Incorporation of an Endogenous Neuromodulatory Lipid

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Incorporation of an endogenous neuromodulatory lipid, oleoylethanolamide, into cubosomes: nanostructural characterization Mohammad Younus, Richard N Prentice, Andrew N Clarkson, Ben J. Boyd, and Shakila B Rizwan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02395 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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Incorporation of an endogenous neuromodulatory lipid, oleoylethanolamide, into phytantriol cubosome lipid bilayer 338x190mm (96 x 96 DPI)

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Incorporation of an endogenous neuromodulatory lipid, oleoylethanolamide, into cubosomes: nanostructural characterization Mohammad Younus1, Richard N Prentice1, Andrew N Clarkson2,

3, 4, 5

,

Ben J Boyd6 and

Shakila B Rizwan*1, 3

1

New Zealand’s National School of Pharmacy; 2Department of Anatomy, Otago School of

Medical Sciences, 3Brain Health Research Centre, and 4Brain Research New Zealand University of Otago, Dunedin, 9054, New Zealand, 5Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, 2006, Australia and 6Monash Institute of Pharmaceutical Sciences and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University (Parkville Campus), Parkville VIC 3052 Australia.

ABSTRACT Oleoylethanolamide (OEA) is an endogenous lipid with neuroprotective properties, and fortification of its concentration in the brain can be beneficial in the treatment of many neurodegenerative disorders. However, OEA is rapidly eliminated by hydrolysis in vivo, limiting its therapeutic potential. We hypothesise that packing OEA within a nanoparticulate system such as cubosomes, which can be targeted towards the blood-brain barrier (BBB) will protect it against hydrolysis and enable therapeutic concentrations to reach the brain. Cubosomes are lipid-based nanoparticles with a unique bicontinuous cubic phase internal structure. In the present study the incorporation and chemical stability of OEA in cubosomes was investigated. Cubosomes containing OEA had a mean particle size less than 200 nm with low polydispersity (polydispersity index 0.05). Physical stability of formulations stored at ambient temperature was investigated over a week. No significant changes in the size and PDI were observed with the exception of 50% w/w OEA formulations in which some accumulation of lipid at the water/air interface was evident a day after formulation. However, the loose aggregates could be redispersed by vortex mixing.

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Figure 3. Effect of varying the ratio of phytantriol to OEA (expressed as % w/w OEA relative to phytantriol) on mean particle size (Z-average, blue symbols) and polydispersity index (PDI, red symbols) of dispersions stabilised by Pluronic F127 (triangles) or Tween 80 (squares). Data presented are the mean ± SD of three independent experiments. 3.3. Influence of OEA on nanoparticle structure Cryo-TEM was used to analyse the structure of selected OEA-containing dispersions. The results are summarised in representative micrographs shown in Figure 4. Tween 80stabilised dispersions containing 10% w/w OEA (Panel A) show cubosomes with characteristic periodic internal structure and ‘vesicular coat’, without any independent vesicles, as has been recently reported for Tween 80-stabilised phytantriol cubosomes.22 This suggests that no significant morphological changes occurred with incorporation of OEA into the nanoparticles at 10% w/w concentration. However, with an increase in the concentration of OEA up to 50% w/w, vesicle formation (Figure 5B and C) and a vesicular coat on the cubosomes was more evident (Figure 5C, arrows). In contrast, Pluronic F127-stabilised dispersions containing 10% w/w OEA (Figure 5D), showed a small population of vesicles alongside cubosomes, which is in line with a previously published study on Pluronic F127stabilised phytantriol cubosomes.34 These vesicles became more frequent at 50% w/w OEA (Figure 5F), indicating that increasing OEA content in the formulation was resulting in the disruption of the internal structure. The structure of the vesicles were similar (arrow heads) to that of liposomes observed under TEM.35 Based on the observation of hundreds of micrographs, greater numbers of vesicles were evident in Pluronic F127-stabilised dispersions as compared to dispersions stabilised with Tween 80. Fast Fourier Transforms (FFTs) of selected nanoparticles (Figures 5a’-d’) show cubic symmetry.


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Figure 4. Cryo-TEM images of phytantriol dispersions stabilised with Tween 80 (panels A-C) and Pluronic F127 (panels D-F) containing 10% w/w (A, C), 30% w/w (B, D) and (C, F) 50% w/w OEA. Panel A shows the formation of cubosomes only, whereas co-existing vesicles (arrows), liposomes (arrow head) along with cubosomes are evident in panels B-F. Panels a–d show an enlarged view of a representative cubosome selected from the micrographs (marked with *), with their respective FFT showing cubic symmetry in panels a′–d′. Scale bar =

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The internal nanostructure of the dispersed particles was further interrogated using SAXS (Figure 5). Phytantriol cubosomes stabilised with Pluronic F127 showed peaks with relative positions at ratios √2: √3: √4: √6. This is in agreement with the Pn3m bicontinuous cubic structure indicating cubosomes with D-type cubic nanostructure. The lattice parameter of 71.5 ± 0.21 nm is also consistent with previously published work.26 In contrast, Tween 80 stabilised cubosomes showed three peaks with relative positions at ratios √2: √4: √6, which indicates nanoparticles with Im3m spacing. The lattice parameter of Tween 80-stabilised cubosomes was 123.15 ± 0.61 nm, which is in line with previous work.22 The SAXS profiles for nanoparticles prepared with 10% w/w and 30% w/w OEA stabilised with either Pluronic F127 or Tween 80 also indicated the presence of cubic phases albeit with an increase in the lattice parameter as the concentration of OEA was increased, indicating incorporation of OEA into the lipid structure. Nanoparticles containing 50% w/w OEA stabilised with either stabiliser showed peaks with relative positions at √2: √4: √6, indicating cubosomes with P-type nanostructure. In addition to these, sharp crystalline peaks (indicated by *) were observed in formulations containing the highest amount of OEA (50% w/w) and are most likely caused by precipitation of excess OEA.


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* A 4 6

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4 6

*

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50% 30%

4 6

10% Tween 80

2

4 6

Arbitrary intensity units

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0%

*

6

2

4

*

* 50%

2 3 4

6

2

30% 3 4

6

Pluronic 10% F127

2 3

4

6

0%

0.0

0.1

0.2

0.3 q(Å-1)

0.4

0.5

0.6

Figure 5. (A) SAXS profiles of phytantriol dispersions stabilised with Pluronic F127 (lower curves) or Tween 80 (upper curves), containing increasing amounts OEA (% w/w relative to phytantriol). Relative peak positions for each formulation is shown. Note that the scattering profiles are displaced on the vertical axis for clarity of presentation. Sharp crystalline peaks indicated by * were observed in formulations containing 50% w/w and are most likely caused by precipitation of excess OEA. (B) Change in lattice parameter as a function of the concentration of OEA for dispersions stabilised with Tween 80 (triangles) or Pluronic F127 (squares). 15 ACS Paragon Plus Environment

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3.4. Encapsulation and chemical stability of OEA in nanoparticles The chemical stability of OEA was investigated qualitatively by monitoring changes in amide group absorbance (~1640 cm-1) and the presence of the absorbance band of the carboxylic acid group (~1710 cm-1) of oleic acid (a hydrolytic product) using ATR-FTIR spectroscopy. The infrared spectra of the pure lipids (phytantriol, OEA and oleic acid) and formulations are presented in Figure 6. Regions where the amide and carboxylic acid bands show characteristic absorbance bands are highlighted in blue and are magnified on the right side of Figure 6. Standard OEA and standard oleic acid show absorbance bands at ~1640 cm-1 (characteristic of the amide bond) and ~1710 cm-1 (characteristic of the carboxylic acid bond). On the other hand phytantriol, which contains neither functional groups in its structure does not show any absorbance bands in this region. All formulations investigated, show only a single absorbance band at ~1640 cm-1 while standard OEA spiked with standard oleic acid (20% w/w of OEA) showed bands at ~1640 cm-1 and ~1710 cm-1. There was no observable difference between the spectra of samples analysed after seven days storage at ambient temperature (dashed lines).


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Figure 6. FTIR spectra of pure components; phytantriol, OEA, oleic acid (OA) and OEA + OA (lower curves). Spectra for formulations containing 30% w/w OEA stabilised with Tween 80 (a) or Pluronic F127 (b); 50% w/w OEA stabilised with Tween 80 (c) or Pluronic F127 (D) (upper curves). Samples analysed after one week of storage at ambient temperature are shown as dashed lines on the IR spectra. The IR region where the amide (1710 cm-1) and carboxylic acid (1640 cm-1) bands show characteristic absorption peaks has been highlighted (blue rectangle) and is magnified on the right side of the figure. In order to gain further insight into the stability of OEA after encapsulation into cubosomes, the content of OEA and its degradation by product oleic acid, in cubosomes was quantified using a validated HPLC method. OEA and oleic acid were eluted separately at 6.5 min and 10.1 min, respectively. The assay was validated for accuracy and precision of both OEA

and

oleic acid simultaneously (chromatograms,

Supplementary Figure

1).

Approximately 90% of the total OEA could be recovered from all formulations investigated


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as shown in Table 1. Furthermore, no oleic acid was detected in any of the formulations suggesting that OEA loaded into cubosomes remained chemically stable. The amount of OEA encapsulated in nanoparticles was determined using PD-10 columns filled with Sephadex G-25. Studies with free OEA showed that very little OEA was eluted in water eluents and approximately 85% OEA was eluted in the organic phase. Nanoparticles were mostly eluted in the first three water fractions. Studies with free OEA in presence of nanoparticles showed the separation of free OEA from nanoparticles (Supplementary Information Figures 2 and 3). Encapsulation efficiency was determined as the ratio between the amount of OEA recovered in the nanoparticle fraction and the initial amount of OEA in the original sample. Approximately 99% of OEA was found to be encapsulated in nanoparticles (Table 1). Table 1. The chemical stability of OEA and the percentage of OEA encapsulated in nanoparticles. The chemical stability of OEA is represented as the percentage of OEA found in freshly prepared dispersions (Day 1) and after at least a week of storage at room temperature. Percentage of OEA encapsulated in nanoparticles after passing the formulations through PD-10 columns (encapsulation efficiency). Data presented are the mean ± SD of three independent experiments. Formulation Stabiliser

Tween 80

F127

Entrapment

Retention

Chemical stability

(% OEA)

(% OEA)

(% OEA)

10

100 ± 0.0

99.8 ± 0.3

102.1 ± 7.6

30

99.2 ± 1.4

99.7 ± 0.1

92.6 ± 5.3

50

99.6 ± 0.1

99.7 ± 0.2

90.3 ± 3.2

10

99.6 ± 0.7

99.8 ± 0.2

100.3 ± 7.4

30

99.9 ± 0.1

99.7 ± 0.2

94.5 ± 6.9

50

99.5 ± 0.1

99.1 ± 0.3

97.5 ± 6.7

OEA (%w/w)


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4.

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DISCUSSION In this study the incorporation of the biologically active molecule, OEA, into

cubosomes was investigated. The key challenge of incorporating any therapeutic molecule into cubosome structures is the sensitivity of the self-assembling structure to the addition of other molecules, which may offset the curvature of the lipid bilayer required to form the cubic phase. The effects of amphiphilic molecules on the cubosome bilayer have been investigated previously, but the aims of these studies have been to model the effects of molecules with different chemical and structural properties on the stability of cubic phase36, 37, to increase cellular interaction in vitro38, to fluorescently label cubosomes39, or to determine how the introduction of a particular amphiphile may be used to fine tune the nanoparticles to undergo phase changes under simulated physiological conditions40, 41. In this study a novel approach was taken in that the amphiphilic lipid we set out to incorporate was an endogenous, bioactive ligand, meaning the successful incorporation of it into cubosomes would be a step towards a therapeutically useful delivery system. In addition to the therapeutic usefulness of OEA, it has also been reported to self-assemble into a bulk cubic phase in excess water above 40 ºC12, 13. This highlights the potential for OEA to become more than just a therapeutic molecule encapsulated into cubosomes, but to become an essential component of the nanoparticle structure itself. In this study, OEA–phytantriol dispersions were prepared in the presence of Pluronic F127 and Tween 80. Particle size analysis with DLS showed both the stabilisers have similar stabilising effect and homogenous distribution of nanoparticles (low PDIs). In contrast, cryoTEM revealed an increased number of vesicles as OEA content was increased. The increased appearance of vesicles can be attributed to destabilization of phytantriol cubic structure due to the replacement of phytantriol molecules with OEA in the lipid bilayer. The addition of amphiphilic lipids to liquid crystalline forming lipids has been previously reported to result in 19 ACS Paragon Plus Environment

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the formation of cubosomes along with vesicles, as observed upon addition of 1,2dipalmitoylphophatidylserine (DPPS) to phytantriol38, 42, and phosphatidyl choline, diglycerol monooleate43 or sodium cholate44 to GMO. The vesicles were more apparent with Pluronic F127 than Tween 80. While cryo–TEM showed heterogeneity in 30% w/w and 50% w/w formulations with cubosomes and vesicles of various sizes, with DLS it is not possible to distinguish between vesicles and cubosomes and it showed homogeneity with low PDIs. Existence of vesicles alongside cubosomes in Pluronic F127 stabilised cubosomes has been a long standing issue. The underlying mechanisms reported include production method, use of co-solvents, concentration of stabiliser, and the interaction of a stabiliser with a particular lipid29, 45-49. Due to the large variability in these factors in the literature where cryoTEM images are provided, the exact cause for vesicles in different formulations is debatable. Azhari et al. reported formation of phytantriol cubosomes without any vesicles, but it was not clear whether stabilization with Tween 80 or use of the propylene glycol co-solvent precursor method (as used in this paper) or both were responsible22. The formation of more vesicles with Pluronic F127 might be due to the differences in interaction between Pluronic F127 and cubosome forming lipids as compared with Tween 80. SAXS showed that the addition of OEA resulted in increased lattice parameter, indicating the enlargement (swelling) of the cubic phase structure. This swelling further increased as OEA concentration increased to 30% w/w. It is apparent that overall the OEA molecule favours the formation of lamellar structures and adding it to the phytantriol induces swelling but also increases the co-existence of vesicle structures, as indicated by cryo–TEM. While this occurred in formulations containing both stabilisers, the increase in lattice parameter was more predominant in Tween 80 cubosomes, suggesting the additional oleate group from Tween 80 was causing even greater swelling of the lipid bilayer. At 50% w/w OEA (or at some point before that), the maximum accommodation of OEA was reached,


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resulting in the transformation of the Pn3m cubic phase to an Im3m cubic phase with Pluronic F127, and Im3m to swollen Im3m with Tween 80, and co-existing solid crystalline phase was present with both of the stabilisers. Fraser et al.,40 reported that the addition of increasing amounts of the mono-unsaturated, negatively charged, C18 amphiphilic lipid oleic acid (the hydrolytic by-product of OEA) to phytantriol cubosomes stabilised with Pluronic F127 resulted in an increase in negative curvature, as shown by a 7.5 mol % concentration of oleic acid resulting in the conversion of the cubic phase to a hexagonal phase. In contrast, GMO, a neutral, mono-unsaturated, C18 amphiphilic lipid decreased the negative curvature, resulting in an increase in the lattice parameter up to the 7.5 mol % concentration measured. Likewise, OEA is a neutral, mono-unsaturated, C18 amphiphilic lipid, which decreased the negative curvature and increased the lattice parameter without resulting in any complete phase transitions, even at higher concentrations. OEA will most likely be vulnerable to enzymatic and chemical hydrolysis due to the presence of amide group, causing degradation into oleic acid and ethanolamine. Rizwan et al., have previously reported that cubosomes formed with only GMO transitioned from a cubic to hexagonal phase upon storage of the aqueous dispersion for two weeks and attributed this to the chemical hydrolysis of GMO to produce ionised oleic acid.29 Fraser et al., showed that addition of only 7.5 mol% oleic acid into phytantriol cubosomes induced a phase change to hexosomes.40 Du et al., later produced a full phase diagram for phytantriol and oleic acid systems at varying pH and showed that anything greater than 5% oleic acid content for pH 27 and >20% oleic acid content for pH 7-9, would result in significant structural deviation away from the bicontinuous cubic phase.41 The maintenance of the cubic phase even up to 50% w/w OEA as observed in this study, suggests that OEA was successfully protected from chemical hydrolysis to oleic acid within the aqueous dispersion. This observation was further supported by ATR-FTIR and HPLC analysis demonstrating that significant hydrolysis of


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OEA to oleic acid in the dispersion was not detectable even a week after formulation. The stability of OEA in cubosomes can be attributed to relatively low susceptibility of the amide group to hydrolysis in aqueous conditions compared to the ester group present in GMO. However, the susceptibility of the amide group of OEA to hydrolysis in the presence of amidases in plasma is yet to be explored and will be the subject of our future studies. The encapsulation efficiency studies showed approximately 99% incorporation of OEA in nanoparticles in all formulations, even a week after production. Neither the DLS, nor the encapsulation efficiency studies, showed the presence of aggregated crystals of OEA that were detected for 50% w/w formulations by SAXS. However, it should be noted that stored 50% w/w dispersions were typically vortexed for 5-10 min before analysis to return them to a free-flowing state (without any visible aggregates), and it is possible that these formulations may not have been fully redispersed prior to SAXS analysis. However, formulations with 10% w/w and 30% w/w OEA showed consistent results, demonstrating that up to 30% w/w OEA can be incorporated into cubosomes without significant disruption of the internal structure. The highly lipophilic nature of OEA means that a high encapsulation in the lipid environment afforded by the cubosomes was expected in any case. All biological effects of OEA reported after systemic administration appear to have required high doses, whereas it has been shown to stimulate the PPARα receptor at nanomolar concentrations.3 The high doses were likely necessary to overcome the effects of rapid hydrolytic degradation in vivo, which is a recognised issue with OEA administration, as evidenced by the investigation of synthetic hydrolysis-resistant OEA analogues11 and FAAH inhibitors50, 51 as potential solutions to overcome it. These strategies have their limitations, however, with synthetic hydrolysis-resistant analogues possibly having relatively less affinity for target receptors and FAAH inhibitors simultaneously raising the levels of other endogenous substrates (N-acylethanolamines) for the enzyme, which can produce different


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physiological effects to OEA alone. The possibility of encapsulating therapeutic levels of OEA, whilst maintaining the structural stability of nanoparticles and the ability of the nanoparticles to maintain OEA in its active form is sufficiently compelling for the planning of future intravenous disposition studies in an animal model.

5.

CONCLUSIONS In this paper, we have reported the co-formulation of OEA with phytantriol to form

cubosomes, which may potentially serve to protect OEA from rapid in vivo hydrolysis and aid therapeutic delivery. We demonstrated that a high proportion of OEA (up to 30% w/w relative to phytantriol) can be incorporated into phytantriol-based cubosomes without significant loss of the internal cubic structure and that the chemical stability of OEA is preserved in these dispersions. This was achieved with both Pluronic F127 and Tween 80stabilised cubosomes, demonstrating the potential to modify the stabiliser and possibly influence the in vivo distribution and targeting capacity of the cubosomes, which will be the subject of future studies.

6.

ACKNOWLEDGEMENTS We would like to acknowledge Mr. Richard Easingwood from the Otago Centre for

Electron Microscopy for help with acquisition and analysis of the electron microscopy data. This work was supported by funding from Lottery Health NZ. MY and RP are supported by a Doctoral Scholarship from the University of Otago. SAXS studies were conducted on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia. BB is the recipient of an ARC Future Fellowship.


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