Emanuel Fleige

Stimuli-responsive polymeric nanocarriers for the controlled transport of active compounds: Concepts and applications ☆

The use of polymeric nanocarriers to transport active compounds like small-molecular drugs, peptides, or genes found an increased attention throughout the different fields of natural sciences. Not only that these nanocarriers enhance the properties of already existing drugs in terms of solubility, bioavailability, and prolonged circulation times, furthermore they can be tailor-made in such a manner that they selectively release their cargo at the desired site of action. For the triggered release, these so-called smart drug delivery systems are designed to react on certain stimuli like pH, temperature, redox potential, enzymes, light, and ultrasound. Some of these stimuli are naturally occurring in vivo, for example the difference in pH in different cellular compartments while others are caused by the disease, which is to be treated, like differences in pH and temperature in some tumor tissues. Other external applied stimuli, like light and ultrasound, allow the temporal and spatial control of the release, since they are not triggered by any biological event. This review gives a brief overview about some types of stimuli-responsive nanocarriers with the main focus on organic polymer-based systems. Furthermore, the different stimuli and the design of corresponding responsive nanocarriers will be discussed with the help of selected examples from the literature.

Penetration of normal, damaged and diseased skin — An in vitro study on dendritic core–multishell nanotransporters

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.

Position-dependent effects of fluorinated amino acids on the hydrophobic core formation of a heterodimeric coiled coil.

Systematic model investigations of the molecular interactions of fluorinated amino acids within native protein environments substantially improve our understanding of the unique properties of these building blocks. A rationally designed heterodimeric coiled coil peptide (VPE/VPK) and nine variants containing amino acids with variable fluorine content in either position a16 or d19 within the hydrophobic core were synthesized and used to evaluate the impact of fluorinated amino acid substitutions within different hydrophobic protein microenvironments. The structural and thermodynamic stability of the dimers were examined by applying both experimental (CD spectroscopy, FRET, and analytical ultracentrifugation) and theoretical (MD simulations and MM-PBSA free energy calculations) methods. The coiled coil environment imposes position-dependent conformations onto the fluorinated side chains and thus affects their packing and relative orientation towards their native interaction partners. We find evidence that such packing effects exert a significant influence on the contribution of fluorine-induced polarity to coiled coil folding.

pH-responsive dendritic core-multishell nanocarriers.

In this paper we describe novel pH-responsive core-multishell (CMS) nanocarrier (pH-CMS), obtained by introducing an aromatic imine linker between the shell and the core. At a pH of 5 and lower the used imine linker was rapidly cleaved as demonstrated by NMR studies. The CMS nanocarriers were loaded with the dye Nile red (NR) and the anticancer drug doxorubicin (DOX), respectively. The transport capacities were determined using UV/Vis spectroscopy, and the sizes of the loaded and unloaded CMS nanocarriers were investigated using dynamic light scattering (DLS). We could show that CMS nanocarriers efficiently transported NR in supramolecular aggregates, while DOX was transported in a unimolecular fashion. After cellular uptake the DOX-loaded pH-responsive nanocarriers showed higher toxicities than the stable CMS nanocarriers. This is due to a more efficient DOX release caused by the cleavage of the pH-labile imine bond at lower pH within the intracellular compartments.

Nanodynamics of dendritic core-multishell nanocarriers.

The molecular dynamics of polymeric nanocarriers is an important parameter for controlling the interaction of nanocarrier branches with cargo. Understanding the interplay of dendritic polymer dynamics, temperature, and cargo molecule interactions should provide valuable new insight for tailoring the dendritic architecture to specific needs in nanomedicine, drug, dye, and gene delivery. Here, we have investigated polyglycerol-based core-multishell (CMS) nanotransporters with incorporated Nile Red as a fluorescent drug mimetic and CMS nanotransporters with a covalently bound fluorophore (Indocarbocyanine) using fluorescence spectroscopy methods. From time-resolved fluorescence depolarization we have obtained the rotational diffusion dynamics of the incorporated dye, the nanocarrier, and its branches as a function of temperature. UV/vis and fluorescence lifetime measurements provided additional information on the local dye environment. Our results show a distribution of the cargo Nile Red within the nanotransporter shells that depends on solvent and temperature. In particular, we show that the flexibility of the polymer branches in the unimolecular state of the nanotransporter undergoes a temperature-dependent transition which correlates with a larger space for the mobility of the incorporated hydrophobic drug mimetic Nile Red and a higher probability of cargo-solvent interactions at temperatures above 31 °C. The measurements have further revealed that a loss of the cargo molecule Nile Red occurred neither upon dilution of the CMS nanotransporters nor upon heating. Thus, the unimolecular preloaded CMS nanotransporters retain their cargo and are capable to transport and respond to temperature, thereby fulfilling important requirements for biomedical applications.

Skin penetration enhancement of core–multishell nanotransporters and invasomes measured by electron paramagnetic resonance spectroscopy

In order to cross the skin barrier several techniques and carrier systems were developed to increase skin penetration of topical dermatics and to reduce systemic adverse effects by avoiding systemic application. Ultra-flexible vesicles, e.g. invasomes and core-multishell (CMS) nanotransporters are efficient drug delivery systems for dermatological applications. Electron paramagnetic resonance (EPR) spectroscopic techniques were used for the determination of localization and distribution of the spin label 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA; logP=-1.7) within the carrier systems and the ability of the carriers to promote penetration of PCA into the skin. The results show an exclusive localization of PCA in the hydrophilic compartments of the invasome dispersion and the CMS nanotransporter solution. PCA penetration was enhanced 2.5 fold for CMS and 1.9 fold for invasomes compared to PCA solution. Investigation of penetration depth by step-wise removal of the stratum corneum by tape stripping revealed deepest PCA penetration for invasomes. UV-irradiation of PCA-exposed skin samples revealed that the spin label is still reactive. In conclusion novel polymer-based CMS nanotransporters and invasomes can favor the penetration of PCA or hydrophilic drugs. This offers possibilities for e.g. improved photodynamic therapy.

Tailored dendritic core-multishell nanocarriers for efficient dermal drug delivery: A systematic top-down approach from synthesis to preclinical testing

Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester- and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNFα supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications.

Investigation of cutaneous penetration properties of stearic acid loaded to dendritic core-multi-shell (CMS) nanocarriers.

Dendritic core-multi shell (CMS) particles are polymer based systems consisting of a dendritic polar polyglycerol polymer core surrounded by a two-layer shell of nonpolar C18 alkyl chains and hydrophilic polyethylene glycol. Belonging to nanotransport systems (NTS) they allow the transport and storage of molecules with different chemical characters. Their amphipihilic character CMS-NTS permits good solubility in aqueous and organic solutions. We showed by multifrequency electron paramagnetic resonance (EPR) spectroscopy that spin-labeled 5-doxyl stearic acid (5DSA) can be loaded into the CMS-NTS. Furthermore, the release of 5DSA from the carrier into the stratum corneum of porcine skin was monitored ex vivo by EPR spectroscopy. Additionally, the penetration of the CMS-NTS into the skin was analyzed by fluorescence microscopy using indocarbocyanine (ICC) covalently bound to the nanocarrier. Thereby, no transport into the viable skin was observed, whereas the CMS-NTS had penetrated into the hair follicles down to a depth of 340 μm ± 82 μm. Thus, it could be shown that the combined application of fluorescence microscopy and multi-frequency EPR spectroscopy can be an efficient tool for investigating the loading of spin labeled drugs to nanocarrier systems, drug release and penetration into the skin as well as the localization of the NTS in the skin.

Localization of dexamethasone within dendritic core-multishell (CMS) nanoparticles and skin penetration properties studied by multi-frequency electron paramagnetic resonance (EPR) spectroscopy.

Graphical abstract Figure. No Caption available. Abstract The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer‐based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core‐multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual‐frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3‐carboxy‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxy (PCA) is associated with the CMS. The micro‐environment of the drug loaded to CMS nanoparticles was investigated by pulsed high‐field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g‐, A‐matrices, and spin‐lattice relaxation time) represent specific probes for the micro‐environment. Additionally, the rotational correlation time of spin‐labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown.

Core-multishell nanocarriers: Transport and release of dexamethasone probed by soft X-ray spectromicroscopy

Label-free detection of core-multishell (CMS) nanocarriers and the anti-inflammatory drug dexamethasone is reported. Selective excitation by tunable soft X-rays in the O 1s-regime is used for probing either the CMS nanocarrier or the drug. Furthermore, the drug loading efficiency into CMS nanocarriers is determined by X-ray spectroscopy. The drug-loaded nanocarriers were topically applied to human skin explants providing insights into the penetration and drug release processes. It is shown that the core-multishell nanocarriers remain in the stratum corneum when applied for 100min to 1000min. Dexamethasone, if applied topically to human ex vivo skin explants using different formulations, shows a vehicle-dependent penetration behavior. Highest local drug concentrations are found in the stratum corneum as well as in the viable epidermis. If the drug is loaded to core-multishell nanocarriers, the concentration of the free drug is low in the stratum corneum and is enhanced in the viable epidermis as compared to other drug formulations. The present results provide insights into the penetration of drug nanocarriers as well as the mechanisms of controlled drug release from CMS nanocarriers in human skin. They are also compared to related work using dye-labeled nanocarriers and dyes that were used as model drugs.