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The same nucleoside crosslinking agent to develop hydrogel and organogel platforms
Marion Pitorre
1,2, Guillaume Bastiat
1,2, Le Thuy Trang Pham
1,2and Jean-Pierre Benoit
1,21 LUNAM, MINT, Angers, France, guillaume.bastiat@univ-angers.fr, +33244688531
2 INSERM, U1066, Angers, France, guillaume.bastiat@univ-angers.fr, +33244688531
INTRODUCTION
Gels are attractive pharmaceutical systems when a pathology requires a treatment involving a local administration and/or a drug sustained release. Based on nanotechnology, new drug delivery systems were designed as nanoparticle-loaded gels, combining the gel advantages with the nanoparticle properties: stealthiness, targeting site, decreased toxicity and protection of the encapsulated drug from degradation. In a previous work, Moysan et al observed an unexpected and spontaneous gelling after the formulation of lauroyl gemcitabine- loaded lipid nanocapsules (LNCs) (1), instead of the classical LNC aqueous suspension usually obtained. This hydrogel was due to H-bond interactions between LNCs (form gemcitabine moieties exposed at the LNC surface), forming a network in water. The subcutaneous administration of this hydrogel in mice showed a sustained release of LNCs along with a progressive accumulation in lymph nodes, and allowed to combat mediastinal metastases issued from an orthotopic non- small-cell lung cancer model (2).
We decided to develop the same platform without the use of gemcitabine (highly toxic component), replacing it by an endogenous molecule with similar properties. 4-(N)- palmitoyl cytidine was synthesized. This new molecule allowed a LNC-hydrogel formation, like for the lauroyl gemcitabine; and also permitted the creation of an organogel.
Figure 1. 4-(N)-palmitoyl cytidine structure
MATERIALS & METHODS
Synthesis of 4-(N)-palmitoyl cytidine (Cyt-C16) Cytidine base (4 mmol) was dissolved in water (20 mL) and palmitic anhydride (8 mmol) mixed with dioxane (80 mL) under magnetic stirring at 50°C for 48 h. The residue was purified by silica gel column flash chromatography (elution with a mixture of dichloromethane–ethanol 95/5 v/v). A white product, Cyt-C16 was obtained after evaporation (yield: 28%). The purity of Cyt-C16 was
controlled by 1H-NMR spectra (recorded on an Avance DRX 500 MHz, Bruker Daltonics GmbH, Bremen, Germany) in deuterated dimethylsulphoxide.
Cytidine-C16-loaded LNC preparation
The LNC formulation process was based on a phase- inversion of a microemulsion, as described before (3).
Firstly, Cyt-C16 was dissolved with Labrafac® WL 1349, Span® 80 and acetone. Then, all the compounds were precisely weighed (Kolliphor® HS15, water milliQ, NaCl) and then were mixed together under magnetic stirring. The mixtures were heated up to 75 °C followed by cooling to 45 °C. This cycle was repeated three times.
In the last temperature decrease at the phase-inversion temperature (50°C), 2 g of water at room temperature was added to dilute the emulsion and to generate the LNCs.
Organogel preparation
To obtain an organogel, Cyt-C16 was simply solubilized in an oil phase (Labrafac® WL1349) at 70°C. A gel was formed after cooling.
Rheological properties
The viscoelastic properties of gels were measured using a Kinexus® rheometer (Malvern Instruments S.A., Worcestershire, United Kingdom). LNCs-based hydrogels were studied according to different concentrations of LNCs, the influence of LNC sizes and Cyt-C16 concentrations with a cone plate geometry (diameter 40 mm, angle: 2°). Organogels were studied according to different concentrations of Cyt-C16 with a parallel plate geometry (diameter 20 mm, gap: 800 mm). At constant strains, 0.1% for hydrogel and 0.01% for organogel (in the linear regime), oscillatory frequency sweeps from 0.1 to 100 Hz were obtained, recording G’ (elastic modulus) and G’’ (viscous modulus), to determine the viscoelastic properties. Finally, to determine the solid-to-liquid transition temperature (TSL), temperature sweeps were performed from 25 to 75oC (heating rate 2oC/min), at a constant frequency of 1 Hz and strain of 0.01%.
Cell mortality assay
Human THP1 monocyte cells were differentiated into macrophages and the cytotoxicity of diluted Cyt-C16- loaded LNCs and non-loaded LNCs was determined using the MTT test on the macrophage cell line. Cells were seeded into 96-well plates (15,000 cells per well with
- 2 - 200µL RPMI medium) and incubated at 37°C in a CO2–
air mixture (5/95 v/v) overnight. Various amounts of LNCs (maximum concentration values of 10 mg/mL) were added into the wells, and the cells were incubated for 48 h. The number of living cells was determined using a MTT assay (colorimetric assay based on the conversion of a tetrazolium salt into formazan). Absorbance was measured using a spectrophotometer (Multiscan Ascent MP reader, Thermo Scientifc, Courtaboeuf, France), after exposition to MTT (20 µL per well) for 4 h at 37°C. The relative percentage of died cells (compared to living cells with fresh medium control) was calculated.
Figure 2: (A) Cyt-C16-loaded LNCs: Cytidine-C16 moieties were exposed at the surface. (B) Hydrogel of LNCs (C) Organogel.
RESULTS AND DISCUSSION
The hydrogel of LNCs using an analog of nucleoside was first described by Moysan et al (1). Gemcitabine hydrochloride was modified with a lauroyl chain (grafted in 4N position) on the purpose to encapsulate the prodrug in LNC. This modified drug allowed spontaneous gelling of LNC formulations.
The objective of this study was to develop a similar hydrogel form without gemcitabine which is a highly toxic molecule. Cytidine was selected since its structure is close to gemcitabine. Different sizes of aliphatic chain were tested to synthesize a modified cytidine. Finally, the cytidine modified with palmitic chain was chosen as the best candidate. In fact, Cyt-C16 presented the highest amphiphilic properties and the best synthesis yield (about 30%). LNCs were produced though a phase inversion temperature process (1,3). Cyt-C16-loaded LNCs kept the same size and the same polydispersity than non-loaded LNCs. The hydrogel form was due to H-bond interactions between cytidine moieties exposed at the surface of LNCs. The association of LNCs formed a network in water similar to a tridimensional pearl necklace structure.
Hydrogels were evaluated through their rheological properties. A gel form was obtained at a Cyt-C16 concentration of 1% (w/w Labrafac®), Cyt-C16 presents a better efficiency than the modified gemcitabine (5% w/w Labrafac®). The gel was only obtained for a LNC concentration higher than 0.2 g/mL. and LNC sizes from 50 to 100 nm. With lower concentration or lower size, individual LNCs were in suspension after formulation.
For all the hydrogels, phase transition temperatures were
higher than the body temperature (> 70°C). Furthermore, the use of a syringe and different needle sizes did not alter the gel state of the formulation. Addition of urea (60 mol for 1 mol Cyt-C16) change the rheological properties of the hydrogel without destroying it. Sodium chloride and ethanol did not have any effect on the gel properties. All these results showed hydrogels of LNCs could be subcutaneously administered without modification of the gel state of the formulation.
Cell mortality study on a macrophage cell line (derived from THP-1 cell line) confirmed the safety of Cyt-C16, comparing LNCs with and without Cyt-C16.
Preliminary studies showed that Cyt-C16 was also able to form an organogel. The molecule was simply solubilized in an oil phase at high temperature and a gel was formed after cooling. Organogel was due to interactions between Cyt-C16, forming fiber network in oil. The nature of interaction between molecules remained unknown.
Viscoelastic properties of the organogel were enhanced with increasing Cyt-C16 concentration and phase transition temperature was always higher than 70°C.
CONCLUSION
Cyt-C16 is a very promising crosslinking agent to obtain two gel platforms with opposite properties after subcutaneous administration. The LNC-based hydrogel could allow sustained release of lipophilic drugs (with progressive release of LNCs). Whereas, sustained release of hydrophilic components could be obtained from the organogel.
ACKNOWLEDGEMENTS
Authors wish to acknowledge EuroNanoMed2 ERA-NET.
(Project NICHE) for providing financial support for this work.
REFERENCES
1. Moysan E, González-Fernández Y, Lautram N, Béjaud J, Bastiat G, Benoit J-P. An innovative hydrogel of gemcitabine-loaded lipid nanocapsules: when the drug is a key player of the nanomedicine structure. Soft Matter.
2014 Mar 21;10(11):1767–77.
2. Wauthoz N, Bastiat G, Moysan E, Cieślak A, Kondo K, Zandecki M, et al. Safe lipid nanocapsule- based gel technology to target lymph nodes and combat mediastinal metastases from an orthotopic non-small-cell lung cancer model in SCID-CB17 mice. Nanomedicine Nanotechnol Biol Med. 2015 Jul;11(5):1237–45.
3. Heurtault B, Saulnier P, Pech B, Proust J-E, Benoit J-P. A novel phase inversion-based process for the preparation of lipid nanocarriers. Pharm Res. 2002 Jun;19(6):875–80.
