Nicolas Tsapis

Ultrasound-triggered drug delivery for cancer treatment using drug delivery systems: From theoretical considerations to practical applications.

Ultrasound-triggered drug delivery is now becoming a mature technology with first patients enrolling in clinical trials. Having a clear overview of the field is complicated as it mixes ultrasound physics and biological effects, particle formulation, and pharmacokinetics and biodistribution. The scope of this review is to move from basics to the latest developments of combined techniques using ultrasound for triggering drug release. Generalities on ultrasound are first given to better understand the parameters on which the clinician can operate to modulate the amount of delivered energy. Ultrasound effects on biological tissues such as thermal effects, mechanical effects and radiation forces are also presented. The second part of this review deals with the combination of ultrasound and drug delivery systems to enhance the efficacy of current cancer treatment. The in vivo behavior of drug delivery systems and how ultrasounds can be combined to improve treatment efficacy are detailed. The example of ThermoDox®, a new formulation of thermosensitive liposomes undergoing a phase III clinical trial, is particularly discussed on the basis of the available clinical data. Through the present article, researchers will be able to better grasp the different levels of complexity when designing an efficient formulation to be combined with ultrasound.

Disintegration of nano-embedded microparticles after deposition on mucus: A mechanistic study

The conversion of colloidal drug carriers/polymeric nanoparticles into dry microparticulate powders (e.g., by spray-drying) is a prominent approach to overcome the aerodynamic limitations of these formulations for delivery via inhalation. However, to what extent such nano-embedded microparticles disintegrate into individual/intact nanoparticles after contacting relevant physiological media has so far not been addressed. Polymeric nanoparticles were spray-dried into nano-embedded microparticles (NEMs) using different amounts of trehalose as embedding matrix excipient. Formulations were characterized and then evaluated for their disintegration behavior after aerosolization onto model mucus. Although a rapid and complete aqueous redispersion was observed for specific excipient/nanoparticle weight ratios (i.e., greater than 1/1), the same formulations revealed no disintegration after deposition onto a static mucus layer. Double-labeled NEMs powders (i.e., dual color staining of polymeric nanoparticles and trehalose) demonstrated rapid matrix dissolution, while the nanoparticle aggregates persisted. When deposited onto agitated mucus, however, sufficient disintegration of NEMs into individual polymeric nanoparticles was observed. These findings indicate that mechanical forces are necessary to overcome the attraction between individual nanoparticles found within the NEMs. Thus, it remains questionable whether the lung mechanics (e.g., breathing, mucociliary clearance) acting on these formulations will contribute to the overall disintegration process.

Paclitaxel-Loaded PEGylated Nanocapsules of Perfluorooctyl Bromide as Theranostic Agents.

We optimize the encapsulation of paclitaxel (PTX) into nanocapsules made of a shell of poly(lactide-co-glycolide)-polyethylene glycol and a core of perfluorooctyl bromide (PFOB) to serve as theranostic agents. Two main challenges were met: keeping the imaging moiety (PFOB) encapsulated while loading the polymer shell with a hydrophobic drug very prone to crystallization. Encapsulation is performed by a modified emulsion-evaporation method leading to 120nm diameter nanocapsules with a drug loading compatible with tumor treatment. The optimized formulation tested in vitro on CT-26 colon cancer cells yields a similar IC50 as the generic Taxol® formulation. In vivo, 19F-MRI shows that PTX encapsulation does not modify the ability of nanocapsules to accumulate passively in CT-26 tumors in mice by the enhanced permeation and retention (EPR) effect. This accumulation leads to a promising and statistically significant twofold reduction in tumor growth as compared with negative control and generic Taxol® group. Altogether these results advocate for an interesting potential of these paclitaxel-loaded theranostic agents.

PEGylated nanocapsules of perfluorooctyl bromide: Mechanism of formation, influence of polymer concentration on morphology and mechanical properties

PEGylated nanocapsules containing a liquid core of perfluorooctyl bromide (PFOB) were formulated by an emulsion-evaporation process to be further used as ultrasound contrast agents (UCAs). In an attempt to modulate their acoustic response, related to their shell thickness-to-radius ratio, the initial concentration of polymer was varied in the formulation. Indeed, thinner shells may lead to higher echogenicity. PEGylated nanocapsules morphology was studied by electron microscopy, Small Angle Neutron Scattering and (19)F NMR spectroscopy and related to their mechanical properties to allow a better understanding of their mechanism of formation. We show that the variation of polymer concentration in the formulation impacts the formation mechanism of nanocapsules, and consequently their morphology and mechanical properties. Using low concentration of Poly(ethylene glycol)-b-poly(dl-lactide-co-glycolide) (PLGA-b-PEG), it is impossible to reduce the shell thickness of the UCA, most probably due to dewetting of the polymer layer at the PFOB/water interface. This leads to the coexistence of thick shells along with free PFOB droplets. On the other hand, for high polymer concentration, PEGylated nanocapsules with thick shells were produced with high encapsulation efficiency.

Wound healing effects of collagen-laminin dermal matrix impregnated with resveratrol loaded hyaluronic acid-DPPC microparticles in diabetic rats

Graphical abstract Figure. No Caption available. Abstract An alternative formulation for the treatment of diabetic foot wounds that heal slowly is a requirement in pharmaceutical field. The aim of this study was to develop a dermal matrix consisting of skin proteins and lipids with an antioxidant that will enhance healing and balance the oxidative stress in the diabetic wound area due to the high levels of glucose. Thus a novel three dimensional collagen‐laminin porous dermal matrix was developed by lyophilization. Resveratrol‐loaded hyaluronic acid and dipalmitoylphosphatidylcholine microparticles were combined with this dermal matrix. Characterization, in vitro release, microbiological and in vivo studies were performed. Spherical microparticles were obtained with a high RSV encapsulation efficacy. The microparticles were well dispersed in the dermal matrix from the surface to deeper layers. Collagenase degraded dermal matrix, however the addition of RSV loaded microparticles delayed the degradation time. The release of RSV was sustained and reached 70% after 6 h. Histological changes and antioxidant parameters in different treatment groups were investigated in full‐thickness excision diabetic rat model. Collagen fibers were intense and improved by the presence of formulation without any signs of inflammation. The highest healing score was obtained with the dermal matrix impregnated with RSV‐microparticles with an increased antioxidant activity. Collagen‐laminin dermal matrix with RSV microparticles was synergistically effective due to presence of skin components in the formulation and controlled release achieved. This combination is a safe and promising option for the treatment of diabetic wounds requiring long recovery.

Polysaccharide-coated liposomes by post-insertion of a hyaluronan-lipid conjugate

Hyaluronan (MW: 1.5 MDa) was linked to a phospholipid (dipalmitoyl phosphatidylethanolamine, DPPE) by an amidification procedure to obtain novel macromolecules (HA-DPPE) able to coat liposomes. Liposomes made of dipalmitoyl phosphatidylcholine and cholesterol (DPPC/Chol: 95/5 molar ratio), with a mean size around 100nm, were incubated with HA-DPPE at 55°C, allowing the insertion of DPPE moieties in the liposomal bilayer and leading to hyaluronan-coated liposomes (HAsomes) as evidenced by several techniques including dynamic light scattering and differential scanning calorimetry. The amount or HA-DPPE coating liposomes was quantified by different methods among which capillary electrophoresis and their stability in serum was finally compared to that of plain liposomes. As a conclusion, we provide insight into the physico-chemical characterization of HA-DPPE and of HAsomes demonstrating that easy coating of phospholipid vesicles can be achieved by post-insertion of a lipid derivative of hyaluronan. This approach represents an innovative strategy for coating vesicular systems to confer them simultaneously with long circulation properties and selective targeting towards HA-receptors.

End-chain fluorination of polyesters favors perfluorooctyl bromide encapsulation into echogenic PEGylated nanocapsules

Perfluorinated end-capped polylactides (PLAs) with various perfluorinated chain lengths from C3F7 to C13F27 were synthesized and formulated into PEGylated nanocapsules of perfluorooctyl bromide (PFOB) to be used as ultrasound contrast agents (UCAs). We show that the perfluorinated end groups do not reduce the interfacial tension between PFOB and the organic solvent used during formulation and do not allow a significant reduction of shell thickness (Small angle neutron scattering (SANS) experiments). However, the PFOB encapsulation efficiency increases with the fluorinated chain length until C8F17. This suggests the possible presence of favorable fluorophilic interactions between PFOB and perfluorinated end groups. In addition, nanocapsules formulated with different fluorinated polymers do not promote any specific toxicity in vitro compared to non-fluorinated PLAs. Ultrasound imaging performed on samples presenting the lowest thickness values, namely nanocapsules made from 50% PLA-C6F13/50% polylactide-b-poly(ethylene glycol) (PLA-PEG) and pure PLA-PEG nanocapsules, shows that fluorinated nanocapsules exhibit a higher ultrasound contrast enhancement in vitro most probably thanks to the higher PFOB content and density arising from polymer fluorination. This highlights the benefit of fluorination for improving the echogenicity of nano-sized ultrasound contrast agents.

Ultrasound-induced mild hyperthermia improves the anticancer efficacy of both Taxol® and paclitaxel-loaded nanocapsules

&NA; We study the influence of ultrasound on paclitaxel‐loaded nanocapsules in vitro and in vivo. These nanocapsules possess a shell of poly(dl‐lactide‐co‐glycolide)‐poly(ethylene glycol) (PLGA‐PEG) and a liquid core of perfluorooctyl bromide (PFOB). In vitro experiments show that mechanical effects such as cavitation are negligible for nanocapsules due to their small size and thick and rigid shell. As the mechanical effects were unable to increase paclitaxel delivery, we focused on the thermal effects of ultrasound in the in vivo studies. A focused ultrasound sequence was therefore optimized in vivo under magnetic resonance imaging guidance to obtain localized mild hyperthermia with high acoustic pressure. Ultrasound‐induced mild hyperthermia (41–43 °C) was then tested in vivo in a subcutaneous CT‐26 colon cancer murine model. As hyperthermia is applied, an inhibition of tumor growth for both paclitaxel‐loaded nanocapsules and the commercial formulation of paclitaxel, namely Taxol® have been observed (p < 0.05). Ultrasound‐induced mild hyperthermia at high acoustic pressure appears as an interesting strategy to enhance cytotoxic efficacy locally. Graphical abstract Figure. No caption available.

Echogenicity enhancement by end-fluorinated polylactide perfluorohexane nanocapsules: Towards ultrasound-activable nanosystems

Polylactide (PLA) polymers containing five distinct lengths of fluorinated (from C3F7 to C13F27) and non-fluorinated (C6H13) end-groups were successfully synthesized by ring-opening polymerization of d,l-lactide. Fluorination was expected to increase the encapsulation efficiency of perfluorohexane (PFH). 150u202fnm nanocapsules were obtained and 19F nuclear magnetic resonance revealed that nanocapsules formulated with fluorinated polymers increased by 2-fold the encapsulation efficiency of PFH compared with non-fluorinated derivatives, without any effect of fluorine chain length. Fluorination of the polymers did not induce any specific in vitro cytotoxicity of nanocapsules towards HUVEC and J774.A1 cell lines. The echogenicity of fluorinated-shelled nanocapsules was increased by 3-fold to 40-fold compared to non-fluorinated nanocapsules or nanoparticles devoid of a perfluorohexane core for both conventional and contrast-specific ultrasound imaging modalities. In particular, an enhanced echogenicity and contrast-specific response was observed as the fluorinated chain-length increased, probably due to an increase of density and promotion of bubble nucleation. When submitted to focused ultrasound, both intact and exploded nanocapsules could be observed, also with end-group dependency, indicating that PFH was partly vaporized. These results pave the way to the design of theranostic perfluorohexane nanocapsules co-encapsulating a drug for precision delivery using focused ultrasound.nnnSTATEMENT OF SIGNIFICANCEnWe have synthesized novel fluorinated polyesters and formulated them into nanocapsules of perfluorohexane as ultrasound contrast agents. This nanosystem has been thoroughly characterized by several techniques and we show that fluorination of the biodegradable polymer favors the encapsulation of perfluorohexane without producing further reduction of cell viability. Contrary to nanocapsules of perfluoroctyl bromide formulated with the fluorinated polymers [32], the presence of the fluorinated moieties leads to an increase of echogenicity that is dependent of the length of the fluorinated moiety. Morevover, the ability of nanocapsules to explode when submitted to focused ultrasound also depends on the length of the fluorinated chain. These results pave the way to theranostic perfluorohexane nanocapsules co-encapsulating a drug for precision delivery using focused ultrasound.

Comb-like fluorophilic-lipophilic-hydrophilic polymers for nanocapsules as ultrasound contrast agents

Imaging the enhanced permeation and retention effect by ultrasound is hindered by the large size of commercial ultrasound contrast agents (UCAs). To obtain nanosized UCAs, triblock copolymers of poly(ethylene glycol)-polylactide-poly(1 H,1 H,2 H,2 H-heptadecafluorodecyl methacrylate) (PEG-PLA-PFMA) with distinct numbers of perfluorinated pendant chains (5, 10, or 20) are synthesized by a combination of ring-opening polymerization and atom transfer radical polymerization. Nanocapsules (NCs) containing perfluorooctyl bromide (PFOB) intended as UCAs are obtained with a 2-fold increase in PFOB encapsulation efficiency in fluorinated NCs as compared with plain PEG-PLA NCs thanks to fluorous interactions. NC morphology is strongly influenced by the number of perfluorinated chains and the amount of polymer used for formulation, leading to peculiar capsules with several PFOB cores at high PEG-PLA-PFMA20 amount and single-cored NCs with a thinner shell at low fluorinated polymer amount, as confirmed by small-angle neutron scattering. Finally, fluorinated NCs yield higher in vitro ultrasound signal compared with PEG-PLA NCs, and no in vitro cytotoxicity is induced by fluorinated polymers and their degradation products. Our results highlight the benefit of adding comb-like fluorinated blocks in PEG-PLA polymers to modify the nanostructure and enhance the echogenicity of nanocapsules intended as UCAs.