, which may have implications for local toxicities of PLGA-based nanomedicine, and provided additional insight in cytotoxic effects of internalized nanoparticles.

Drug-induced nanocarrier assembly as a strategy for the cellular delivery of nucleotides and nucleotide analogues.

The natural nucleotide adenosine triphosphate (ATP) and nucleotide analogues such as azidothymidine triphosphate (AZT-TP) display important pharmacological activities for the treatment of ischemia and HIV infections, respectively. Their clinical use is, however, limited mostly due to their hydrophilicity, which highly restricts their diffusion into the target cells. Few nanocarriers have been proposed to address the challenge of ATP/AZT-TP cellular delivery, but the loading efficiency, preparation complexity, and efficient cellular delivery remain important barriers to their development. In this study, we propose an original, straightforward and versatile design of nucleotide and nucleotide analogue nanocarriers based on the natural polysaccharide chitosan (CS). We show that the drugs ATP and AZT-TP can induce ionotropic gelation of CS, leading to CS/ATP and CS/AZT-TP nanoparticles with high drug entrapment efficiency and loading rate-up to 44%. Such nanocarriers release ATP and AZT-TP in physiological media and allow an efficient in vitro cellular delivery of these molecules down to the cell cytoplasm.

Near infrared labeling of PLGA for in vivo imaging of nanoparticles

To introduce optical imaging among methods available to follow nanoparticle biodistribution, we have evaluated the concept of covalently labeling poly(lactide-co-glycolide) (PLGA) with a near-infrared (NIR) dye to obtain stable NIR fluorescent nanoparticles. PLGA was coupled with the NIR dye (DY-700, Dyomics) by an amide bond with 38% efficiency. Incorporating 1% of this conjugate into PLGA nanoparticles stabilised by polyvinyl alcohol (PVA) leads to stable nanoparticles (NPs) without affecting their colloidal characteristics (average diameter, polydispersity and zeta potential). In addition, nanoparticles remain strongly fluorescent and display good storage stability for 4 weeks at 4 °C or over one week at 37 °C. Nanoparticle cytotoxicity evaluated using HUVEC, NIH/3T3 and J774.A1 cell lines was similar for unlabeled or labeled NPs. Fluorescent nanoparticles and free dye were injected intravenously into mice and their biodistribution was followed for 24 h by NIR imaging, in vivo and ex vivo. Nanoparticles were found mainly in the liver whereas the free dye was not accumulating preferentially in this organ. The DY-700 NIR conjugate incorporated into PLGA NPs shows good performance both in vitro and in vivo, thus paving the way to finely traceable PLGA nanosystems for in vivo administration.