Michał R. Radowski

Nanoparticles for skin penetration enhancement--a comparison of a dendritic core-multishell-nanotransporter and solid lipid nanoparticles.

Nanosized particles are of growing interest for topical treatment of skin diseases to increase skin penetration of drugs and to reduce side effects. Effects of the particle structure and size were studied loading nile red to dendritic core-multishell (CMS) nanotransporters (20-30 nm) and solid lipid nanoparticles (SLNs, 150-170 nm). Interaction properties of CMS nanotransporters with the dye molecules--attachment to the carrier surface or incorporation in the carrier matrix--were studied by UV/Vis and parelectric spectroscopy. Pig skin penetration was studied ex vivo using a cream for reference. Interactions of SLN and skin were followed by scanning electron microscopy, internalisation of the particles by viable keratinocytes by laser scanning microscopy. Incorporating nile red into a stable dendritic nanoparticle matrix, dye amounts increased eightfold in the stratum corneum and 13-fold in the epidermis compared to the cream. Despite SLN degradation at the stratum corneum surface, SLN enhanced skin penetration less efficiently (3.8- and 6.3-fold). Viable human keratinocytes showed an internalisation of both nanocarriers. In conclusion, CMS nanotransporters can favour the penetration of a model dye into the skin even more than SLN which may reflect size effects.

Influence of nanocarrier type and size on skin delivery of hydrophilic agents

The nanoparticulate carrier systems solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters gained interest for the topical treatment of skin diseases as they facilitate the skin penetration of loaded lipophilic drugs. Here, we studied if these carrier systems are also suitable drug delivery systems for more hydrophilic agents using the dye rhodamin B as model compound. Furthermore, the influence of the particle size on the skin penetration was investigated. Loading rhodamin B onto SLN (250-340 nm) and CMS nanotransporters (20-30 nm), the dye amount increased significantly in viable epidermis and dermis as compared to a conventional cream. CMS nanotransporters were most efficient. Creating nanoparticles of 50-200 nm demonstrated only marginal size effect for the skin penetration. Therefore, the superiority of the CMS nanotransporters seems to be attributed to the character of the nanoparticles and not to its smaller size.

Dendritic multishell architectures for drug and dye transport

Here we present the efficiency and versatility of newly developed core-multishell nanoparticles (CMS NPs), to encapsulate and transport the antitumor drugs doxorubicin hydrochloride (Dox), methotrexate (Mtx) and sodium ibandronate (Ibn) as well as dye molecules, i.e., a tetrasulfonated indotricarbocyanine (ITCC) and nile red. Structurally, the CMS NPs are composed of hyperbranched poly(ethylene imine) core functionalized by alkyl diacids connected to monomethyl poly(ethylene glycol). In order to evaluate their transport in aqueous media in vitro, we have used and compared SEC, UV, ITC, and NMR techniques. We observed that the CMS NPs were able to spontaneously encapsulate and transport Dox, Mtx and nile red in both organic and aqueous media as determined by SEC and UV-VIS spectroscopy. For the VIS transparent Ibn Isothermal Titration Calorimetric (ITC) experiments show an exothermic interaction with the CMS NPs. The enthalpic stabilization (DeltaH) upon encapsulation was in the order of approximately 7 kcals/mol which indicates stable interaction between Ibn and nanoparticles. A T(1) inversion recovery NMR experiment was carried out for 31P and 1H nuclei of Ibn and an increment of spin-lattice relaxation time for respective nuclei was observed upon encapsulation. CMS NPs were also found to encapsulate ITCC dye with stoichiometry of 6-8 molecules/nanocarrier. For in vivo imaging studies the dye loaded CMS NPs were injected to F9 teratocarcinoma bearing mice and a strong contrast was observed in the tumor tissues compared to free dye after 6 h of administration.

A New Family of Nonionic Dendritic Amphiphiles Displaying Unexpected Packing Parameters in Micellar Assemblies

In this paper we report on the synthesis of a new family of nonionic dendritic amphiphiles that self-assemble into defined supramolecular aggregates. Our approach is based on a modular architecture consisting of different generations of hydrophilic polyglycerol dendrons [G1-G3] connected to hydrophobic C(11) or C(16) alkyl chains via mono- or biaromatic spacers, respectively. All amphiphiles complex hydrophobic compounds as demonstrated by solubilization of Nile Red or pyrene. The structure of the supramolecular assemblies as well as the aggregation numbers are strongly influenced by the type of the dendritic headgroup. While the [G1] amphiphiles form different structures such as ringlike and fiberlike micelles, the [G2] and [G3] derivatives aggregate toward spherical micelles of low polydispersity clearly proven by transmission electron microscopy (TEM) measurements. In the case of the biaromatic [G2] derivative, the structural persistence of the micelles allowed a three-dimensional structure determination from the TEM data and confirmed the aggregation number obtained by static light scattering (SLS) measurements. On the basis of these data, molecular packing geometries indicate a drastic mass deficit of alkyl chains in the hydrophobic core volume of spherical micelles. It is noteworthy that these highly defined micelles contain as little as 15 molecules and possess up to 74% empty space. This behavior is unexpected as it is very different from classical detergent micelles such as sodium dodecyl sulfate (SDS), where the hydrophobic core volume is completely filled by alkyl chains.

Influences of opioids and nanoparticles on in vitro wound healing models

For efficient pain reduction in severe skin wounds, topical opioids may be a new option - given that wound healing is not impaired and the vehicle allows for slow opioid release, since long intervals of painful wound dressing changes are intended. We investigated the influence of opioids on the wound healing process via in vitro models, migration assay and scratch test. In fact, morphine, hydromorphone, fentanyl and buprenorphine increased the number of migrated HaCaT cells (spontaneously transformed keratinocytes) twofold. In the scratch test, morphine accelerated the closure of a monolayer wound (scratch). As possible slow release application forms are nanoparticulate systems like solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters, we evaluated the effect of unloaded nanoparticles on HaCaT cell migration, too. CMS nanotransporters did not inhibit migration, SLN even enhanced it (twofold). Applying morphine plus unloaded nanoparticles reduced morphine effects possibly due to uptake into CMS nanotransporters and adsorption to the surface of SLN. In contrast to SLN, TGF-beta1 was taken up by CMS nanotransporters, too. Both nanoparticles are tolerable by skin and eye as derived from Episkin-SM(TM) skin irritation test and HET-CAM assay. No acute toxic effects were observed either. In conclusion, opioids as well as the investigated nanoparticulate carriers conform the essential conditions for topical pain reduction.

Cellular Copper Import by Nanocarrier Systems, Intracellular Availability, and Effects on Amyloid β Peptide Secretion

Studies in animals have reported that normalized or elevated Cu levels can inhibit or even remove Alzheimers disease-related pathological plaques and exert a desirable amyloid-modifying effect. We tested engineered nanocarriers composed of diverse core-shell architectures to modulate Cu levels under physiological conditions through bypassing the cellular Cu uptake systems. Two different nanocarrier systems were able to transport Cu across the plasma membrane of yeast or higher eukaryotic cells, CS-NPs (core-shell nanoparticles) and CMS-NPs (core-multishell nanoparticles). Intracellular Cu levels could be increased up to 3-fold above normal with a sublethal dose of carriers. Both types of carriers released their bound guest molecules into the cytosolic compartment where they were accessible for the Cu-dependent enzyme SOD1. In particular, CS-NPs reduced Abeta levels and targeted intracellular organelles more efficiently than CMS-NPs. Fluorescently labeled CMS-NPs unraveled a cellular uptake mechanism, which depended on clathrin-mediated endocytosis in an energy-dependent manner. In contrast, the transport of CS-NPs was most likely driven by a concentration gradient. Overall, nanocarriers depending on the nature of the surrounding shell functioned by mediating import of Cu across cellular membranes, increased levels of bioavailable Cu, and affected Abeta turnover. Our studies illustrate that Cu-charged nanocarriers can achieve a reasonable metal ion specificity and represent an alternative to metal-complexing agents. The results demonstrate that carrier strategies have potential for the treatment of metal ion deficiency disorders.

Synthesis and transport properties of new dendritic core–shell architectures based on hyperbranched polyglycerol with biphenyl-PEG shells

A new core–shell type of nano-architectures based on hyperbranched polyglycerol (hPG) has been designed by attaching a mono(methoxy)polyethylene glycol (mPEG) shell either directly or through a hydrophobic biphenyl spacer to the hPG scaffold. Alternatively the hPG core was decorated with hydrophobic segments specifically located around the hPG and mPEG as the shell. The constructed structures were compared and contrasted for their ability to solubilize guest molecules of different polarity indices to their corresponding non-solvent for possible drug delivery applications. UV/Vis spectroscopy and Scanning Force Microscopy (SFM) techniques have been used to characterize the host–guest complex. Highly hydrophilic nanocarriers composed of an hPG–mPEG arrangement were found to be very efficient in transporting hydrophilic molecules to an organic environment with almost no encapsulation of the hydrophobic guests. Introduction of biphenyl fragments as hydrophobic spacers between hPG and mPEG, or near the hPG core, substantially increased the hydrophobic guest encapsulation efficiency of the resulting system. The encapsulation and transport properties were found to critically depend on the Mn of hPG, degree of functionalization with hydrophilic and/or hydrophobic fragments and length of mPEG chains, either alone or in combination with each other. SFM images revealed that the size of the nanocarriers is within the range of 10 nm as single particles and 50 nm as aggregates, with the sizes substantially increased upon interaction with the guest species.

Increased cutaneous absorption reflects impaired barrier function of reconstructed skin models mimicking keratinisation disorders

The aim of this study was to assess a recently established 3D model of congenital ichthyosis, representing severe epidermal barrier function defects, for skin penetration and permeation. We have generated disease models by knock‐down of either TGM1 or ALOXE3 in primary human keratinocytes, and using keratinocytes and fibroblasts from patients with congenital ichthyosis. The results indicate disturbed barrier function as demonstrated by increased permeation of testosterone and caffeine particularly in TGM1 knock‐down models compared to control models. In addition, enhanced penetration of the model dye nile red incorporated into solid lipid nanoparticles and core‐multishell nanotransporters, respectively, was evident in disease models. Thus, in vitro skin disease models reproduce differences in barrier permeability and function seen in congenital ichthyosis and pave the way to personalised disease models. Furthermore, our findings indicate that nanocarriers may be useful in new, topical therapeutic approaches for the currently very limited treatment of congenital ichthyosis.