Evaluation of cell and hemocompatibility of Cucurbiturils
Introduction
Cucurbiturils (CB) are macrocyclic molecules consisting of methylen-bridged glycoluril units (Fig. 1). The most common representatives of the cucurbituril family are CB[5], CB[6], CB[7], CB[8], and CB[10] (Lagona et al., 2005a) whereas the number indicates the number of glycoluril units. All CB consist of a hydrophobic cavity, surrounded by ureido carbonyl groups, forming a portal. Although CB have varying width and volumes, all CB share a common depth of 9.1 Å. The structure enables the molecules to complex a variety of cationic and hydrophobic guests like metal ions, dyes and amino acids (Lagona et al., 2005a; Jaheon Kim, 1999). CB were first characterized by Robert Behrend (1905), who described them as a white and weakly soluble substance. These polymers resulted from the condensation of glycoluril and formaldehyde under acid conditions (Robert Behrend, 1905). Mock and Shih (1986) elucidated the structure of CB. Because of their pumpkin-like shape, the authors named them `cucurbiturils`, since the pumpkin belongs to the Cucurbitacea family.
CB are structurally related to the complex-building cyclodextrins, which are already used as delivery systems (Vyas et al., 2008). There is a wide range of applications for CB. Choi et al. (2007) demonstrated that CB derivatives are able to kill bacteria by forming a core that leads to disruption of the bacterial membrane. Another promising field of application is the utilization of CB as molecular drug containers (Shchepotina et al., 2011; Ghosh and Nau, 2012). Beneficial characteristics of CB are the strong binding of guest molecules by a combination of hydrophobic effect and ion dipole interactions (Mock and Shih, 1986). The encapsulation of drugs like antitumor agents by CB can enhance their stability. So the low therapeutic effect caused by quick degradation of the drugs by peptides and proteins in vivo can be improved (Wheate, 2008). The release of complexed guests can be triggered by a wide range of stimuli e.g. pH (Mock and Pierpont, 1990), photochemistry (Pischel, 2016) etc. (Lagona et al., 2005b). Furthermore, CB show enzyme like properties such as reaching a limit value of the rate of reaction or inhibition by substrate analogues (Lagona et al., 2005b). Carlqvist and Maseras (2007) described a 5.5 × 104 increase of catalytic rate of a dipolar cycloaddition within CB[6]. As reviewed by Hennig et al. (2007) CB are also able to prevent enzymatic cleavage by steric hindrance.
Currently there is a lack of information about the cellular effects of CB. Former cytotoxicity studies on CB focused on different cell lines like ovarian, kidney, liver or macrophages. Oun et al. (2014) investigated the neuro, myo- and cardiotoxic potential of CB[6] and CB[7] in which none of the CB showed neurotoxic activity whereas myo- and cardiotoxic effects were observed for both CB.
CB have a high potential for medical and dermal application hence, better knowledge of the effects of these compounds on human skin cells is required. Therefore, cytotoxic effects of CB on HaCaT keratinocytes were investigated by determining the ATP content after incubation with CB as well as secretion of pro-inflammatory cytokines. The type of cell death was subsequently examined by using different apoptotic assays based on flow cytometry. For biomedical application of CB with potentially direct contact with blood, hemocompatibility of these compounds is decisive. So, hemolytic effects of CB on human erythrocytes were investigated.
Section snippets
Materials
CB [5], [6], and [7] were purchased from Strem Chemicals Inc. (France). Stock solutions of the complexes were prepared in cell culture medium. All solutions were sterilized by filtration (0.2 µm filter, Sartorius, Stedim Biotech, Germany).
PBS (phosphate buffered saline), DMEM (Dulbecco's modified Eagle medium), FCS (fetal calf serum) and Trypsin-EDTA for cell culture were obtained from PromoCell (Germany).
Annexin V-FITC, 7-amino-actinomycin D (7-AAD) staining solution, Annexin V binding buffer
Determination of cytotoxicity by ATP measurement
Cell viability and proliferation of HaCaT keratinocytes incubated for 24 h with increasing concentrations of CB[5], CB[6], and CB[7] were determined by detection of the cellular ATP content. As shown in Fig. 2, a dose-dependent decline of cell viability was observed. The cytotoxicity of the CB seems to be structure-dependent. CB[6] had low influence on cell viability even at concentrations of up to 30 mg/mL (IC50 nd.). For CB[5] no cytotoxic effects were observed after 24 h incubation with
Discussion
CB have a high potential for future biomedical applications due to the specific complexing properties. This will require an excellent biocompatibility, which has been insufficiently explored. So far, only little has been reported about the cytotoxic properties of CB, especially on human keratinocytes. Hence, in the present study the cell and hemocompatibility of three different CB on human HaCaT keratinocytes and erythrocytes were investigated in vitro. Good cell compatibility could be shown
Conclusions
In conclusion, a very good biocompatibility of the CB[5] and CB[6] was demonstrated. Viability of HaCaT keratinocytes after incubation with these compounds was unaffected, even at concentrations up to 15 mg/mL for CB[6]. In addition, no hemolytic effect on erythrocytes was observed after incubation with CB in contrast to the structural analogues cyclodextrins. However, CB[7] exhibited significant cytotoxic effects on HaCaT keratinocytes and caused apoptotic cell death. In accordance with these
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Sarah Fink: Writing - original draft, Formal analysis, Investigation, Visualization. Kirsten Reddersen: Methodology, Validation, Investigation, Writing - review & editing. Cornelia Wiegand: Conceptualization, Writing - review & editing, Project administration, Funding acquisition. Peter Elsner: Writing - review & editing, Funding acquisition. Uta-Christina Hipler: Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
None.
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