Sirolimus Encapsulated Liposomes for Cancer Therapy

Article pubs.acs.org/molecularpharmaceutics

Sirolimus Encapsulated Liposomes for Cancer Therapy: Physicochemical and Mechanical Characterization of Sirolimus Distribution within Liposome Bilayers Ichioma Onyesom,† Dimitrios A. Lamprou,‡ Lamprini Sygellou,§ Samuel K. Owusu-Ware,† Milan Antonijevic,† Babur Z. Chowdhry,† and Dennis Douroumis*,† †

School of Science, University of Greenwich, Medway Campus, Chatham Maritime, Kent ME4 4TB, U.K. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, Glasgow G4 0NR, Scotland § Foundation for Research and Technology Hellas, Institute of Chemical Engineering Sciences (FORTH/CE-HT), 26504 Patras, Greece ‡

ABSTRACT: Sirolimus has recently been introduced as a therapeutic agent for breast and prostate cancer. In the current study, conventional and Stealth liposomes were used as carriers for the encapsulation of sirolimus. The physicochemical characteristics of the sirolimus liposome nanoparticles were investigated including the particle size, zeta potential, stability and membrane integrity. In addition atomic force microscopy was used to study the morphology, surface roughness and mechanical properties such as elastic modulus deformation and deformation. Sirolimus encapsulation in Stealth liposomes showed a high degree of deformation and lower packing density especially for dipalmitoyl-phosphatidylcholine (DPPC) Stealth liposomes compared to unloaded. Similar results were obtained by differential scanning calorimetry (DSC) studies; sirolimus loaded liposomes were found to result in a distorted state of the bilayer. X-ray photon electron (XPS) analysis revealed a uniform distribution of sirolimus in multilamellar DPPC Stealth liposomes compared to a nonuniform, greater outer layer lamellar distribution in distearoylphosphatidylcholine (DSPC) Stealth liposomes. KEYWORDS: stealth liposomes, sirolimus, mechanical properties, atomic force microscopy, X-ray photon electron analysis

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INTRODUCTION Sirolimus (rapamycin) is a potent immunosuppressive agent clinically approved for the prevention of organ transplant rejection and restenosis.1,2 Other sirolimus related compounds are in phase I−III clinical trials for the treatment of cancer.3 Sirolimus inhibits a subset of mammalian target of rapamycin (mTOR) signaling functions leading to a growth-inhibitory effect against a wide range of human cancers. mTOR is a 289 kDa serine/threonine kinase, termed PI3K-related kinase,4 which is regulated by the upstream molecules PI3K/Akt, and subsequently phosphorylates two downstream substrates leading to initiation of protein translation.5,6 Furthermore, it has been shown that mTOR inhibition is an effective approach for the treatment of ErbB2-positive breast cancers. Human breast cancers often overexpress mutationally activated epidermal growth factor (RGF) receptors such as ErbB27 and sirolimus decreases the coupling efficiency between these receptors and the PI3K signaling cascade leading to tumor suppression.8 Liposome drug delivery formulations for cancer treatment have been extensively investigated for the last 30 years for the © 2013 American Chemical Society

improved delivery of a wide range of pharmaceutically active agents including antibiotics (doxurobicin, daunorobicin), alkaloids (paclitaxel, vincristine), pyrimidine antagonists (fluorouracil) or alkylating drugs (cisplatin).9 The aim of any drug carrier is to modulate the pharmacokinetics and/or the tissue distribution, achieve controlled release or enable drug targeting to specific tumor sites and subsequently to increase the therapeutic index of the drug while minimizing its side effects.10,11 Liposomes are the most commonly used carriers for both hydrophilic and hydrophobic anticancer drugs due to their versatile in vitro and in vivo physicochemical behavior. The use of conventional (first generation) or long circulating (second generation, Stealth) liposome−drug formulations results in a wide variety of liposome shapes and sizes with improved pharmacokinetic properties (AUC, t1/2, Cmax, plasma clearance), decreased toxicity and immunogenicity, less fluctuation Received: Revised: Accepted: Published: 4281

June 21, 2013 September 5, 2013 October 7, 2013 October 7, 2013 dx.doi.org/10.1021/mp400362v | Mol. Pharmaceutics 2013, 10, 4281−4293

Molecular Pharmaceutics

Article

The lipidic films were hydrated with deionized water and then passed through an extruder (Lipex extruder by Northern lipids Inc.) 20 times via a 400 and then a 200 nm polycarbonate filter (Nucleopore) at a set temperature of 5 °C above the phase transition temperature of the lipid mixtures. The liposome formulations used in this study are depicted in Table 1.

in plasma drug concentrations and increased chemical stability of the drug(s) used relative to the drug alone.12 It is known that the encapsulation of the anthracycline doxorubicin into liposomes with attached polyethylene glycol (PEG) substantially alters its in vivo performance.13,14 The half-life of liposome encapsulated drugs depends on the size and the composition of the liposomes, with Stealth liposomes showing slower uptake by the reticuloendothelial system and reduced leakage while in circulation. Nevertheless, considerable progress has been made in engineering liposome based drug formulations with acceptable patient compliance and therapeutic effectiveness of drugs by improving the aforementioned features.15−17 Sirolimus liposome formulations18 can be used as an alternative for the treatment of breast cancer compared to the free drug preparation19 and have demonstrated significant efficacy in suppressing tumor growth and metastasis. For such purposes it is critical to engineer suitable liposome formulations with the appropriate encapsulated drug loading, particle size distribution and lipid composition. Thus, the physicochemical characterization of liposomes incorporating sirolimus can provide useful insights in relation to liposome stability, fluidity, morphology, drug-model biomembrane interactions and drug distribution/incorporation within the lipid bilayers. A detailed understanding of the foregoing parameters, using contemporary analytical techniques, will facilitate further engineering of the sirolimus liposomal formulations prior to clinical trials, thus maximizing the future clinical performance of sirolimus for chemotherapy of breast cancer patients. In the current study a variety of techniques such as differential scanning calorimetry (DSC), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) have been used to determine the characteristics of empty and sirolimus loaded conventional/Stealth liposome formulations. AFM measurements of the shape, morphology and mechanical properties can provide important information in relation to liposome elasticity and successful drug incorporation.20 DSC was used to investigate lipid thermotropic phase behavior and possible drug−liposome interactions.21 Finally, XPS is introduced as a novel approach to confirm sirolimus−liposome interactions and characterize the distribution of the drug within the lipid bilayers.

Table 1. Particle Size and Zeta Potential of Extruded Liposome Formulations (n = 3) particle size (nm) months

empty

1 3 6

177.0 181.4 192.8

1 3 6

161.1 166.4 174.1

1 3 6

197.0 201.4 204.8

1 3 6

180.5 181.7 182.6

drug loaded

zeta potential (mV) empty

DPPC/Chol −0.87 ± 1.11 ± 5.1 190.8 ± 7.4 ± 6.0 195.6 ± 6.8 0.17 ± 0.49 ± 5.8 202.1 ± 8.3 0.01 ± 0.96 DSPC/Chol ± 3.2 128.1 ± 4.5 0.73 ± 0.21 ± 4.7 132.7 ± 4.8 0.55 ± 0.13 ± 4.3 132.4 ± 5.1 0.70 ± 0.10 DPPC/Chol/DSPE-MPEG-2000 (Stealth) ± 4.3 160.8 ± 5.8 0.91 ± 0.45 ± 3.7 161.6 ± 5.5 −0.08 ± 0.01 ± 4.1 163.1 ± 6.3 0.70 ± 0.13 DSPC/Chol/DSPE-MPEG-2000 (Stealth) ± 3.0 138.2 ± 3.0 0.59 ± 0.78 ± 4.8 138.8 ± 4.0 −0.47 ± 0.52 ± 3.1 141.3 ± 2.7 1.71 ± 0.12

drug loaded −6.14 ±7.74 −7.25 ± 4.69 −5.36 ± 6.31 −8.02 ± 5.20 −7.46 ± 6.73 −7.98 ± 4.61 −13.15 ± 5.45 −13.78 ± 3.72 −12.01 ± 3.09 −18.22 ± 3.10 −19.66 ± 4.55 −19.44 ± 4.88

The sirolimus encapsulation efficiency (EE) was estimated by using the following equation: EE =

amount of the drug encapsulated in the NPs × 100 amount of the drug added in the process

Briefly, the drug amount encapsulated in the liposome dispersions was measured by high performance liquid chromatography (HPLC). An Agilent Technologies 1200 series with a quaternary pump, an autosampler and a detector used at 278 nm was used for HPLC assay. A Lichrocart 250-4-RP 18 5 μm (Merck) column was used with mobile phase 60:40 acetonitrile:water. The column was maintained thermostatically at 50 °C. The flow rate was 1.0 mL/min and the injection volume 10 μL. Calibration curves were constructed using standard solutions of known concentrations (3−50 ng/mL). The Chemstation software calculated the peak area of each standard solution and sample automatically. Freeze-dried liposome formulations (3 mg) were dissolved in 1 mL of DCM followed by solvent evaration and the addition of 3 mL of mobile phase. The solution was filtered by 0.45 mm PVDF syringe filter for HPLC analysis. Particle Size and Zeta Potential Analysis. The particle size distribution and the zeta potential of the liposome preparations were determined by dynamic light scattering (PCS) using a Malvern Zetasizer Nano-ZS (Malvern, U.K.). Measurements were carried out at 25 °C at a fixed angle 90°. The sizes quoted are the z-average means (dz) of the liposomal hydrodynamic diameters (nm). The Nano-ZS instrument can be used for particle size determination in the 0.6 nm to 6 μm range. All samples were stored at 4 °C, and the particle size/zeta potential was measured at one, three and six month time intervals in order to determine their stability.

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MATERIALS AND METHODS Materials. Sirolimus was obtained as a gift from Phoqus Pharmaceuticals Ltd. (West Malling, U.K.). Distearoylphosphatidylethanolamine-methyl-polyethylene glycol (DSPE-MPEG2000), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Avanti Lipids Inc.) were kindly donated by Lipoid GmbH (Ludwigshafen, Germany). Calcein, Triton X-100, serum FBS and dichloromethane (DCM) were purchased from Sigma (Gillingham, U.K.). Methods. Liposome Preparation and Encapsulation Efficiency (EE). Oligolamellar (OLV) liposome vesicles were prepared following the thin film hydration method, as previously described by Bangham et al.22 In brief, conventional and Stealth liposome formulations were produced by dissolving the appropriate amounts of lipid mixtures (DPPC, DSPC, DSPE-MPEG-2000 and cholesterol) in the presence or absence of sirolimus in chloroform. The lipid mixtures were subsequently evaporated under a vacuum in a round-bottomed flask connected to a rotor evaporator to obtain thin lipid films. 4282

dx.doi.org/10.1021/mp400362v | Mol. Pharmaceutics 2013, 10, 4281−4293


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Figure 1. Membrane integrity of the liposome formulations using percentage latency over a period of 24 h.

Evaluation of Liposome Membrane Integrity. The membrane integrity of liposome formulations, comprising different lipid compositions after incubation at 37 °C, was evaluated by calculating the percentage retention latency of the liposome encapsulated calcein formulations. Briefly, calcein encapsulated liposome formulation fractions (1 mL) were incubated with serum proteins (80% FBS) at 37 °C in a shaker bath, and samples were collected at different time intervals for 24 h. The retention of calcein in the liposome formulations was estimated by mixing 20 μL of the collected samples and 4 mL of PBS, pH 7.4. The calcein fluorescence of the samples was measured (at excitation and emission wavelengths of 490 and 520 nm, respectively) before and after the addition of Triton X100 (final concentration of 1%). The percentage latency of the calcein was determined using the expression % latency =

scanning data into a digital leveling algorithm (Bruker Image Analysis Nanoscope Analysis software V 1.40). AFM images were collected from two different samples by random spot surface sampling (at least five areas). Differential Scanning calorimetry (DSC). A Mettler-Toledo 823e (Greifensee, Switzerland) differential scanning calorimeter (DSC) was used to conduct DSC scans of sirolimus as well as unloaded and loaded liposome formulations. Each sample (2−5 mg) was placed in a sealed aluminum pan and heated at 10 °C/ min from 0 to 220 °C under an atmosphere of dry nitrogen. An empty pan was used as a reference. All DSC data was normalized to a sample mass of 1 g. X-ray Photoelectron Spectroscopy (XPS). The surface analysis studies were performed in a UHV chamber (P < 10−9 mbar) equipped with a SPECS LHS-10 hemispherical electron analyzer. The XPS measurements were carried out at room temperature using a non-monochromatized Al Kα radiation source under conditions optimized for maximum signal (constant ΔE mode with a pass energy of 97 eV giving a full width at half-maximum (fwhm) of 1.7 eV for the Au 4f7/2 peak). The analyzed area was a rectangle with dimensions of 2.5 × 4.5 mm2. To improve the energy resolution the O1s and C1s spectra were also obtained with pass energy of 36 eV giving a fwhm of 0.9 eV for the Au 4f7/2 peak. The XPS core level spectra were analyzed using a fitting routine, which can decompose each spectrum into individual mixed Gaussian− Lorentzian peaks after a Shirley background subtraction. The liposome samples were in freeze-dried form, pressed on a freshly cut indium support.

1.1(FAT − FBT) × 100 1.1FAT

where FBT and FAT represent the fluorescence intensities of calcein before and after the addition of Triton X-100, respectively. Atomic Force Microscopy (AFM). For AFM experiments, the Stealth loaded and unloaded DPPC and DSPC samples were diluted 50 times with saturated drug, and then 3 μL of the liposome sample was deposited onto a freshly cleaved mica surface (G250-2 mica sheets 1 in. × 1 in. × 0.006 in.; Agar Scientific Ltd., Essex, U.K.), and left to dry for 1 h before AFM imaging. The images were obtained by scanning the mica surface, in air, under ambient conditions using a PeakForce QNM scanning probe microscope (Digital Instruments, Santa Barbara, CA, USA; Bruker Nanoscope analysis software Version 1.40). The AFM measurements were obtained using ScanAsystair probes, and the spring constant (0.67 N/m; nominal 0.4 N/ m) and deflection sensitivity were calibrated, but not the tip radius (a nominal value of 2 nm was used). The following settings were used for all samples: aspect ratio 1, amplitude set point 250 mV, and drive amplitude 1500 mV. Surface roughness (Ra) values were determined by entering surface

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RESULTS AND DISCUSSION Liposome Particle Size, Stability and Membrane Integrity. The particle size and zeta potential of the produced conventional and Stealth liposome formulations were measured immediately after preparation and for a period of six months after storage at 4 °C (Table 1). All formulations showed very good polydispersity (conventional 0.165; Stealth 0.149), and the zeta potential was approximately zero for the non-drug loaded liposome samples. The incorporation of sirolimus 4283



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Table 2. Mechanical Properties of Stealth Liposomes Measured by AFM (n = 20 Random Particles) mechanical properties

DSPC-empty

DSPC-drug

DPPC-empty

DPPC-drug

height (nm) diameter (nm) surface roughness/nm density (μm−2) force (pN) DMT modulus (MPa) deformation (pm) dissipation (eV)

12.0 ± 1.0 176.0 ± 26.0 5.0 ± 1.0 3.97 179.0 ± 31 235.0 ± 27 88.6 ± 36.0 721.0 ± 76.0

14.0 ± 3.0 200.0 ± 45.0 5.0 ± 2.0 0.63 348 ± 36 49.0 ± 24.0 2648.0 ± 715.0 701.0 ± 85.0

11.0 ± 1.0 185.0 ± 35.0 5.0 ± 1.0 3.67 158.0 ± 58.0 230 ± 27 97.0 ± 45.0 744.0 ± 73.0

12.0 ± 2.0 187.0 ± 44.0 5.0 ± 1.0 0.85 292.0 ± 82.0 51.0 ± 30.0 3747.0 ± 661.0 737.0 ± 51.0

have also demonstrated that the lipid composition has a significant effect on liposome membrane permeability due to the differences in their phase transition temperatures.29 In addition to these findings, a difference in membrane rigidity is also found between the two different Stealth formulations of which DPPC-Stealth liposomes demonstrated decreased membrane integrity compared to DSPC-Stealth liposomes. It is, however, important to consider the permeability of the liposome formulations in the context of the encapsulation of the drug for delivery into cancer cells. A very permeable liposome membrane formulation suggests greater release of the drug from the liposome matrix, but this could also mean a less stable formulation. Atomic Force Microscopy of Liposome Formulations. Scanning probe microscopy has long been recognized as a useful tool for measuring mechanical properties of materials, but until recently it has been impossible to achieve truly quantitative high-resolution mapping. The atomic force microscope is one kind of scanning probe microscope, and with the mode PeakForce QNM AFM instrument it is possible to identify material properties at high resolution across a topographic image such as surface roughness, density, adhesion, DMT modulus, deformations and dissipation. The mechanical values for the individual spherical lipid nanoparticles (all the particles are separated from each other and do not form aggregates) were calculated by AFM QNM mode,30 and the results are shown in Table 2. For all liposome formulations investigated by AFM the overall particle diameter (estimated from the width of the peak at the baseline in sectional height profiles) was estimated, as shown in Figure 2. The AFM studies showed spherically shaped vesicles, which were flattened as a result of their adsorption on the mica surface. However the average diameters of the liposomes are slightly larger than those determined by DLS, which may be attributed to the slight deformation of the liposomes caused by adsorption.31 The height (≈13 nm) is usually underestimated due to the high surface density of the liposomes preventing the tip from accessing the substrate and the width overestimated because of tip broadening effects.32 The surface roughness, determined from the images, for all the liposomes was ≈5 nm (rms), which means the liposomes are very smooth and without any detectable pores. Furthermore, the individual liposomes appear to be located in different planes, which may be caused by rearrangements of the liposomes during imaging, causing a reduction in image resolution.33 The DMT modulus of the unloaded liposomes was approximately 5 times larger than that of the drug loaded liposomes. The force of adhesion (Fad) of the drug-loaded liposomes was approximately 2 times larger, and the deformation was approximately 30 times larger, than those of the unloaded liposomes. The energy dissipation does not show significant differences (≈700 eV) between the

induced a negative zeta potential leading to improved particle stability. For conventional liposome formulations no substantial difference in the particle size was observed between unloaded and loaded dispersions, with only a slight increase due to sirolimus addition. The particle size obtained for unloaded conventional and Stealth liposomes showed a small particle size decrease for the latter of approximately 22 nm. This could possibly be attributed to the different molar ratios of DSPC:chol and DPPC:chol and the presence of DSPEMPEG-2000. More importantly, the data in Table 1 shows that sirolimus loaded conventional and Stealth liposomes display size reductions of around 35 nm compared to the respective unloaded liposome formulations. The additional particle size reduction was initially attributed to possible drug interactions with the lipid bilayers, as previously reported.23,24 However, further characterization of the dispersions revealed that drug−liposome interactions are negligible and the size reduction is related to the drug distribution within the liposome bilayers. Particle size measurements of the stored liposomes demonstrated excellent stability even after six months with only a slight increase in the size (2−12 nm) for some of the formulations. Similarly, the zeta potential remained unchanged for the same time period. The overall stability of both the conventional and Stealth formulations was attributed not only to the composition of the formulations but also to the optimized sirolimus loading at 2 mg/mL. The formulations were also evaluated in terms of drug loading (data not shown) for 1−5 mg/mL where particle aggregation was noticed for loadings above 2 mg/mL. These results are in agreement with studies conducted by Rouf et al.,25 who observed similar stability properties of sirolimus using different liposome formulations. The sirolimus EE was estimated for all formulations, and it was found to be 95 − 98%. Calcein encapsulation and release from liposomes was used to study the integrity of conventional and Stealth formulations. The possibility of increasing the membrane integrity and physical stability of the liposome dispersions by coating their surface with polyethylene glycol molecules was also evaluated. Calcein release across the phospholipid bilayer membrane is a simple but efficient assay often used to elucidate the influence of different factors on membrane permeability or rigidity.26,27 The data in Figure 1 demonstrates that conventional DPPCChol liposome possesses a less rigid membrane followed by DSPC-Chol liposomes as assessed by the percentage of encapsulated calcein after 24 h. In contrast, Stealth liposomes appear to be more rigid compared to the conventional formulations. Piperoudi et al.28 reported that pegylation of liposome formulations increases membrane rigidity, and this is observed in the membrane integrity difference between the Stealth and conventional formulations. Previous investigations 4284



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Figure 2. AFM images of sirolimus loaded DPPC/Chol/DSPE-MPEG-2000 (A, B) and DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes (C, D).

data). This can be potentially explained by two phenomena. First, sirolimus loading may result in liposomes with a lower phospholipid packing density (PPD) due to the molecular dimensions of sirolimus. The lower PPD results in less stabilizing intermolecular forces between phospholipids and therefore a lower stiffness. Second, the intermolecular forces between sirolimus−phospholipid and sirolimus− sirolimus are not as significant as phospholipid−phospholipid, phospholipid−cholesterol and cholesterol−cholesterol interactions and as such liposome stiffness is decreased.

loaded/unloaded liposomes and the mica surface at the imaging set point employed. The mechanical data clearly shows that sirolimus significantly influences liposome stiffness (or perhaps “solidness”). Liposomes produced from either DSPC or DPPC without sirolimus loading show similar mechanical properties (force, DMT modulus and deformation), which is unsurprising given the similarity in chemical structure (a difference of two CH2 groups in the phospholipid acyl chain). Loading liposomes with sirolimus results in a significant reduction in liposome stiffness (particularly seen in the DMT modulus and the deformation 4285



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Table 3. DSC Transition Parameters Obtained for the Unloaded and Loaded DPPC Liposome Formulations DPPC conventional (empty) 2nd event

DPPC Stealth (empty)

transition

1st event

3rd event (Tm)

peak (°C) ΔH (J/g)

47.6 ± 0.2 15.1 ± 4.0

transition

1st event

2nd event

3rd event (Tm)

1st event


46.0 ± 2.2 12.3 ± 0.8

67.8 ± 0.1 5.7 ± 0.5

90.4 ± 0.4 2.8 ± 0.2

51.5 ± 1.1 30.3 ± 1.0

70.2 ± 0.4 90.4 ± 0.1 7.9 ± 0.3 2.2 ± 0.4 DPPC conventional (loaded)

1st event

2nd event

51.3 ± 0.4 102.9 ± 15.6

3rd event (Tm) 89.5 ± 0.4 2.2 ± 1.1

DPPC Stealth (loaded) 2nd event

3rd event (Tm) 83.5 ± 0.4 0.8 ± 0.1

Table 4. DSC Transition Parameters Obtained for the Unloaded and Loaded DSPC Liposome Formulations DSPC conventional (empty) transition

1st event


65.1 ± 0.1 22.4 ± 2.8

2nd event

DSPC Stealth (empty)

3rd event (Tm)

1st event

94.6 ± 0.7 8.1 ± 1.7

46.2 ± 0.5 16.8 ± 2.3

DSPC conventional (loaded) transition

1st event


65.6 ± 0.2 17.6 ± 1.7

2nd event

2nd event 58.9 ± 0.6 18.7 ± 1.0 DSPC Stealth (loaded)

3rd event (Tm) 78.5 ± 0.4 2.4 ± 0.1

3rd event (Tm)

1st event

2nd event

3rd event (Tm)

94.4 ± 0.1 8.6 ± 0.1

46.8 ± 0.6 6.5 ± 1.5

59.2 ± 0.5 18.1 ± 0.4

74.6 ± 0.6 0.6 ± 0.3

Differential Scanning Calorimetry Analysis (DSC). It is well accepted that, upon dehydration of liposome formulations, the main transition temperature (Tm), which represents the transition from the gel-to-liquid crystalline state or the acyl chain melt, increases. This change in Tm has been attributed to the decrease in phospholipid headgroup spacing in the bilayer, resulting in an increase in van der Waals interaction between phospholipid molecules.34 In this study the Tm observed for freeze-dried DPPC liposomes is 99.7 ± 0.6 °C, which is comparable with previously reported data for a similar system.35 There were no significant differences observed in the Tm for conventional DPPC and DSPC liposomes (Tm = 99.7 ± 0.6 and 100.4 ± 0.3 °C, respectively). By incorporating cholesterol into the DPPC liposomes (conventional liposome formulation), the Tm decreased to 90.4 ± 0.1 °C (Table 3). In addition, significant differences in the enthalpy change associated with this chain melting transition (ΔHTm) were observed (19.3 ± 1.8 and 2.2 ± 0.1 J/g for DPPC only and DPPC conventional formulations, respectively). Again the observed ΔHTm for the DPPC conventional formulation is very similar to that reported previously.36 Similar behavior is observed for the DPPC and DSPC conventional liposome formulations, i.e., the Tm for the DSPC liposome decreases to 94.6 ± 0.7 °C in the presence of cholesterol and the ΔHTm decreases from 17.6 ± 1.5 J/g (DSPC liposome) to 8.1 ± 1.7 J/g (DSPC conventional). The change in temperature and magnitude of the chain melting transition in the presence of cholesterol has been attributed to weakened intermolecular interactions between the phospholipids and increased motional freedom.37 This suggests that in the presence of cholesterol the liposomal bilayer is in a more fluid state in comparison to the bilayer without cholesterol. This can be explained by considering the packing properties of the phospholipids in liposome formulations. In the absence of cholesterol the phospholipids in the liposome bilayers are able to pack tightly together, hence restricted motional freedom. However, when cholesterol is added to the formulation, this packing ability is hindered because of the multiring, rigid and nonlinear structure of cholesterol, which wedges between

phospholipid molecules and prevents tight packing of lipid molecules. When comparing the DPPC and DSPC conventional liposomes, it is clear that the DSPC conventional liposome formulations exhibit better stability, as both the Tm and ΔHTm are greater (Tm = 94.6 ± 0.7 °C, ΔHTm = 8.1 ± 1.7 J/g) than the conventional DPPC formulation (Tables 3 and 4). This is due to the differences in the carbon chain length and hence molecular weight of the DSPC (C18 and 789 g/mol) in relation to the DPPC (C16 and 733 g/mol), i.e., DSPC has greater acyl chain interactions due to the extra (CH2)4 groups. The presence of cholesterol has a relatively lower disruptive influence on the packing of DSPC molecules, hence the need for more energy to disrupt the phospholipid interactions in the bilayer, resulting in the observed higher Tm and ΔHTm values, in addition to the differences in Tm. There are also significant differences in the pre-Tm transitions observed for DPPC and DSPC. DPPC exhibits two pre-Tm transitions at 46.0 ± 2.2 °C and 70.2 ± 0.4 °C (Table 3), while DSPC exhibits only one pre-Tm transition at 65.1 ± 0.1 °C. The origins of the endothermic events observed below the Tm’s described above for the unloaded DPPC and DSPC conventional liposome formulations are not entirely clear. However, previous studies have suggested that these transitions (in addition to the main transitions described earlier) may be due to uneven distribution of cholesterol in the liposome formulations.36 Ohtake et al. explained that the lower temperature transitions might actually be chain-melting transitions, resulting from “cholesterol-rich” domains. For example, the first event observed at 46.0 ± 2.2 °C and 65.1 ± 0.1 °C for DPPC and DSPC conventional liposomes, respectively, arises from the chain melt of “cholesterol-rich” domains in the formulation. This implies that the Tm values at 90.4 ± 0.1 °C and 94.6 ± 0.7 °C result from the chain melt of “cholesterol-poor” domains for conventional DPPC and DSPC liposomes, respectively. The incorporation of molecules (drugs) into liposomes has been reported to affect the thermal behavior of its phase transitions, which results in reduction in the main transition temperature. However studies have shown that a more 4286



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Figure 3. DSC thermograms of unloaded and loaded conventional DPPC/Chol liposomes.

pronounced effect can also be observed in the pre-Tm transitions, and in some cases an effect can only be observed in the enthalpy change.38 Papahajopoulos et al.39 classified the interaction of molecules with phospholipids into three major groups which comprise (i) surface adsorption only, (ii) partial embedding into the bilayer of the lipid and (iii) penetration into the core of the anionic or zwitterionic lipids bilayers. Depending on the molecule (drug) interaction with the lipid bilayer, the following observations are seen in DSC, with the first interaction resulting in an increase in the enthalpy change accompanied by either an increase or no change in the transition temperature (Tm). The other two interaction processes result in the reduction of enthalpy with a decrease or no change in the transition temperature. In this study the encapsulation of sirolimus into different liposome formulations resulted in either the decrease of Tm or the enthalpy change depending on the drug distribution and interaction with the lipid bilayers. The addition of sirolimus to the conventional DPPC liposome formulation showed significant differences in the temperature and enthalpy changes of the pre-Tm transitions (Figure 3). This is not surprising if we consider these pre-Tm transitions to result from “cholesterol-rich” domain phospholipid chain melts. In theory, the incorporation of the drug into the bilayer increases the distance between the phospholipids in the bilayer (this is conditional on the fact that the drug molecules occupy sites

between phospholipid molecules, rather than between the lipid monolayers of the bilayer) and therefore causes a further increase in motional freedom of the phospholipid molecules. This in turn results in a decrease in transition temperatures and enthalpies of the phospholipid molecules in the bilayer. What is observed here, however, is that a decreased fraction of DPPC exists in the drug loaded formulations, hence in the “cholesterol-rich” domains there are relatively lower amounts of DPPC molecules in a liposomal bilayer in comparison to the bilayer which gives a Tm of 90.4 °C. As a result the presence of both the cholesterol and drug causes greater disruption of the DPPC packing in the bilayer, and this further results in a greater motional freedom. This causes the reduction of the enthalpy change associated with the transition and a slight decrease in temperatures (Table 3). Interestingly there were no significant differences in the temperatures and enthalpies changes of the Tm for loaded and unloaded DPPC conventional formulations (Table 3). It can only be assumed that the disruption of packing of the DPPC molecules by the cholesterol and drug was negligible due to, again if we consider cholesterol distribution in the bilayer, a lower proportion of cholesterol. These observations indicate two things: (1) sirolimus is incorporated in the lipid bilayers of the conventional DPPC formulations, and (2) attention need not be focused only on the Tm values when analyzing liposome formulations. The preTm transitions are also good indicators of drug incorporation 4287



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Figure 4. DSC thermograms of unloaded and loaded DPPC/Chol/DSPE-MPEG-2000 liposomes.

into lipid bilayers, particularly when studying nano size liposome formulations. There are no significant differences in phase transition temperatures and the enthalpy changes of the Tm between the DSPC conventional loaded and unloaded formulations (Table 4). However, there were significant differences in the enthalpy change of the pre-Tm transitions (22.4 ± 2.8 J/g for DSPC conventional unloaded and 17.6 ± 7 J/g for DSPC conventional loaded). This is also due to the reduction in the fractional composition of the DSPC in the conventional loaded formulation. Similar findings have also been recently reported by Tabbakhian and Rogers40 whereby the incorporation of insulin and polymers into DPPC liposome causes no significant change in the phase transition temperature of the liposome, but, however, decreased enthalpy of the transition was observed in some cases. The addition of DSPE-MPEG 2000 in the unloaded Stealth formulation for DPPC showed no significant difference in the characteristics of the Tm when compared with the unloaded DPPC conventional liposome formulation, i.e., the Tm for unloaded DPPC Stealth formulation is 89.5 ± 0.4 °C and ΔH of 2.2 ± 1.1 J/g and that observed for the unloaded DPPC conventional formulation is 90.4 ± 0.1 °C and ΔH of 2.2 J/g. However, below this chain melt transition significant differences exist between the conventional and Stealth formulations. Only one transition (at 51.3 ± 0.4 °C with ΔH of 102.9 ± 15.6 J/g)

is observed below the Tm for the Stealth formulation. This is a broad transition spanning a temperature range from 40 to 80 °C that consists of several events, which may include transitions resulting from “cholesterol-rich” domains of the liposome formulations. The most important contribution, however, is that from the melting of PEG2000, which in the pure form occurs at ∼53 °C.38 It is important to remember that in the Stealth formulations there is a mixture of phospholipids (DPPC and DSPE which is conjugated with MPEG2000). The differences in the head groups could be causing the small differences in the Tm values observed for the DPPC conventional and Stealth formulations (Table 3). Drug loading of the DPPC Stealth formulation (Figure 4) causes a significant change in the temperature and magnitude of the chain melt transition. The Tm for this transition decreases from 89.5 ± 0.4 °C to 83.5 ± 0.2 °C and the ΔH decreases from 2.2 ± 1.09 J/g to 0.8 ± 0.1 J/g when the DPPC Stealth formulation is loaded with sirolimus. The foregoing parameters confirm the interaction of sirolimus in the lipid bilayer and thus the incorporation of the drug into the lipid bilayer. Interestingly the peak temperature of the transition observed at 51.3 °C for the Stealth formulation (Table 3) does not change significantly when the formulation is loaded with sirolimus. However, the ΔH for this transition decreases from 102.9 ± 15.6 J/g to 30.3 ± 1.0 J/g. This is likely to be the result of the reduction in the fractional composition of the DPPC and DSPE-MPEG 2000 in 4288



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Figure 5. DSC thermograms of unloaded and loaded DSPC/Chol/DSPE-MPEG-2000 liposomes.

However, the ΔH reduction of the first pre-Tm transition of the DSPC Stealth liposomes is lower compared to the DPPC Stealth, suggesting a farther disordered state of the latter. The differences observed with the addition of DSPE-MPEG 2000 for both the DPPC and DSPC Stealth formulations are most likely due to steric hindrance caused by the molecules of the MPEG 2000 near the conjugation site of the DSPE-MPEG 2000. This could prevent closer packing of the DSPE and the DPPC or DSPC head groups, which in turn affects the packing ability of the lipid acyl chains;33 hence, the lower temperatures and ΔH changes observed for the Stealth formulations in comparison to the conventional. This could also be an additional explanation as to why greater differences were observed between loaded and unloaded Stealth formulations in comparison to the conventional formulations. X-ray Photoelectron Analysis (XPS) of Stealth Liposomes. Figure 6 shows the XPS spectra recorded for all samples, where the presence of the following elements (hydrogen is not accessible for analysis by XPS) was detected: In, O, C, P and N. Based on the binding energy (BE) values of a strong photoelectron peak, information about the chemical state for each element can be obtained. A correction to account for binding energy shift due to electrostatic charging was applied based on In3d BE at 443.6 eV.41 The indium substrate is composed of a superficial thin indium oxide layer and carbon contamination due to exposure to the atmosphere. Figure 7

the loaded formulation. However, this behavior also supports the notion that the formulation exists in a state of increased motional freedom driven by the lipid bilayer, rather than the external PEG2000 molecules. As a result the liposomes in the loaded DPPC Stealth formulation are in a relatively more disordered state in comparison to the unloaded Stealth formulation. Significant differences are observed in the Tm transition between the conventional and Stealth DSPC formulations (94.6 ± 0.7 °C, ΔH of 8.1 ± 1.7 J/g and 78.5 ± 0.4 °C, ΔH of 2.4 ± 0.1 J/g, respectively). In addition, the DSPC Stealth formulation exhibits two pre-Tm transitions at 46.2 ± 0.5 and 58.9 ± 0.6 °C. This is very interesting because it is the reverse of what is observed for the DPPC, i.e., in the DPPC the conventional formulation exhibited two pre-Tm transitions while the Stealth formulation exhibited only one. No explanation can be offered for this observation, apart from the differences in interaction of the DSPE-MPEG2000 with the DPPC and DSPC molecules. Drug loading of the DSPC Stealth formulation (Figure 5) causes a significant shift in the Tm transition temperature and enthalpy change (Table 4). On the other hand, the temperature and ΔH of the pre-Tm at 58.9 ± 0.6 °C remain similar. The first pre-Tm transition exhibits no significant difference in temperature, but the ΔH changes from 16.8 ± 2.3 J/g to 6.5 ± 1.5 J/g when the DSPC Stealth liposomes are loaded with drug. 4289



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Figure 6. XPS wide spectra of unloaded and loaded DPPC/Chol/ DSPE-MPEG-2000 and DSPC/Chol/DSPE-MPEG-2000 Stealth liposomes.

shows the P2p and N1s core level spectra of all samples. The BE of P2p is 133.8 ± 0.1 eV, which was identified as the P5+ state resulting from the PO4 moiety of the lipid headgroup.42 The BE of N1s is 402.8 ± 0.1 eV, which is assigned to positively charged nitrogen atoms.43 In addition, Figure 8 shows the C1s peaks for unloaded and loaded Stealth DSPC samples while Figure 9 shows the C1s peaks for unloaded and loaded Stealth DPPC samples, respectively. The peak for the unloaded samples and for the Stealth DPPC loaded is analyzed into 3 components corresponding to the C−C/C−H component at

Figure 8. C1s XPS core level spectra of DSPC/Chol/DSPE-MPEG2000 unloaded and loaded liposomes.

285.0 ± 0.1 eV, C−O(H) groups at 286.6 ± 0.1 eV and O− CO acrylate or ester groups at 289.5 ± 0.1 eV. In Stealth

Figure 7. P2p and N1s XPS core level spectra of Stealth liposomes. 4290



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Figure 10. O1s XPS core level spectra of DSPC/Chol/DSPE-MPEG2000 unloaded and loaded liposomes.

Figure 9. C1s XPS core level spectra of DPPC/Chol/DSPE-MPEG2000 unloaded and loaded liposomes.

DSPC loaded samples the main peak for the (C−C/C−H) component centered at 284.5 eV shows a significant shift from the 285.25 eV of the unloaded liposomes. This shift is attributed to the presence of increased C−O/C−C(H) moieties on the liposome surface (e.g., sirolimus). Furthermore, the data shown in Figure 10 depict the O1s peaks for unloaded and loaded Stealth DSPC samples while Figure 11 the Stealth DPPC unloaded and loaded samples, respectively. The O1s peak is divided into three components where the first is centered at 530.6 eV corresponding to O2− of In2O3. The second at 532.5 ± 0.1 eV is due to CO groups (ketones) peak I, which are present in sirolimus, and the third at 533.8 ± 0.1 eV corresponds to C−O−C or C−OH bonds (peak II), which are present in the liposomes. Using the total peak area of N1s, P2p and C1s peaks, the sum of peak I and peak II of O1s peak in each case, the appropriate sensitivity factors (based on Wagner’s collection and adjusted to the transmission characteristics of analyzer EA10) and physicochemical parameters, the average relative atomic concentration in the analyzed region, normalized with respect to carbon, can be determined. The peak intensities are corrected in relation to the thickness of liposome samples. The data in Table 5 shows the relative atomic concentrations, the P/N ratio and the intensity ratio of the two O1s peaks I(peak I)/I(peak II). The theoretical atomic concentrations for the unloaded liposome samples (based on the molar ratios) are C:O:N:P = 1:0.19:0.016:0.015 for the Stealth DSPC and C:O:N:P = 1:0.20:0.017:0.016 for the DPPC. The experimental atomic ratios and the P/N ratio are, within the experimental error of the XPS technique (10%), close to the theoretical (0.93) value. As shown in the data in Table 5 the P/N ratio of Stealth DPPC

Figure 11. O1s XPS core level spectra of DPPC/Chol/DSPE-MPEG2000 unloaded and loaded liposomes.

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Table 5. Surface Atomic Concentrations, and Intensity Ratios of I(peak I)/I(peak II) and IP2p/IN1s for Liposome Formulations

Stealth Stealth Stealth Stealth

DSPC empty DSPC loaded DPPC empty DPPC loaded

C:O:N:P

I(peak I)/I(peak II)

P/N

1:0.19:0.012:0.014 1:0.17:0.0092:0.010 1:0.19:0.014:0.016 1:0.18:0.014:0.016

0.62 2.1 0.55 0.76

1.17 1.09 1.14 1.14

REFERENCES

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loaded and unloaded nanoparticles remains identical. This means that the distribution of N near or at the surface did not increase with the presence of the drug. As a result it can be concluded that sirolimus is concentrated inside the nanoparticles with an even and random distribution. This is also confirmed by the small increase of the I(peak I)/I(peak II) ratio which is related to the increase in the C−OH(R) groups of sirolimus. In contrast, the decrease in the P/N ratio for the Stealth DSPC liposomes indicates a greater outer lamellar layer of sirolimus on the liposome surface. This becomes more evident by the significant increase of the I(peak I)/I(peak II) ratio of the Stealth DSPC liposomes. The significant increase in the C−OH groups of sirolimus suggests the drug distribution on the liposome surface during nanoparticle formation. The results of sirolimus colocalization are very interesting as the drug was expected to distribute within the liposome bilayers due to its high lipophilicity. However, this was observed only for the Stealth DPPC liposomes and not for the Stealth DSPC liposomes. The XPS analysis provided a comprehensive understanding of sirolimus distribution in Stealth liposomes in complement with the AFM and DSC studies. The analysis of the mechanical properties (AFM) and the thermal behavior of the sirolimus Stealth DPPC liposomes showed a lower packing density and more disordered state compared to the Stealth DSPC liposomes which is attributed to the different drug distribution along the lipid bilayer. Stealth DPPC liposomes showed lower packing density and higher bilayer distortion due to the more uniform distribution of sirolimus within the nanoparticles.

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CONCLUSIONS Conventional and Stealth liposome nanoparticles were loaded with the anticancer drug sirolimus and characterized using a range of analytical techniques. All formulations were found to be stable over six months with Stealth liposomes demonstrating better membrane integrity. It was shown that sirolimus encapsulation in liposomes has a significant effect on the packing density of the bilayers leading to a distorted state of the nanoparticles. Indeed, the incorporation of sirolimus into liposomes resulted in a uniform distribution of sirolimus in multilamellar DPPC Stealth liposomes compared to a nonuniform outer layer lamellar distribution in DSPC Stealth liposomes. The techniques employed in this study can be a valuable tool for the characterization and development of anticancer liposome formulations.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +44 2083319805. Tel: +44 2083318440. E-mail: d. [email protected]. Notes

The authors declare no competing financial interest. 4292



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