Characterization of Capecitabine Loaded Stealth Liposomes by Nuclear Magnetic Resonance

Introduction

A liposome is a spherical shaped vesicle with a membrane made up of a phospholipid and cholesterol bilayer used for the delivery of drug.^1,2 Liposomes are mostly used for drug delivery and biosensing applications.³⁻⁵ Liposomes form spontaneously when particular lipids are hydrated in aqueous media. Liposomes are ideal candidates for biological applications due to their intrinsic biocompatibility and chemical versatility.^6,7 Liposomes can accommodate both water and lipid soluble drugs.^8,9 While designing liposomes several parameters have to be considered and they can be differentiated based on the method of preparation and their physical characteristics.¹⁰ In all the different physical characteristics of liposomes, vesicle lamellarity is very important as it affects the efflux rate and encapsulation efficiency of the liposomal encapsulated drugs which have the implications on drug delivery and stability of the liposomes.^11,12

Liposomes can be classified based on their structural features as, small unilamellar vesicles (SUVs) with the diameter in the range of 20-40 nm, large unilamellar vesicles (LUVs) with the diameter larger than 100 nm, and the multilamellar vesicles (MLVs) whose size can be ranging from 0.1 to several μm.¹³ SUVs can be characterized both by the high curvature and by the marked packing asymmetry of the inner and outer monolayers.^14-16 Because of these structural properties, SUVs are significantly different from the biological cell membranes. LUVs constitute a good model for the biological membranes due to their smaller curvature and also are good candidates for therapeutic delivery applications.¹⁷ MLVs have very low curvature, extremely near to that of the real biological membranes, and therefore are useful for membrane characterizations.^18,19 The most dynamic tool for evaluation of liposomal formulations is Nuclear magnetic resonance (NMR).^20,21 NMR applications are more beneficial because this method is based on the use of intrinsic reporter moieties (the various nuclei, i.e., 13C, 31P, 1H, 15N, 195Pt). In case of capecitabine-loaded liposomes, NMR provides important information on the liposomal phospholipids.^22-24 By using NMR study, we characterized the physical and chemical nature of capecitabine in the liposomal formulations.

NMR resolution differs among liposomal vesicles because of their different characteristic structures. SUVs, show fast molecular tumbling averages, and thus allow classical NMR spectra with good resolution.^25,26-28 Molecular tumbling is slow in LUVs and MLVs to average out anisotropic interactions and therefore the NMR spectra appears to be broad and unresolved.

Advanced techniques are required to determine the degree of multilamellarity. Among these, Phosphorus NMR (³¹P NMR) has been used to estimate vesicle lamellarity. Different methods are available in the liposome formulation and each of these methods produce a different system with respect to vesicle lamellarity and size distribution. The thin film hydration method described for liposome preparation involves formation of dried lipid film in a round-bottom flask, which is then allowed for hydration with phosphate buffer, followed by sonication.^29,30,31 This method yields multilamellar vesicles in the micron-size range with a heterogeneous size distribution. Size reduction techniques are used to obtain homogeneous small unilamellar vesicle populations such as extrusion through a membrane by using pore size below 200 nm or sonication. Particle size of liposomes, hydration level have significant effect on the NMR spectra.³²

Materials and Methods

For the preparation of liposomal formulation, capecitabine was received as a gift sample from Mylon Laboratories, Bangalore, India. DSPC (Distearoylphosphatidyl choline) and Polyethylene glycol–phosphatidylethanolamine (PE- PEG) 2000 were purchased from Lipoid Germany. Solvents and reagents used were of laboratory reagent (LR) grade. All other reagents and chemicals used were of the analytical grade.

Preparation of capecitabine encapsulated stealth liposomes

PEGylated liposomes of capecitabine were prepared by thin film hydration method from various combinations of phospholipids. The weighed quantity of drug, lipids and cholesterol was dissolved in organic solvents like ethyl acetate and ethanol (2:1) in a round bottom flask. Round bottom flask was attached to a rotary evaporator by means of a clip, and vacuum was applied through a vacuum pump, rotated at 80 rpm and subjected to evaporation to obtain a thin dry lipid film. Lipid film was completely dried overnight to drain residual organic solvent by using vacuum pump. The film was allowed to hydrate using Phosphate-buffered saline (PBS) pH 7.4 above transition temperature by hand shaking for 15 minutes and further kept at room temperature and subjected to sonication. The non-entrapped drug was separated by centrifugation. Final liposomal dispersion was packed in sterile glass vials and covered with special stoppers for lyophilization. Liposomal dispersion was preserved by addition of sodium azide.³³

Nuclear Magnetic Resonance

Solid State NMR Analysis

Capecitabine-loaded liposomes were studied by 31P NMR. Capecitabine loaded liposomal formulations were formulated and analyzed using solid-state 31P NMR. The analyses were performed at a spinning speed of 10 KHz. 31P measurements were carried out at room temperature using a 5 mm computer-switchable probe. The 31P chemical shifts were measured relative to phosphoric acid set at 0 ppm. Data were collected with broad-band decoupling of the protons, with spectral width of 10 KHz, and an acquisition time of 1.6 s. The liposomes were composed of DSPC (Distearoylphosphatidyl choline), PE 18:0/18:0 PEG 2000 and cholesterol. Samples was prepared by mixing 700 Wl of the original liposome sample with 70 Wl of 2H2O. The 31P-NMR is the most accurate method for determining the lamellarity of liposomes; the broad peak spectrum designates the presence of MLV while a narrow peak spectrum indicates SUV. Using paramagnetic ions such as Mn2+, Co2+, and Pr3+ shifts the individual peaks to either downfield or upfield because of ionic interactions towards the phosphate backbone.³⁴

Results

Solid State NMR Studies

To analyze the localization of the capecitabine in the liposomal formulations, 31P solid state NMR studies were carried out. Solid state 31P NMR studies provide valuable information regarding the structure and dynamics of the lipid membrane and the phase in which the lipid molecules are present in, and any possible interaction between the drug molecule and the membrane. Figure 1 shows the solid state 31P NMR spectra for the capecitabine-loaded stealth liposomes. Phospholipid structure DSPC constituent used for the formation of liposomes is represented in Figure 1(a). Apart from the DSPC, PE-PEG and cholesterol were observed in Figure 1 (b & c). The spectra for the capecitabine loaded liposome states that capecitabine does not interact with the hydrophobic fatty acid region of the liposome bilayer. Considering Figure 1, signals observed at 1.00, 0.980, 7.103 and 2.027, 0.994, 0.612, 1.336, 2.027, 8.166, 0.157, 1.00, 7.013 ppm are corresponding to R -NH2, CH3, Ar-OH, R-NH2, R-OCH3, RCH3, Ar-phenyl oxo, Phosphate (PO42) RCH3, R-CH2, HC-OH, respectively. In the capecitabine-loaded liposomes, broadening of the peak suggests that capecitabine localizes near the phosphate moiety. At room temperature, the 31P peak observed was very broad.

Discussion

Liposomal formulations must be stable and should have a long circulation time to improve therapeutic action. In the present research work, we used 31P-NMR to determine the concentration of encapsulated capecitabine liposomes, and any other phosphorus containing drugs in the aqueous liposomal core. Vesicle size of the liposomes should be small enough to extravasate into the tissue. Through the application of NMR techniques, 31P NMR, capecitabine-containing liposome preparations were observed to have identical lipid concentrations and similar size distribution. This suggests that no possible interactions occurred between the capecitabine and the phospholipid. The use of 31P-NMR in combination with chemical shifting reagents has been extensively described. Encapsulated capecitabine has a characteristic peak that was slightly shifted downfield. This shift was caused by the physicochemical environment induced by the interior lipid bilayer membrane, resulting in de-shielding of the phosphorus nuclei shifting its resonance downfield.

Conclusion

It can be concluded that NMR is a powerful tool in the characterization of drug-lipid interactions and for the characterization of physical state of capecitabine inside the liposomes. Even after the drug encapsulation, capecitabine remained unaffected during the loading process. Encapsulated capecitabine resembles, with minor differences, the parent compound dissolved in the aqueous phase. That is, the drug within the liposomes did not precipitate or crystallize. Drug encapsulated in the liposomes was very stable with no efflux of drug or influx of medium components observed. The formulation was stable and no leakage of the hydrophilic capecitabine was detected under the experimental conditions. Improved capecitabine solubility by the contribution of the membrane grafted PE-PEG 2000 and possibly by the small size of the internal aqueous compartment was observed.

Conflict of Interest

None

Acknowledgments

I would like to Heart fully thank my guide Dr. B A Vishwanath for his continuous, enormous support and encouragement.