Voltammetric and biological studies of folate-targeted non-lamellar lipid mesophases
Introduction
The growing interest in lipidic cubic and hexagonal mesophases observed over the last decade is associated with their promising applications in medicine. Such systems display structures similar to those observed in biological membranes and their ordered organization allows for protection of the carried biological compounds [1,2]. They present a high drug delivery potential as they can be accumulated in cancer cells due to enhanced permeability and retention effect (EPR). Therefore, the chemotherapeutic can be accumulated at a desired site and released on demand via an external stimulus. The greater selectivity of drug delivery may be achieved by design of targeted materials able to increase the affinity for specific tumor cells. Utilization of lipid non-lamellar liquid crystalline nanostructures (LLC NPs) as drug delivery systems (DDS) offers advantages of high cargo loading capacity and controlled release through appropriate functionalization [[2], [3], [4]].
Lipids, when placed in water, can adopt different structural organizations, the most common ones being the inverted cubic (V2) and inverted hexagonal (H2) phase [1]. The H2 phase consists of closed reverse micellar cylinders that are arranged in a 2D hexagonal lattice. 3D-ordered structures include micellar cubic (I2) and bicontinuous cubic phase (V2) structures (including the primitive (P, Im3m), diamond (D, Pn3m) and gyroid (G, Ia3d) cubic phases) [1]. The inverted bicontinuous V2 mesophase consists of two interpenetrating, but unconnected, aqueous channels surrounded by a lipid bilayer. Such compartmentalization in lipid mesophases can be used to introduce cargos of hydrophilic, lipophilic or amphiphilic properties. The release rate of drug from liquid crystalline systems can be manipulated by changing variables such as temperature, pressure, pH or their composition [[5], [6], [7], [8], [9]]. Therefore, drug release from the bicontinuous cubic phase may be significantly faster than from the other mesophases [10,11]. In addition, the structural parameters of mesophases can be adjusted and targeted towards the specific requirements of the drug release by changing their curvature to affect the bilayer thickness, water channel diameter, and unit cell size. These factors can be predicted based on Israelachvili's concept [12] known as the critical packing parameter (CPP = v/a × l, where v is the effective volume, a is the head-group area and l is the chain length). Amphiphiles with the smaller hydrophilic heads (CPP≫1) should lead to the formation of highly negatively curved hexagonal (H2) or micellar cubic mesophases (I2). Formation of more swollen cubic systems can be obtained by additives with CPP<1, which promote the fine flattering of the membrane's curvature [13,14].
Overall, nanoparticles, which actively target tumor cells and increase the efficiency of cancer treatment, can be obtained by conjugation of the nanoparticles with ligands that bind to specific receptors overexpressed on the tumor cells. Cubosomes (CUB) and hexosomes (HEX), nanoparticles containing the inverse cubic phase or the inverse hexagonal phase, respectively, are promising strategies for the development of such targeted lipid-based DDS, as they can be functionalized with e.g. biotin or folic acid (FA) and used for imaging and active targeting of the receptors overexpressed by cancer cells [[15], [16], [17], [18], [19], [20]]. Also, paclitaxel (PX)-loaded cubosomes, functionalized with an antibody presented high affinity for an epidermal growth factor receptor (EGFR) and showed significantly higher cytotoxicity than a free drug formulation against ovarian cancer cells [16]. Moreover, PX-loaded biotinylated cubosomes presented decreased drug toxicity due to enhanced efficacy of PX against tumor cells. This was caused by the biotin ligand-promoted drug uptake by cancer cells via receptor-mediated endocytosis [17]. Other recent studies on the preparation of folate-modified cubosomes or hexosomes showed that the folate-modified cubosomes containing etoposide or camptothecin present faster uptake as a result of receptor-ligand interactions [[18], [19], [20]].
In our previous studies, we investigated application of lipidic cubic phases for controlled delivery of doxorubicin (DOX) - an anticancer drug [[21], [22], [23]]. We used electrochemical methods to determine diffusional properties of DOX and other compounds in different cubic mesophases. More recently, we showed that hexosomes are promising DOX-delivery vehicles, characterized by a prolonged release capability [24]. In this work, we extended our previous studies and performed a more detailed analysis, applying a range of in vitro assays to evaluate the effect of folate-functionalized cubosomes and hexosomes on the DOX uptake and viability of tumor cells. The structure of the folate-conjugated nanoparticles was verified by means of small angle X-ray scattering, while electrochemical techniques were used to determine the kinetics and drug release properties. The biological studies were carried out on three cancer-derived cell lines exhibiting different expressional levels of folate receptor protein (FR). We found that efficiency of DOX uptake depends not only on the release capability of the applied mesophase, but also on the level of folate receptor protein present in the cells. Moreover, it was demonstrated that exposure of the cells to an excess concentration of folic acid effectively saturated the cell's FR, resulting in the inhibition of the binding properties of folate-modified phases. Finally, we established that DOX-loaded folate-modified hexosomes induce the apoptotic state in tumor cells via prolonged drug release. The obtained data indicates that FA-mediated acquisition of drug encapsulated in the lipid mesophases can be considered as a potential strategy for targeted delivery of chemotherapeutics to FR-positive cells.
Section snippets
Materials
Monoolein (1-oleoyl-rac-glycerol) purity ≥99% (GMO), tetradecane (TD), 2-(N-morpholino) ethanesulfonic acid (MES), doxorubicin hydrochloride (DOX) and Pluronic F108 (PF108), used for the synthesis of the mesophases, were purchased from Sigma-Aldrich (USA). Schemes of the compounds used are shown in S1. All solutions were prepared with Milli-Q water (18.2 MΩ cm−1; Millipore, USA).
Preparation of nanostructures (NPs)
To produce folate-functionalized LLC nanoparticles, folic acid was conjugated with a stabilizer, Pluronic F108. Thus,
Sample characterization
All samples were analysed using SAXS technique, which offers qualitative information about the internal structure of each mesophase. The internal structure of LLC NPs, in terms of mesophase structure and lattice parameter, can be affected by various factors. High amounts of stabilizer, with respect to the amount of lipid, may change the lattice parameter. This also depends on the type of stabilizer, as in some cases, a more swollen Im3m phase may be formed. The commonly used Pluronic F108 was
Conclusion
Folate receptor is overexpressed in many tumor types, and thereby, is considered as a potential molecule for development of targeted cancer therapies. It can be exploited to deliver therapeutic compounds directly to cancerous tissues. Here, we evaluated the efficacy of folate-targeted lipid mesophases in three human-derived cell lines exhibiting different expressional levels of folate receptor protein. Phase identity and structural parameters of folate-targeted mesophases were verified through
Acknowledgements
Mirosław Salamończyk is acknowledged for help with SAXS experiments. This work was financially supported by the National Science Centre, Poland (Project No. 2016/23/B/ST4/03295). EN acknowledges support from the National Science Centre, Poland (Project No. 2017/25/B/ST4/02817).
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