Selective Sequence for the Peptide-Triggered Phase Transition of

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Selective sequence peptide-triggered phase transition of lyotropic liquid crystalline structures Qingtao Liu, Yao-Da Dong, and Ben J. Boyd Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00547 • Publication Date (Web): 05 May 2016 Downloaded from http://pubs.acs.org on May 8, 2016

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Selective sequence peptide-triggered phase transition of lyotropic liquid crystalline structures Qingtao Liu, †

†,‡

†

Yao-Da Dong and Ben J Boyd*,

†,‡

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash

University (Parkville Campus), 381 Royal Parade, Parkville, VIC, 3052, Australia ‡

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of

Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia

ABSTRACT: A novel concept of using mixed lipids to construct selective peptide sequence-sensing lyotropic liquid crystalline (LLC) dispersion systems was investigated. The LLC systems were constructed using a mixture of phytantriol, a lipid that forms lyotropic liquid crystalline phases, and a novel synthesized peptide-lipid (peplipid) for sensing a target peptide with RARAR sequence. The internal structure of the dispersed LLC particles converted from the lamellar structure (liposomes) to the inverse bicontinuous cubic phase (cubosomes) in the presence of the target peptide. Addition of common human proteins did not induce any structural change, indicating high selectivity of interaction with the target peptide. The concept has potential for the design of targeted controlled release drug delivery agents.

Keywords: peptide, lyotropic liquid crystalline, cubosomes, liposomes, drug release

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ACS Paragon Plus Environment

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INTRODUCTION Lipid-based lyotropic liquid crystalline systems (LLCs) are spontaneously formed when certain amphiphilic lipids are exposed to aqueous environments. LCCs have received much attention due to their potential to encapsulate and sustain the release of actives with different physiochemical properties, from conventional drug molecules to proteins.1, 2, 3 The LLCs of current research interest as potential drug delivery systems are the inverse hexagonal (H2), inverse bicontinuous cubic (V2), inverse discontinuous cubic (I2) and lamellar (Lα) phases. The structure formed depends on the critical packing parameter of the lipid composition, as well as water concentration and temperature.4 With the presence of steric stabilisers, such as Pluronic® F127, the bulk liquid crystalline mesophases can be dispersed to form submicron particles that retain the internal nanostructure of the parent bulk phase. The particles with V2 and H2 internal nanostructure, are termed cubosomes and hexosomes, respectively. Studies have shown that the different phases have very different drug release properties. For example, drugs encapsulated in the V2 phase are released more rapidly than from the H2 and reverse micellar (L2) structures.2 The LLCs can be constructed to be sensitive to external stimuli such as light and electric field, which induce phase transitions and consequently changes in drug release rate.5 Such stimuli-responsive LLCs provide the capability for non-invasive “on-demand” drug delivery applications.6, 7 LLCs can also be made to be sensitive towards chemical variations such as changes in pH,8 salt concentration9 and specific ion species.7, 10, 11, 12 These systems open up further potential for self-regulation and targeting based on the intrinsic physiological and pathological triggers within the body. For example, pathological conditions such as cancer or inflammation can induce changes in the body such as a decrease in pH or over expression of specific peptides or proteins.13, 14 LLCs that are sensitive to these pathological markers can potentially target and interact with the area of disease and the marker-induced 2


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structural change then allows for the effective delivery of the encapsulated therapeutic agents. However, the differences in pH or ion concentration between pathological state and normal physiological state are often narrow and non-specific. As such, LLCs as triggered drug delivery agents based on variations in pH15, 16 or ion concentration17 have limited potential due to lack of specificity and sensitivity. In comparison, the expression of biological markers including specific peptides,18 proteins,19,

20

enzymes21 and nucleic acids22 in pathological conditions often differs significantly from the normal physiological state. As such, LLCs that are constructed with specific bio-marker sensitivity may have greater potential as targeted stimuli-responsive drug delivery agents. In this study, the concept of peptide-sensitive LLCs as a potential drug delivery system is explored by constructing particles using mixtures of a liquid crystalline forming lipid, phytantriol, and a novel peplipid (DVDVDK(Myr)-Myr, in Figure 1).

Figure 1. Chemical structures of the novel peplipid, phytantriol and the target pentapeptide.

Phytantriol has been shown to form the inverse bicontinuous cubic phase with Pn3m spacegroup in excess water23,

24

and has been used as the main geometric

scaffold to ensure the formation of liquid crystalline structure.9 The novel peplipid consists of the peptide DVDVD headgroup as the recognition element, and two myristate aliphatic chains (Myrs) which allows the intercalation of the peplipids into the phytantriol-based bilayers within the LLCs. The DVDVD headgroup is known to 3


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interact with a target pentapeptide RARAR, and induces the formation of beta sheets based on cooperative interaction including electrostatic attraction and hydrophobic interactions.25, 26, 27 It was hypothesized that the geometric change due to peptide interaction with the peplipid would induce sufficient change in the curvature of the peplipid+phytantriol-based lipid-bilayers to enable a phase transition to occur that could be used to control drug release.

EXPERIMENTAL METHODS Materials Phytantriol was purchased from DSM Nutritional products (Grenzach-Wyhlen, Germany). The target pentapeptide RARAR and the peplipid DVDVDK(Myr)-Myr were purchased from GL Biochem Ltd (Shanghai, China). Pluronic® F127, sodium chloride, calcium chloride dihydrate, sodium hydroxide, dextran-FITC (Mw=4000 Da), albumin from human plasma, fibrinogen from human plasma, immunoglobulin G from human serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were all purchased from Sigma-Aldrich (St Louis, MO). Chloroform and DMF were purchased from Merck (Kilsyth, Australia). Milli-Q water (0.05 µS cm-1 at 25 °C) was purified through a Millipore system (Sydney, Australia).

Preparation of dispersed systems. Lipid mixtures were prepared firstly by weighing appropriate amounts of phytantriol and the peplipid, DVDVDK(Myr)-Myr to 5 mL Covaris® glass vials (Covaris Inc, Massachusetts, USA). An appropriate volume of DMF was used to dissolve the lipid mixtures under sonication. The DMF was then removed from the mixture using vacuum at 25 °C. Unless stated otherwise, HEPES buffer solution (40 mM, pH 7.4, 1.8 mL) containing 1.5% Pluronic® F127 (w/w) was added to the dried lipid mixture, and the lipid mixture was immediately dispersed by ultrasonication (Covaris S220X, Covaris Pty. Ltd. Woburn, MA) for 100 cycles (90 s for every cycle) at average power output 4


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of 41 mW at 18 °C. For peptide-triggered experiments, the liquid crystalline dispersions were mixed with HEPES buffers containing various concentrations of the target pentapeptide RARAR to induce phase transition. For the blank samples, the liquid crystalline dispersions were mixed with HEPES buffer at same volume ratio as for the peptide-triggered samples. Blank HEPES buffer or buffer containing various proteins from human plasma were used as negative controls and for testing the selectivity of the triggered LLC system. The final dispersions contained 5% lipid (w/w), 0.75% Pluronic® F127 (w/w) and various concentrations of the target pentapeptide RARAR or proteins from human plasma, in 20 mM HEPES buffer.

Particle size and zeta potential. Particle size and zeta potential were measured using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK) at 25 °C assuming a viscosity of pure water. The dispersion samples were diluted 20-fold (v/v) with Milli-Q water prior to particle size and zeta potential measurement for optimal measurement sensitivity. The particle size measurements were carried out using automated settings in low-volume cuvettes. The

zeta potential measurements were carried out using the Helmholtz–Smoluchowski mode and automated settings.

Small-angle X-ray scattering (SAXS). SAXS measurements were conducted at the SAXS/WAXS beamline of the Australian Synchrotron28 (Victoria, Australia) to define the internal particle structures at various mole ratios of peplipid in phytantriol and the pentapeptide RARAR. For the SAXS measurements, samples of the dispersions (250 µL) or gel-like bulk phases, were added to the wells of a clear 96-well plate (PerkinElmer), which was mounted vertically in the beam path.29 The 2D SAXS patterns were collected using a Pilatus 1 M (170 mm × 170 mm) detector which was located 650 mm from the sample position, with 1 s exposure and X-ray wavelength of 1.03 Å. 5


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Cryogenic transmission electron microscopy (cryo-TEM). Cryo-TEM images were conducted at CSIRO (Parkville, VIC, Australia).5 A drop of lipid dispersion was placed on a TEM grid blotted with filter paper, and the sample was vitrified by rapidly plunging the grid into liquid ethane. The samples were stored in liquid nitrogen at −170 °C and transferred to a TEM (FEI Tecnai 20) operating at 120 kV for imaging.

RESULTS AND DISCUSSION 1. Effect of peplipid on self-assembly of phytantriol in excess water Impact on particle surface charge - The presence of aspartic acid (D) residues in the peplipid dictates that at physiological pH, the peplipid will have an overall negative charge. Figure 2A shows the changes in zeta potential of phytantriol-based particles with increasing peplipid concentration. The inclusion of peplipid at 5% w/w in phytantriol significantly increased the zeta potential from ~ -5 mV to between -25 ~ -30 mV indicating that the peplipids were integrated into the particles and affected the apparent surface charge. However, increasing the peplipid concentration beyond 5% (w/w) did not lead to a further decrease in zeta potential.

Figure 2. Average zeta potential of the phytantriol-based particles in HEPES buffer (pH 7.4) with (A) increasing peplipid (% w/w) concentration in phytantriol, in the absence of the target RARAR pentapeptide. (B) phytantriol-based particles with fixed peplipid concentration (% w/w) and increasing concentration of RARAR pentapeptide expressed as the mole ratio of the pentapeptide to the peplipid. The gray dashed line 6


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represents zeta potential of particles formed by only phytantriol.

The presence of arginine groups (R) in the RARAR peptide impart an overall positive charge to the peptide at physiological pH. The electrostatic interaction between the overall negatively-charged DVDVD sequence in free form and the overall positively charged RARAR peptide is known to form the β-sheet configuration.25,

26, 27

Figure 2B summarizes the change in zeta potential of the

phytantriol-peplipid particles, with increasing concentration of the RARAR peptide added to the dispersions. Increasing the RARAR peptide concentration in solution induced a decrease in zeta potential towards neutral values. This indicates that the conjugation of lipid chains to the DVDVD sequence integration into the phytantriol-dominant particles did not significantly impact on the ability of the DVDVD sequence to interact with the RARAR peptide. The overall trend in zeta potential with increasing RARAR concentration was towards neutral charge for all particles. The particles with 7.5% and 15% peplipid showed fluctuation in the zeta potential values with increasing peptide concentration. It is speculated that for these particles, the change in peptide concentration also induced significant structural change which may also influence the surface charge of the particles. Impact on particle internal structure - The internal structure of the particles with increasing peplipid concentration in phytantriol and dispersed in HEPES buffer-only was investigated using small angle X-ray scattering (SAXS) and cryo-TEM. The X-ray scattering profiles are summarized in Figure 3A. The structures of the corresponding non-dispersed parent bulk phases were also investigated using crossed polarized light microscopy and presented in Figure 3B-G. At lower peplipid concentration (1.5 % w/w in lipid), the scattering profile in Fig 3A indicated that the particles retained the inverse cubic (V2) phase with Pn3m spacegroup formed by phytantriol in excess water. The non-birefringent appearance of the corresponding bulk phase in contact with excess water under the crossed polarized light (Fig 3B) also indicated the presence of the cubic phase (or at least an 7


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absence of anisotropic structure). Increasing peplipid concentration to 5% w/w induced the formation of the more swollen inverse cubic (V2) with Im3m spacegroup according to the SAXS profile, and at 7.5 – 10% w/w, the formation of the lamellar phase was indicated. The structural change was confirmed using cryo-TEM where the lipid dispersion containing 10% w/w peplipid to phytantriol showed particles with vesicular structures only (Figure 5A). Furthermore, the birefringent mosaic appearance of the corresponding bulk phase also indicated the presence of lamellar structures (Figure 3 D-G). The visual appearance of the dispersions of lipid mixtures also turned from opaque to transparent in appearance with increasing peplipid concentration consistent with a transition from cubosomes to liposomes (See Supporting Information Figure S5). The progressive transition from Pn3m cubic phase, to Im3m cubic phase and then lamellar phase with increasing peplipid content in the phytantriol-based matrix indicates that the presence of the peplipid induces a positive curvature effect on the lipid bilayers as expected upon addition of charged amphiphiles.9 At higher peplipid concentrations, the structures formed could not be determined using SAXS due to lack of sufficient Bragg peaks. However based on the birefringent appearance of the corresponding bulk phase and the size of the particles (300 – 600 nm), it is speculated that the structure formed are highly swollen multilamellar vesicular structures rather than micelles.

8


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Figure 3. The SAXS profiles of peplipid+phytantriol-based LLC dispersions (A) and polarized optical images of the LLCs bulk phases (B-G) with increasing peplipid concentration in the lipid mixtures, in excess HEPES buffer. The peplipid concentrations (w/w) in the lipid mixture used in the polarized optical images are 1.5% (B), 5% (C), 10% (D), 15% (E), 17.5% (F) and 22.5% (G), respectively.

2. Detection of the pentapeptide RARAR The ability of the peplipid-based LLC dispersions to sense the target pentapeptide RARAR in HEPES buffer was investigated using SAXS, cryo-TEM and zeta potential measurements. The SAXS profiles of phytantriol+peplipid-based dispersed particles in HEPES buffer with increasing concentration of the target RARAR pentapeptide are summarized in Figure 4. At 1.5% w/w peplipid to phytantriol concentration, with increasing RARAR concentration, the dispersed particle retained the V2(Pn3m) phase with minimal change to the lattice spacing (Figure 4A). When the peplipid content was increased to 5% w/w, increasing the RARAR concentration induced slight deswelling of the V2(Im3m) phase lattice and conversion to the V2(Pn3m) phase at high RARAR concentrations (Figure 4B). The specific interaction between the negatively charged peplipid 9


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intercalated in the cubic phase bilayer and the positively charged RARAR pentapeptide countered the effect of addition of the peplipid alone, inducing a more negative curvature effect on the internal structure and the consequent observed phase transition. At higher peplipid contents (10 - 22.5% w/w in lipid mixture), increasing the RARAR concentration induced a decrease in the lattice spacing of the lamellar structure and phase transition to the V2(Im3m) phase and then the V2(Pn3m) phase (Figure 4C – 4F). At the highest peplipid concentration (30% w/w to phytantriol) and high RARAR concentrations, the Bragg peaks representing V2(Im3m) and V2(Pn3m) phases were very weak, this suggests that the system may contain a significant proportion of mixed structures.

Figure 4. SAXS profiles of phytantriol+peplipid dispersions with increasing target pentapeptide RARAR concentrations expressed as mol ratio relative to peplipid present. Peplipid concentrations are 1.5 % (A), 5% (B), 10% (C), 15% (D), 22.5% (E) and 30% (F), respectively. The dependence of the lattice constants on the ratio of target pentapeptide to peplipid in lipid mixture, is inserted in the appropriate SAXS profiles. The molar ratios of target pentapeptide to peplipid in the lipid dispersions were increased from 0 up to 4 folds. 10


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The structures formed by peplipid+phytantriol in the presence of the target RARAR peptide were further confirmed using cryo-TEM (Figure 5). In the absence of the RARAR peptide, the dispersion containing 10% peplipid in phytantriol showed only vesicles (Figure 5A).30 At an RARAR to peplipid mole ratio of 1:1, square facetted cubosomes were clearly observed, coexisting some liposomes and fibrous structures (Figure 5B). At mole ratio 4:1, cubosomes with a less-square silhouette are observed (Figure 5C), possibly indicating the shift from cubosomes with V2(Im3m) spacegroup at 1:1 ratio (from the SAXS data in Figure 4C) to those with V2(Pn3m) spacegroup at 4:1 ratio. Co-existing vesicles with cubosomes were also observed in the dispersion system, however at high RARAR concentrations, the vesicular particles were less clearly defined, possibly indicating a state of transition into more cubic phase structures.

Figure 5. Cryo-TEM images of phytantriol dispersions containing 10% peplipid in lipid mixture in HEPES. (A) without target pentapeptide RARAR; (B) 1:1 molar ratio of target pentapeptide RARAR to peplipd in dispersion; (C) 4:1 molar ratio of target pentapeptide RARAR to peplipid in dispersion.

Based on the above results, the hypothesized model of interaction between the peplipid+phytantriol LLC system and the RARAR peptide, and the consequent structural changes observed are summarized schematically in Figure 6. In the absence of peptide, the peptide part (DVDVD) of the peplipid is collapsed into the headgroup region to allow the charged aspartic acids (D) to orient towards the aqueous phase, and the hydrophobic valines (V) to associate with hydrophobic regions of the 11


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phytantriol-based bilayer.31, 32, 33 The collapsed configuration of the peplipid decreases the critical packing parameter (CPP) of the peplipid, inducing an overall positive effect on the bilayer curvature, inducing the phase transition from the inverse cubic phase to the lamellar phase at higher peplipid to phytantriol molar ratios.34, 35, 36, 37 In the presence of the RARAR peptide, the DVDVD proportion of the peplipid interacts with the RARAR peptide, forming the β-sheet configuration. This is proposed to reduce the headgroup cross-sectional area, increasing the CPP of the peplipid, inducing a negative curvature effect on the lipid bilayer, and resulting in the transition back to the inverse cubic phase. An alternative possible mechanism of interaction for the RARAR induced phase transition was through selective electrostatic interaction between the positively charged RARAR and negatively charged DVDVD in peplipids. In this model, one RARAR peptide attracts two or more adjacent DVDVD sequences of the peplipids, to induce a negative effect on the bilayer curvature and consequent phase transition from lamellar phase to the inverse cubic phase.

Figure 6. Illustration of the hypothesized models of interaction between target pentapeptide RARAR with the phytantriol+peplipid-based bilayer of the LLC system, and the consequent triggered phase transition from lamellar to inverse cubic phase.

12


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A similar triggered phase transition via only electrostatic interactions was previously observed between LLC particles containing charged amphiphiles and increasing electrolyte or polyelectrolyte concentration.38 However, this is the first time that liquid crystalline phase transitions induced by selective peptide interactions has been observed. The coordinated hydrophobic and electrostatic interaction between the RARAR peptide and DVDVD of the peplipids imparts significantly greater responsiveness

to

the

LLC

system

compared

to

previously

reported

electrolyte-sensitive LLC systems. At higher RARAR concentrations, the potential formation of ordered β-sheet between RARAR and DVDVD sequence of the peplipid reduces the effective hydrophilic volume to trigger the phase transition due to alteration of the packing parameter. This concentration-dependent phase transition of the LC system was also observed in a previous study using novel specific cadmium-sensitive LLC systems.12 As mentioned above, the selectivity of this LLC system is based on the coordinated hydrophobic and electrostatic interaction between the RARAR peptide and DVDVD. Other short peptide sequences with positively charged and/or hydrophobic residues could also interact with the peplipid, but the strength of the interaction

is

anticipated

to

be

much

lower

compared

with

the

positive-hydrophobic-positive-hydrophobic-positive sequence intercting with the DVDVD sequence. Although selective for specific ions and not a specific peptide, selectivity of a peplipid to the target was previously shown using cadmium-sensitive LLC systems.12 Therefore, the recognition of the peplipid+phytantriol LLCs system to target pentapeptide RARAR is high selective. The short pentapeptide sequence, while enabling a ready demonstration of this concept was not amenable to use of a ‘scrambled’ peptide which might be typically employed as a negative control in studies with longer peptide sequences. Such a peptide would be expected to interact with the peplipid but as discussed is likely to have a reduced strength of interaction and therefore less likely to trigger the transition observed for this selective pair. Hence we chose whole common proteins as a pragmatic negative control, in order to provide a scenario reflective of what may happen in vivo, described further below. 13


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3. Selectivity of the RARAR-triggered phase transition The potential application of stimuli-responsive systems as drug delivery agents depends on their sensitivity and selectivity to the specific triggering factors. The human blood contains a large variety of components such as electrolytes, proteins, blood cells, etc. Figure 7 compares the X-ray scattering profiles of 15% peplipid in phytantriol system dispersed in HEPES buffer and in the presence of various proteins commonly present in human blood, and compared to in the presence of the targeting RARAR peptide. The concentrations of targeting RARAR peptides and proteins (4.24 mg/mL) were significantly higher than typically found in human blood. Except for RARAR, none of the human proteins induced any phase change. This indicates that the peptide-induced phase change was highly selective. The structure of the peplipid-phytantriol dispersion was also examined with proteins at lower and physiologically relevant concentrations. Similarly, no protein-induced phase change was detected. (See Supplementary Information)

Figure 7. SAXS profiles for 15% peplipid in phytantriol dispersions with addition of various proteins or the target pentapeptide RARAR. The concentrated target pentapeptide RARAR and proteins albumin (Human), fibrinogen (Human) and globulin (Human), all were fixed at 4.24 mg/mL. Lastly, the concept of triggered control over drug release by the target RARAR pentapeptide was demonstrated for a model drug FITC-dextran (Mw = 4000 Da) as 14


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demonstrated in the Supplementary Information. A clear change in release rate is evident with and without the peptide present. Further drug release studies with real drug in systems designed with biologically relevant peptide pairs are underway.

CONCLUSIONS A novel specific peptide sensing liquid crystalline system was investigated. The internal liquid crystalline structure of the novel system was sensitive to the target peptide sequence which induced structural transition from lamellar phase (vesicle in dispersion) to reverse cubic phase (cubosomes in dispersion). The peptide-induced phase change also altered the release rate of the encapsulated model drug. Furthermore, the triggered phase change is highly selective to the targeting peptide and resistant to potential interference from strong buffer salt and common proteins in human blood. Application of the concept to therapeutically relevant pairs of peptides is anticipated to provide analogous behavior, indicating great potential as targeted controlled drug delivery agents and in new diagnostic approaches.

ASSOCIATED CONTENT Supplementary information HPLC chromatograms and mass spectra of pep-lipid DVDVDK(Myr)-Myr and target pentapeptide RARAR. The optical images, particle size and polydispersity index of peplipid-based LC dispersions. SAXS profiles of bulk phase of peplipid-based LC bulk phases before and after trigger. SAXS profiles of peplipid-based LC dispersions with human blood proteins. The control and RARAR-triggered release profiles.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Phone: +61399039112. Fax: +61399039583.

ACKNOWLEDGEMENTS 15


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The authors acknowledge the Australian Research Council for funding ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. B.J.B. acknowledges the Australian Research Council for a Future Fellowship. Part of this work was conducted at the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia. Lynne Waddington (CSIRO) is thanked for cryoTEM imaging.

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Selective Sequence for the Peptide-Triggered Phase Transition of

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