Core-Multishell Nanocarriers for the Topical Delivery of Pharmacophores

This work explored the possibilities to design CMS nanocarriers for two different types of problems, the encapsulation of small hydrophobic drugs and subsequent transport into skin or the retention and targeted release of a cationic analgesic. The aim of the first part was to design a set of nanocarriers for the encapsulation and penetration enhancement of these drugs and thereby fulfilling the following criteria: Ease of synthesis, non-toxic products and building blocks, good loading capacity, degradability, and degradation-based release and high penetration enhancement. To simplify the synthesis, the dendritic building block, which poses the focal point in the nanocarrier architecture, was exchanged. Instead of hPG-NH2, unfunctionalized hPG was used. This change in the synthesis process had two main impacts. First, this strategy spared three synthetic steps thereby making the procedure less time consuming. Secondly, the potentially toxic building block hPG-NH2 was avoided and thus could not harm the organism post-application as a degradation product. This change was the next step in decreasing the number of amine groups in the core and thus the toxicity ranging from PEI over hPG-NH2 to hPG.[83,85] The inner shell’s building block was varied in length to study the impact on encapsulation, melting temperature and penetration enhancement. Aliphatic diacids with a number of carbon atoms between 12 and 19 as well as a branched version of the one with 18 carbon atoms were used as inner shell building blocks. While, in previous studies, the length of the inner shell had already been altered with bigger steps (six C-atoms),[83] here the focus shifted more onto details and other structural features. The different systems showed an increase of the melting temperature (Tm), of the inner shell with increasing chain length. And while an amide bond in the structure significantly increased the Tm, the introduction of side arms led to its decrease. While the rigid molecule dexamethasone (DXM) showed no apparent correlation between chain length and loading capacity, the situation was different for the bulkier tacrolimus. Here, an optimum was found for a C15 inner shell. Interestingly, while the additional branching did not lead to results different from the linear ester-based architecture, the loading capacity plummeted when an amide-based nanocarrier was used. All examined nanocarriers were degraded by a lipase to a degree of more than 70% in two weeks, which was similar to other ester-based nanotransporters.[124,125] The degradation of DXM-loaded CMS nanotransporters led to the loss of their function as a solubilizer, more than 80% of the loading was released. Thus, the degradation can serve as a possible release mechanism. Various assays were carried out to determine toxicity. Of the investigated ester-based CMS architectures, CMS-E15 showed the least cytotoxicity. The others caused a decrease in cell viability, at least to some extent. Of the building blocks, the aliphatic diacids were only toxic after prolonged exposition, but of the PEG-conjugated diacids, cytotoxicity increased from C12 to C18, which was attributed to interaction with the cell membrane. The most toxic building block was C18-PEG350, also with regard to genotoxicity. The CMS-E18 nanocarrier, the product of this building block, also exhibited some ROS generation and genotoxicity. The overall least toxic, ester-based nanocarrier was CMS-E15, which was selected for in vivo testing and was nontoxic when applied to the skin of Sprague Dawley rats. After establishing the biocompatibility of the different candidates, the CMS nanocarriers were tested for skin penetration and penetration enhancement of several guest molecules on various skin models. Nile red-loaded and DXM-loaded CMS-nanocarriers were used to study the penetration enhancement on excised human skin. All nanocarriers proved to be superior over a formulation with base cream (NR) or LAW cream (DXM), which was in accordance with previous results.[85] The nanotransporters showed no dependence on inner shell chain length or type of bond for attachment. CMS-E15 was then loaded with dexamethasone and applied on an inflammatory skin model which had an upregulated interleukin 8 (IL-8) and IL-1β expression as part of their inflammatory condition. The application of the DXM-loaded nanocarriers reduced the expression of both ILs more effectively than a LAW cream with equal DXM content. The CMS nanocarrier was not only tested for application on skin, but for oral mucosa as well. Here, biocompatibility was of even higher importance, because the constant flow of saliva eventually flushed the CMS nanocarriers into the GI tract if not taken up by the mucosa. Along the penetration experiment, toxicity of the CMS nanotransporters was determined for gingival epithelial cells and found to be only cytotoxic at very high concentrations. The penetration enhancement of PCA-labeled dexamethasone encapsulated in CMS-E15 and the penetration of ICC-labeled, amide based CMS nanocarriers (CMS-ICC) was measured on buccal and masticatory mucosa. While penetration of unloaded CMS-ICC nanocarriers was limited to the stratum corneum on skin,[122] a small fraction also penetrated more deeply into viable tissues, in masticatory slightly more than in buccal mucosa. Although the conditions were not identical, this result might have indicated a higher penetrability for oral mucosa compared to skin, which might be related to a difference in the structure of both tissues. For the penetration enhancement of spin-labeled DXM (DXM-PCA) by CMS-E15, similar results were found as in skin. Even after a washing step, which simulated the flow of saliva in the oral cavity, penetration enhancement of the labeled drug was found. The CMS nanotransporters enhanced the penetration of DXM-PCA more than a cream formulation, and buccal mucosa generally seemed more penetrable than masticatory mucosa for DXM-PCA. Additionally, X-band electron paramagnetic resonance spectroscopy revealed that the guest molecule left its vehicle. This observation was in accordance with previous observations,[122] which indicated that nanocarrier and guest molecule penetrated up to different depths. Taken together, these three studies describe a step-wise selection process at which out of six different initial candidates, one was selected by criteria like ease of synthesis, drug loading capacity, and biocompatibility. The nanocarrier with the best overall performance and thus most promising for future applications was CMS-E15. The aim of the second part of this work was to design a nanocarrier for the encapsulation and controlled release of the drug U 50,488H (U50), which is a strong analgesic for the post-operational pain treatment but elicits side effects when it crosses the blood brain barrier. A formulation into a nanocarrier can help to increase circulation time and facilitate targeted release. After surgery, the tissue is inflamed and thereby acidified and fenestrated. To use these conditions for the targeted release of U50, a nanocarrier was synthesized that offered the possibility to encapsulate U50 efficiently and release it in a pH-dependent manner. As basic architectures, CMS-E15 and CMS-A18, which were the most promising candidates in previous studies, were used and extended by an anchor moiety. It was needed, because there was no possibility to bind the drug covalently to the nanocarrier. For this task, N-Z-L-protected aspartic acid was utilized, because it offered the possibility for ionic interactions, π-π stacking, and hydrogen bonds to U50. Further, it was possible to employ the carboxylic acid groups for a pH-dependent release. While many approaches in literature used a hydrophobic segment that was ionized upon a pH change and thus,[126–128] a lipophilic guest is expelled, here, the opposite is the case. Ionic interactions are interrupted and thus the ionic drug can diffuse out of the nanocarrier. After synthesis, the pKapp of the carboxylic acid groups was determined to be in the physiological range (4.4 for pCMS-A and 5.4 for pCMS-E) and the loading capacity (LC) and encapsulation efficiency (EE) for U50 free base was measured. While the EE at 16 wt% feed already exceeded 10% for all nanocarriers (> 60% EE), the feed was increased to 48 wt%. Under these conditions, the LC of all nanoarchitectures could be increased, especially for the pCMS, which reached up to 28% (> 50% EE). Subsequently, the release kinetics were studied under various conditions to determine the influence of the additional functionalization and factors like pH, the encapsulation of the free base or the methylsulfonate salt, or the presence of physiological salt concentration. While the latter two had no influence on the release kinetics, the functionalization with the binding patch was beneficial for the retention of U50. In the case of pCMS A, there was no difference in this effect at pH 5.5 and 7.4, but for pCMS E, a pH dependence was found. For this reason, pCMS-E was selected for an in vivo study in rats. Here, formulations of U50 with and without pCMS-E were compared regarding prolonged analgesia and reduced side effects. While the latter one was not the case, the formulation with additional pCMS-E resulted in the enhancement and the extension of the analgesic effect, which might indicate a longer blood circulation time caused by the nanocarrier. In a previous prodrug approach, morphine covalently attached to hPG was tested under similar conditions and the absence of a systemic side effect reported.[61] Taken together, these two studies indicated that to optimize post-operational pain treatment, nanocarrier architectures ideally have a sufficiently high molecular weight for a prolonged blood circulation time and bind the analgesic tightly enough to prevent premature release. If possible, this should be realized in a DDS approach, to make the drug interchangeable. When first reported, the core-multishell nanocarrier was termed universal, because it was suitable for both hydrophilic and hydrophobic guest molecules in both aqueous and organic media. Subsequent studies confirmed that and reported the encapsulation of even inorganic nanoparticles and explored the fundamental effects, such as the aggregation of nanoparticles and guest molecules, which were also described theoretically. In a more application-focused approach, many nanocarriers were designed for a certain guest, e.g., copper ions, or target, for instance, the endosome. Ideally both can be achieved, namely, the identification of a promising architecture and further insight into fundamental effects. In this work, this was achieved. In the first part, it could be shown that there is an optimum chain length for the encapsulation tacrolimus and that the addition of one CH2 group to the aliphatic chain can alter the nanocarrier’s properties dramatically. There also was a dependence of cytotoxicity on the length of the hydrophobic part of the amphiphilic double shell, which altogether identified CMS-E15 as the most promising candidate. Addressing oral mucosa with CMS showed that this is an interesting field for DDS like CMS nanocarriers, because even more efficient penetration seems to be possible. In the more application-focused second part, the pH-dependent retention effect that was measured for the best candidate pCMS-E. Even though the encapsulation of hydrophilic guest molecules remains a greater challenge than the formulation of hydrophobic ones, this showed that it was possible even for rather small nanocarrier systems (diameter ~10 nm) to bind a water-soluble drug. Additionally, it could be proven that the chemistry used to attach the anchor moiety influenced the pKapp and thus is crucial for the application. Different anchor molecules could be employed to further fine-tune the pH of release and the retention effect proven in this study might be improved by increasing the nanocarrier’s sizes. One advantage of the DDS-approach is that pCMS are a potential nanocarrier suitable for any aromatic, hydrophobic drug molecule that bears a positive charge, e.g. by an amino group. The past research conducted on CMS nanocarriers has shown that, starting from a universal nanocarrier, of which mainly fundamental effects have been studied, many subclasses and varieties have been realized to meet specific requirements. To be suitable for future applications, a universal nanocarrier will not be able to solve all the problems. It will be necessary to adapt the nanocarrier’s architecture to the requirements of drug and physiological condition. And to achieve that, a library of CMS nanocarriers is needed to study fundamental effects and to select a potential candidate for an application. It is important to design new architectures, because the human body’s complexity demands specialized DDS for specific applications. This work has not only extended the library, but also identified certain novel nanocarriers as promising candidates

Influence of Inner Shell Structure on the Encapsulation Behavior of Dexamethasone and Tacrolimus

We here present the synthesis and characterization of a set of biodegradable core–multishell (CMS) nanocarriers. The CMS nanocarrier structure consists of hyperbranched polyglycerol (hPG) as core material, a hydrophobic (12, 15, 18, 19, and 36 C-atoms) inner and a polyethylene glycol monomethyl ether (mPEG) outer shell that were conjugated by ester bonds only to reduce the toxicity of metabolites. The loading capacities (LC) of the drugs, dexamethasone and tacrolimus, and the aggregate formation, phase transitions, and degradation kinetics were determined. The intermediate inner shell length (C15) system had the best overall performance with good LCs for both drugs as well as a promising degradation and release kinetics, which are of interest for dermal deliver

Radical Stability vs. Temporal Resolution of EPR-Spectroscopy on Biological Samples

Spin-labeling active compounds is a convenient way to prepare them for EPR spectroscopy with minimal alteration of the target molecule. In this study we present the labeling reaction of dexamethasone (Dx) with either TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) with high yields. According to NMR data, both labels are attached at the primary hydroxy group of the steroid. In subsequent spin-stability measurements both compounds were applied onto HaCaT cells. When the signal of Dx-TEMPO decreased below the detection limit within 3 h, the signal of Dx-PCA remained stable for the same period of time

Characterization of an ester-based core-multishell (CMS) nanocarrier for the topical application at the oral mucosa

Objectives Topical drug administration is commonly applied to control oral inflammation. However, it requires sufficient drug adherence and a high degree of bioavailability. Here, we tested the hypothesis whether an ester-based core-multishell (CMS) nanocarrier is a suitable nontoxic drug-delivery system that penetrates efficiently to oral mucosal tissues, and thereby, increase the bioavailability of topically applied drugs. Material and methods To evaluate adhesion and penetration, the fluorescence-labeled CMS 10-E-15-350 nanocarrier was applied to ex vivo porcine masticatory and lining mucosa in a Franz cell diffusion assay and to an in vitro 3D model. In gingival epithelial cells, potential cytotoxicity and proliferative effects of the nanocarrier were determined by MTT and sulphorhodamine B assays, respectively. Transepithelial electrical resistance (TEER) was measured in presence and absence of CMS 10-E-15-350 using an Endohm-12 chamber and a volt-ohm-meter. Cellular nanocarrier uptake was analyzed by laser scanning microscopy. Inflammatory responses were determined by monitoring pro-inflammatory cytokines using real-time PCR and ELISA. Results CMS nanocarrier adhered to mucosal tissues within 5 min in an in vitro model and in ex vivo porcine tissues. The CMS nanocarrier exhibited no cytotoxic effects and induced no inflammatory responses. Furthermore, the physical barrier expressed by the TEER remained unaffected by the nanocarrier. Conclusions CMS 10-E-15-350 adhered to the oral mucosa and adhesion increased over time which is a prerequisite for an efficient drug release. Since TEER is unaffected, CMS nanocarrier may enter the oral mucosa transcellularly. Clinical relevance Nanocarrier technology is a novel and innovative approach for efficient topical drug delivery at the oral mucosa

Drug distribution in nanostructured lipid particles

The targeted design of nanoparticles for efficient drug loading and defined release profiles is even after 25 years of research on lipid-based nanoparticles still no routine procedure. It requires detailed knowledge about the interaction of the drug with the lipid compounds and about its localisation and distribution in the nanoparticle. We present here an investigation on nano-sized lipid particles (NLP) composed of Gelucire and Witepsol as solid lipids, and Capryol as liquid lipid, loaded with Dexamethasone, a glucocorticoid used in topical treatment of inflammatory dermal diseases. The interactions of Dexamethasone, which was spin-labelled by 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (DxPCA), with its microenvironment are monitored by EPR spectroscopy at 94 GHz at low temperatures. The mobility of the spin-labelled drug was probed by X-band EPR at room temperature. In order to relate the magnetic and dynamic parameters deduced from EPR to the local environment of the spin probe in the NLP, investigations of DxPCA in the individual lipid compounds were carried out. The magnetic parameters reflecting the polarity of DxPCA’s environment as well as the parameters describing the mobility of the drug reveal that in the case of colloidal dispersions of the lipids and also the NLP DxPCA is attached to the surface of the nanoparticles. Although the lipophilic drug is almost exclusively associated with the NLP in aqueous solution, dilution experiments show, that it can be easily released from the nanoparticle

Characterization of an Ex Vivo Skin Model for the Assessment of Dexamethasone-Loaded Core Multishell-Nanocarriers

Standard experimental set-ups for the assessment of skin penetration are typically performed on skin explants with an intact skin barrier or after a partial mechanical or chemical perturbation of the stratum corneum, but they do not take into account biochemical changes. Among the various pathological alterations in inflamed skin, aberrant serine protease (SP) activity directly affects the biochemical environment in the superficial compartments, which interact with topically applied formulations. It further impacts the skin barrier structure and is a key regulator of inflammatory mediators. Herein, we used short-term cultures of ex vivo human skin treated with trypsin and plasmin as inflammatory stimuli to assess the penetration and biological effects of the anti-inflammatory drug dexamethasone (DXM), encapsulated in core multishell-nanocarriers (CMS-NC), when compared to a standard cream formulation. Despite a high interindividual variability, the combined pretreatment of the skin resulted in an average 2.5-fold increase of the transepidermal water loss and swelling of the epidermis, as assessed by optical coherence tomography, as well as in a moderate increase of a broad spectrum of proinflammatory mediators of clinical relevance. The topical application of DXM-loaded CMS-NC or DXM standard cream revealed an increased penetration into SP-treated skin when compared to untreated control skin with an intact barrier. Both formulations, however, delivered sufficient amounts of DXM to effectively suppress the production of interleukin-6 (IL-6), interleukin-8 (IL-8) and Thymic Stromal Lymphopoietin (TSLP). In conclusion, we suggest that the herein presented ex vivo inflammatory skin model is functional and could improve the selection of promising drug delivery strategies for anti-inflammatory compounds at early stages of development. View Full-Tex

Nanocrystals for Improved Drug Delivery of Dexamethasone in Skin Investigated by EPR Spectroscopy

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications

Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin

Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e.g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment. Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection. Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry- assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis. Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin