Cisplatin Encapsulation Generates Morphologically Different

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Cisplatin Encapsulation Generates Morphologically Different Multi-Compartments in the Internal Nanostructures of Non-Lamellar Liquid Crystalline Self-Assemblies Intan Diana Mat Azmi, Jesper Ostergaard, Stefan Sturup, Bente Gammelgaard, Arto Urtti, Seyed Moein Moghimi, and Anan Yaghmur Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01149 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Cisplatin Encapsulation Generates Morphologically Different MultiCompartments in the Internal Nanostructures of Non-Lamellar Liquid Crystalline Self-Assemblies

Intan Diana Mat Azmi1,2,3, Jesper Østergaard3, Stefan Stürup3, Bente Gammelgaard3, Arto Urtti4,5, Seyed Moein Moghimi6,7, Anan Yaghmur3,*

1

Centre of Foundation Studies for Agricultural Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

2

Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

3

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

4

Centre for Drug Research, University of Helsinki, Helsinki, Finland

5

School of Pharmacy, University of Eastern Finland, Kuopio, Finland

6

School of Pharmacy, The Faculty of Medical Sciences, King George VI Building, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 7

Division of Stratified Medicine, Biomarkers & Therapeutics, Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK

*

Corresponding author: E-mail address: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Cisplatin (cis-diamminedichloroplatinum (II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in non-lamellar liquid crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E) and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt% phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared with DSPG-containing counterparts

(25-40%)

under similar experimental conditions.

Through

structural and

morphological characterizations involving synchrotron small angle X-ray scattering and cryogenic transmission electron microscopy we further show that varying the phospholipid content of cisplatin-free nanodispersions, triggers an internal phase transition from neat hexagonal (H2) phase to a biphasic phase (internal H2 phase coexisting with lamellar (Lα) phase). However, cisplatin encapsulation in both DPPS- and DSPG-containing nanodispersions generates coexistence of morphologically different multi-compartments in the internal nanostructures comprising vesicles as a core, enveloped by an inverted-type surface bicontinuous cubic Im3m (primitive, QIIP) phase or H2 phase. We discuss the biophysical basis of these drug-induced morphological alterations and provide insights toward the potential development of inverted-type LC nanodispersions for cisplatin delivery. Keywords: Cisplatin, Cryogenic transmission electron microscopy; Cubosomes; Hexosomes; Inverse hexagonal liquid crystalline phase; Inverse bicontinuous cubic phase; Lamellar and nonlamellar liquid crystalline nanodispersions; Synchrotron small angle X-ray scattering


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INTRODUCTION Aqueous nanodispersions of well-ordered internal inverse lyotropic non-lamellar liquid crystalline (LC) mesophases, namely cubosomes (aqueous nanodispersions consisting of nanoparticles with an internal bicontinuous cubic (Q2) phase) and hexosomes (aqueous nanodispersions consisting of nanoparticles with an internal hexagonal (H2) phase) are gaining popularity as carriers of drugs and diagnostic agents.1-11 This is mainly due to the versatility of their internal nanostructures with large interfacial areas (~200 m2/g) capable of accommodating amphiphilic, hydrophobic and hydrophilic molecules.1-9 Despite the versatility and potential pharmaceutical applications of cubosomes and hexosomes1-7, encapsulation of guest molecules in these carriers could potentially induce ultrastructural and morphological alterations1-3,10-12, and these possibilities have not received much attention. Thus, depending on the drug choice and concentration, potentially induced ultrastructural, morphological, and size alterations may dramatically affect the drug release processes and hence the biological performance of cubosomes and hexosomes. Here, we studied the effect of cisplatin [Pt(NH3)2Cl2] encapsulation on the structural and morphological features of a set of inverse LC nanodispersions composed of phytantriol, vitamin E, and a negatively charged phospholipid by synchrotron small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryo-transmission electron microscopy (Cryo-TEM). Figure 1 presents the chemical structures of cisplatin, the copolymer stabilizer Pluronic F127, phytantriol, vitamin E, and the used negatively charged phospholipids. We have chosen cisplatin as a candidate drug, since it is used in nearly 50 % of all cancerous treatments,13-15 but its administration in free form is associated with adverse side effects, including nephrotoxicity and ototoxicity.15-17 Although, cisplatin encapsulation in micelles or liposomal formulations can modulate the drug pharmacokinetics and improve its safety profile18-30, these carriers still show low solubilization capacity for cisplatin in relation to therapeutic studies.26-28


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Here, we not only show a significant increase in cisplatin encapsulation efficiency in inverse LC nanodispersions containing 1.5-4 wt % anionic phospholipid, but further demonstrate that cisplatin, in a concentration-dependent manner, dramatically affects the internal structure and morphological features of the nanoparticles. We elaborate on the biophysical basis of these transformations in relation to the development of pharmaceutically viable formulations.

MATERIALS AND METHODS Materials. Cisplatin (cis-diamminedichloroplatinum (II), CDDP) with 99.99% purity was purchased from Thermo Fisher Scientific Inc. (New Jersey, USA). Phytantriol (3,7,11,15tetramethylhexadecane-1,2,3-triol) with a nominal purity of >96.4% (product specifications by gas chromatography) was a gift from DSM Nutritional Products Ltd. (Basel, Switzerland). αTocopherol (vitamin E) with purity of >96% was purchased from Sigma-Aldrich (St. Louis, USA). Pluronic F127 was a gift from BASF SE (Ludwigshafen, Germany). D-alpha-tocopheryl poly (ethylene glycol) 2000 succinate (TPGS-PEG 2000) with purity of >79% were purchased from Isochem

S.A.S

(Vert-Le-Petit,

France).

Phosphatidylglycerol

(1,2-distearoyl-sn-glycero-3-

phosphoglycerol sodium salt; DSPG) with purity of > 99% and phosphatidylserine (1,2dipalmitoyl-sn-glycero-3-phosphoserine sodium salt; DPPS) with purity of > 97% were purchased from Corden LLC Pharma (Liestal, Switzerland). Endotoxin-free phosphate-buffered saline (PBS) 150 mM was purchased from Sigma-Aldrich (Poole, U.K.), where the pH was adjusted either to 7.4 or to 5.6 with 1 M NaOH or HCl, respectively. PBS at lower chloride concentration (50 mM) at pH 7.4 was prepared in the laboratory. For size exclusion chromatography (SEC), the eluent was prepared based on 20 mM Tris-HCl containing 5% (v/v) of methanol, where Tris (hydroxymethyl) aminomethan (purity of >99.8%) and anhydrous methanol (purity of > 99.8%) were purchased from


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Sigma-Aldrich (Poole, U.K), and hydrochloric acid was purchased from Merck (Darmstadt, Germany). All chemicals were of analytical grade and were used as received.

Sample Preparation Preparation of cisplatin-free reversed LC nanodispersions. Aqueous nanodispersions based on ternary lipid mixture of phytantriol (PHYT)/phospholipid (DSPG or DPPS)/vitamin E (Vit E) were prepared at different weight ratios. In all dispersions, the total lipid concentration was kept constant at 10 wt% (Table 1). PHYT was first melted at ~ 57 °C and weighed into a glass vial followed by the addition of the negatively charged phospholipid (DSPG or DPPS) and Vit E. The copolymer stabilizer Pluronic F127 (1 wt%) or TPGS-PEG 2000 (2 wt%) and PBS buffer (pH 7.4 or 5.6) were then added to the ternary lipid mixture to give 100% of total dispersion weight. The samples were then subjected to the emulsification process via ultrasonication (Qsonica MIS 4417 and Qsonica MIS 4659 4-tip horn) (Qsonica LLC., CT, USA) for 10 min in pulse mode (5 s pulses interrupted by 2 s breaks) at 30% of its maximum power until well-dispersed and stable milky solutions were obtained.

Preparation

of

cisplatin-loaded

reversed

LC

nanodispersions.

Cisplatin-loaded

PHYT/phospholipid (DSPG or DPPS)/Vit E formulations were prepared via passive loading in a two-step procedure. The first step required dissolving cisplatin at a concentration of 3 mg/mL in PBS buffer at either pH 7.4 or 5.6 for 6 h at 65 °C in a dark vial to ensure optimal hydrolysis of cisplatin (formation of mono- and dihydrated cisplatin complexes in aqueous medium).31,32 Cisplatin solution was first mixed with a weighed amount of the negatively charged phospholipid (DSPG or DPPS as presented in Table 1) at 65 °C via vigorous vortexing until a homogeneous


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solution was obtained. In the second step, this mixture was subsequently added to already prepared F127-stabilized PHYT/Vit E nanodispersions (prepared as described in section 2.2.1) at 65 °C until achieving well-homogenized milky formulations. It should be noted that the two-step preparation procedure was selected in the present study to avoid insignificant loading that could occur due to a fast release of cisplatin from the nanoparticles to the surrounding aqueous medium when applying ultrasonication33 (a single-step preparation method). After following the two-step procedure, the prepared cisplatin-loaded nanodispersions were cooled down to room temperature to allow sedimentation of undissolved cisplatin. Free cisplatin was then removed by centrifugation through Amicon® Ultra centrifugal filter (Merck Millipore Ltd., Darmstadt, Germany) at 4000 g using a swinging-bucket rotor for 30 min at 25 °C. The procedure during the preparation procedure was in accordance to the safety guidelines from safety data sheet from Sigma-Aldrich together with the general guidelines applied for cytotoxic drugs.

Encapsulation Efficiency. Free and encapsulated cisplatin were measured using size exclusion chromatography (SEC) coupled on-line to UV (SEC-UV). A Shodex Asahipak GF-310 HQ column (Denko America Inc., New York) was applied and the detection was performed at λ = 222 nm using a flow rate of 0.3 mL/min. The mobile phase consisted of 20 mM Tris-HCl containing 5% (v/v) of methanol at pH 7.4. A calibration curve of cisplatin concentrations in the range of 33–664 ng/mL was developed before analyzing each sample. The nanodispersions were diluted 10 times prior to the analysis. Further, 20 µL of the diluted samples were injected in triplicate to the SEC system. The successful amount of cisplatin loaded into the nanoparticles is called the encapsulation efficiency (EE), which can be calculated as34:


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EE = (

− ) × 100

(1)

where, the concentration of free cisplatin can be determined from the standard curve based on the peak area of an unencapsulated cisplatin in the sample (eluted at ~ 43 min of retention time); whereas the total cisplatin concentration is the initial amount added to the formulation.

Characterizations of Cisplatin-Free and Cisplatin-Loaded Reversed LC Nanodispersions.

Particle size measurements. Particle size analysis was done on representative samples of drug-free and drug-loaded inverted-type LC nanoformulations, which have been diluted 100 times with PBS buffer. The mean size of the particles was determined by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, U.K) at room temperature. This instrument was equipped with a 633 nm laser and 173° detection optics. Data acquisition was performed using Malvern DTS v. 6.34 software (Malvern instruments, Worcestershire, U.K). The value of pure water was used for viscosity and refractive index.

Synchrotron Small-angle X-ray Scattering (SAXS). Structural characterization of drug-free and drug-loaded inverted-type LC nanoformulations was performed at the beamline I911-SAXS (Maxlab synchrotron facility, Lund University, Lund, Sweden) at operating electron energy of 1.5 GeV and a wavelength of 0.91 Å. A two-dimensional (2D) image plate detector (Pilatus 1M, Dectris Ltd, Baden, Switzerland) was implemented by an exposure time of 240 s. The camera was kept under vacuum during data collection in order to minimize the background scattering. All experiments were done at 37 °C (± 0.1 °C) in custom-made glass capillaries with the aid of a Peltier element. Some of the SAXS experiments, including sample measurements at varying temperatures, were


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done at the high flux Austrian SAXS beamline (Elettra-Synchrotron facility, Trieste, Italy) operating at 8 keV. One-dimensional (1D) position sensitive detector (Gabriel type) was applied under exposure time of 20 s. For both beamlines, the q range (q = 4π sinθ/λ, where λ is the wavelength and 2θ is the scattering angle) covered a range of 0.1–5.0 nm-1. Silver behenate [CH3(CH2)20-COOAg) with a d spacing value of 58.4 Å] was used as a standard to calibrate the angular scale of the measured intensity. After subtracting the background scattering, all Bragg peaks were fitted by Lorentzian distributions. The lattice parameter (ɑ) of the internal liquid crystalline nanostructures was gleaned from SAXS reflections by calculating the characteristic distance (d = 2π/q) for every reflection and applying a standard procedure for calculating the lattice parameters of the lamellar liquid crystalline phase, and the inverse bicontinuous cubic (V2) and discontinuous hexagonal (H2) phases.35

Cryo-Transmission Electron Microscopy (Cryo-TEM). The morphological characterization of selected nanodispersions was done in a frozen-hydrated state. The nanodispersions were diluted to a final concentration of 2.5 wt%. An aliquot of 3-4 µL of the samples was withdrawn and applied on the hydrophilized lacey carbon 300 mesh copper grid (Ted Pella Inc., California, USA). The hydrophilization process was performed by glow-discharging the grid (Leica Inc. EM ACE 200, Germany) to obtain better and uniform sample spreading. The excess sample on the grid was then blotted with filter paper using a blotting time 5 s and blotting force 0 at a temperature of 25 °C and 100 % humidity (FEI Vitrobot IV, Holland) and was subsequently plunged into liquid-nitrogen cooled ethane (-180°C), allowing the sample to vitrify. Immediately, the sample was transferred to liquid nitrogen (-165°C) for a short time in order to minimize formation of ice crystals and sample perturbation. A Gatan 626 cryo-holder (Gatan, UK) was used to place the grid containing sample and the observations were done with Tecnai G2 20 transmission electron microscope (FEI, Holland) at a voltage of 200 kV under low-dose rate (~5 e/Å2s). The images were then recorded using a FEI


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Eagle camera 4x4 k at a nominal magnification of 69,000× resulting in a final image sampling of 0.22 nm/pixel.

RESULTS AND DISCUSSION

Encapsulation Efficiency. The data in Table 1 shows the encapsulation efficiency (EE) of cisplatin loaded into reversed LC nanodispersions as determined by Eq. 1. In this study, different formulations based on the ternary mixture of PHYT, Vit E and negatively charged phospholipid (DSPG or DPPS) were prepared at varying lipid compositions and pH values using two different stabilizers (Pluronic F127 or TPGS-PEG 2000). Earlier, it was reported that different EE could be achieved depending on the loading method and the lipid composition of the formulations.36,37 Therefore, it was our primary aim to investigate the optimal experimental conditions for maximum loading of cisplatin into the reversed LC nanodispersions and study the impact of drug solubilization on internal nanostructures. Accordingly, we first compared the EE of DSPGcontaining formulations at different PHYT/DSPG/Vit E ratios prepared with PBS containing 150 mM NaCl (pH 5.6). Increasing the content of DSPG from 1.5 to 3 wt % induced a slight increase in EE from 19 to 28%. Nevertheless, at comparable lipid weight ratios and experimental conditions, the EE of DPPS-containing formulations was greater than that of DSPG-containing nanodispersions, resulting in an increase from 20 to 53% of EE with increasing DPPS concentration from 1.5 to 4 wt%. On the other hand, neutral hexosomes comprising of a binary mixture of PHYT/Vit E showed a negligible cisplatin loading (data not shown). This finding suggests preferable electrostatic interactions among the embedded anionic DPPS molecules at the lipid-water interface with two hydrated cisplatin products (mono- ([PtCl(H2O)NH3)2]+) and divalent cationic ([Pt(H2O)NH3)2]2+) cisplatin molecules). This is presumably occurring as a result of nucleophilic


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displacement of chloride ions by water molecules when cisplatin is dissolved in the aqueous media in nanodispersions.38 Figure 2 presents representative SEC-UV chromatograms of free- and encapsulatedcisplatin samples. The detected hydrolysis products of cisplatin at a concentration of 3 mg L-1 cisplatin in buffer are shown in Figure 2d. These hydrated cisplatin products have been shown to interact with negatively charged phospholipids, but preferentially with phosphatidic acid and phosphatidylserine (PS).39,40 In this context, effective interactions among hydrated forms of cisplatin and DPPS molecules in these nanodispersions may increase cisplatin loading. We also investigated the use of d-α-tocopheryl polyethylene glycol (PEG) 2000 succinate (TPGS-PEG 2000) as a stabilizer for DPPS-containing nanodispersions (lipid mixture of PHYT/DPPS/Vit E at weight ratio of 3:4:3) instead of the Pluronic F127 stabilizer and tested its influence on the solubilization of cisplatin. It should be noted that a higher concentration of TPGSPEG2000 was used than F127 to promote stabilization of the cisplatin-free and loaded nanodispersions. The notion of loading cisplatin in LC nanoparticles stabilized by an amphiphilic lipopolymer such as TPGS-PEG2000 is based on the premise that PEGylation may lead to the development of long-circulating LC nanoparticles.41,42 However, we observed a significant decrease in EE of cisplatin from 53 to 25%, which may be attributed to a shielding effect by surface projected PEG chains of TPGS-PEG2000. Moderate binding of the two hydrated cisplatin products to negatively charged phospholipids was reported to take place at pH 6 and, which can be enhanced significantly at pH 7.4.39 Consequently, we increased pH of the PBS buffer (150 mM NaCl) from 5.6 to 7.4 and tested the influence of such pH change on the EE of TPGS-PEG2000-stabilized non-lamellar LC nanodispersions containing DPPS. At PHYT/DPPS/Vit E weight ratio of 3:4:3, EE was found to drastically increase from 25 to 81% with increasing pH from 5.6 to 7.4 (Table 1). However, EE was


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ascended to a maximum value of 98% (Figure 2b) in the nanodispersion on decreasing the concentration of chloride ions (buffer with 50 mM NaCl) at pH 7.4. These results are consistent with previous studies on liposomal formulations where both low chloride concentration and high pH were found to improve the hydrolysis kinetic of cisplatin38,43. The replacement of DPPS by DSPG at same lipid weight ratio and pH 7.4 induced a lower EE of 40%. As compared to DSPG counterparts, the higher EE in nanodispersions containing DPPS may be attributed to specific DPPS-cisplatin interactions leading to the formation of coordination complexes among the amine and the carboxyl groups of the serine moiety of DPPS with platinum, which is reported not to occur with DSPG39,44.


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Table 1. The lipid composition, encapsulation efficiency (EE), and structural characteristics of cisplatin-loaded nanodispersions prepared at either pH 7.4 or 5.6 using the polymeric stabilizer F127 or TPGS-PEG 2000. All SAXS experiments were performed at 37 °C. Nanodispersion compositions (wt %)

EE (%)

Before loading

After loading LP**, a Space (nm) group

Space group

LP**, a (nm)

20 26 42 46

H2 H2 H2 H2/Lα

4.63 4.91 5.20 5.39/-

89

53

H2

6.14

H2/Lα H2/Lα H2/Lα H2/Im3m H2/trace Im3m

2 2 2

88 88a 88b

25 81 98

H2/Lα H2/Lα H2/Lα

5.79/-

H2/trace Im3m Lα Lα

2 2

F127 1 1

89 89

15 19

H2 H2c

4.59 4.88

2

2

1

89

21

H2c

5.26

2.5 3

2 2

PHYT

DPPS

VIT E

stabilizer

PBS

6.5 6 5.5 5

1.5 2 2.5 3

2 2 2 2

F127 1 1 1 1

89 89 89 89

3

4

3

1 PEGT*

3 3 3

4 4 4

3 3 3

7.2 6.5

DSPG 0.8 1.5

6 5.5 5

H2/Im3m H2/Im3m H2/trace Im3m H2/trace Im3m NM

4.95 5.19 5.25 5.46/17.58 5.63/5.64/-

5.10/19.19 5.27/19.82 5.46/-

1 89 25 H2/Lα 6.54/4.83 5.63/1 89 28 NM PEGT* 3 4 3 2 88a 40 NM NM Most of the lipid nanodispersions were prepared using PBS with 150 mM NaCl and pH 5.6. There are three samples prepared at pH 7.4: two nanodispersions prepared using PBS containing 150 mM NaCla, and a nanodispersion prepared using PBS containing 50 mM NaClb; cThe identified inverse hexagonal (H2) phase is coexisted with an advent of ‘shoulder’ peak, indicating the occurrence of traces of newly formed but not identified phase; NM = not measured; LP = lattice parameter; MLV; multi-lamellar vesicles; PEGT is the stabilizer TPGS-PEG2000.

Size and Structural Characterizations of the Drug-free and Drug-loaded Inverted-type LC Nanodispersions.

Effect of cisplatin loading on the particles size. The results in Table 2 summarizes the hydrodynamic diameter of representative drug-free and drug-loaded LC nanodispersions prepared in PBS (150 mM NaCl) at pH 5.6. In the case of drug-free samples, the DPPS-containing nanodispersion displayed a smaller mean particle size (138.2 ± 2.6 nm) compared with


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nanodispersion containing DSPG (160.2 ± 3.7 nm) at comparable 6:2:2 (PHYT/phospholipid/Vit E) lipid weight ratio. Replacing F127 with TPGS-PEG2000 increased the mean particle size of the drug-free DPPS-containing nanodispersion prepared at PHYT/DPPS/Vit E weight ratio of 3:4:3 despite using higher concentration (2 wt %) of the latter stabilizer. These results indicate that F127 is a more efficient stabilizer than TPGS-PEG2000 and therefore at lower concentrations can sterically stabilize nanodispersions through both enthalpic and entropic contributions. This is consistent with previous studies showing that F127 is one of the most efficient stabilizers for cubosomes, hexosomes, and related nanodispersions.1,9,45-47 Drug loading did not exert any dramatic changes in the mean nanoparticle sizes.

Table 2. Representative particle size analysis on drug-free and drug-loaded nanodispersions prepared in PBS (150 mM NaCl, pH 5.6) Nanodispersions PHYT:DPPS:Vit E 6:2:2a 3:4:3a 3:4:3b PHYT:DSPG:Vit E 6:2:2a

Before loading Mean size (nm) PDI

After loading EE (%) Mean size (nm) PDI

138.2 ± 2.6 155.3 ± 3.5 197.7 ± 7.9

0.134 0.101 0.323

142.7 ± 1.9 159.9 ± 1.2 206.7 ± 4.3

0.10 0.12 0.26

26 53 25

160.2 ± 3.7

0.141

166.7 ± 5.1

0.21

21

a

Aqueous nanodispersions stabilized using 1 wt% F127 b Aqueous nanodispersions stabilized using 2 wt% TPGS-PEG2000

Synchrotron small-angle X-ray scattering (SAXS) measurements on drug-free nanodispersions. Figure 3A,B shows SAXS patterns for DPPS-containing nanodispersions stabilized by either Pluronic F127 or TPGS-PEG 2000. At the lowest DPPS concentration of 1.5 wt%, a SAXS pattern with Bragg peak spacing ratio of 1:√3:√4 was detected, representing the first three characteristic reflections for an inverted-type hexagonal (H2) phase with a lattice parameter, a, of 4.63 nm (Figure 3A). Increasing DPPS content from 1.5 to 2.5 wt% shifted the scattering peaks to lower q


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values. This indicates an increase in the lattice parameter of the internal H2 phase from 4.91 to 5.20 nm. At higher DPPS content (3 wt%), a transition from hexosomes enveloping a neat H2 phase to nanoparticles with internal biphasic nanostructures consisting of an internal H2 phase with slightly larger lattice parameter (5.39 nm) in coexistence with multi-lamellar vesicles (MLVs) was perceived. The two Bragg peaks indicating the formation of MLVs are marked with stars in Figure 3A. Next, the structural characteristics of drug-free nanodispersions based on the ternary PHYT/DSPG/Vit E lipid mixture were investigated. At low DPPG concentration (PHYT/DSPG/Vit E weight ratio of 7.2:0.8:2), an internal neat H2 phase with lattice parameter of about 4.59 nm was detected (Figure 4). The SAXS patterns at higher DSPG content (PHYT/DSPG/Vit E weight ratios of 6.5:1.5:2 and 6:2:2) showed an evident structural effect similar to that of DPPS. Thus, increasing PHYT/DSPG/Vit E weight ratios from 7.2:0.8:2 to 6.5:1.5:2 and 6:2:2 was associated with an enlargement of the hydrophilic nanochannels of the internal H2 phase as reflected with an increase in the lattice parameter to 4.88 and 5.26 nm, respectively. A shoulder peak indicating the occurrence of newly formed coexisting Lα phase (formation of MLVs) was also detected around q of 1.4 and 1.5 nm-1 in the SAXS patterns of the latter two samples (Figure 4). The identification of this coexisting phase was based on the detection of its second characteristic reflection at approximately 2.6 nm-1 on a slight increase in DSPG content (at PHYT/DSPG/Vit E weight ratio of 5.5:2.5:2). These significant structural alterations as illustrated in Figure 5A on the inclusion of negatively charged DPPS or DPPG molecules at the electrically neutral lipid (PHYT)-water interface in the presence of Vit E can be predicted by using the critical packing parameter, CPP, known also the wedge shape factor48:

(2)

CPP = vs / a0 l


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where vs is the hydrophobic chain volume, a0 is the effective headgroup area, and l is the hydrophobic chain length. The electrostatic repulsion forces among DPPS or DSPG molecules embedded at the interfacial film lead to a rise in CPP due a monotonous increase of a0 as discussed in previous studies on “functionalization” of non-lamellar liquid crystalline phases, which is achieved by enlarging the hydrophilic domains of these self assemblies on loading amphiphilic compounds to neutral membranes based on monoglycerides or phytantriol.49-55 This is associated with an increase in the degree of hydration (swelling) of the hydrophilic headgroups of the embedded lipids, resulting as a consequence in a significant enlargement of the hydrophilic nanochannels of the H2 phase of binary PHYT/Vit E mixture in excess water, and leading eventually to a decrease in the negative interfacial curvature with augmenting DPPS (or DSPG) concentration. Thus, a substantial amount of any of these two anionic phospholipids incorporated into the interface exerts its destabilizing effects on the H2 phase, resulting in a structural transition to planar bilayers (the Lα phase). The obtained experimental findings are in good agreement with previous reports on the effect of inclusion the anionic phospholipid DOPG on monoolein (MO), where a structural phase transition in the following order was reported with increasing DOPG concentration: V2 (inverse cubic Pn3m phase) → H2 phase → L3 phase → Lα phase.53,54 It was also suggested using Raman scattering spectroscopy that the increase in a0 on introducing DSPG to MObased cubosomes, which leads to a structural transition from an internal inverse bicontinuous cubic Pn3m to an inverse cubic phase of the symmetry Im3m, is associated with a reduction in the mobility of acyl chains and a simultaneous increase in the number of hydrogen-bonded C=O groups of MO.56 The structural characteristics of lyotropic LC phases can be controlled by finding the right balance between the lipid headgroup repulsions and the lateral pressure in the hydrocarbon chain region.57 Therefore, in an effort to retain the interior H2 nanostructure in DPPS-containing hexosomes


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stabilized by F127 whilst improving the loading efficiency, the concentrations of Vit E and DPPS were subsequently increased to 3 and 4 wt %, respectively, resulting in a final PHYT/DPPS/Vit E weight ratio of 3:4:3 (Figure 3A). At this composition, the SAXS result indicated the formation of hexosomes enveloping an internal H2 phase with a lattice parameter of 6.14 nm. It was also possible to obtain hexosomes based on the ternary PHYT/DPPS/Vit E lipid system at same composition and under similar experimental conditions when utilizing TPGS-PEG 2000 as emulsifying agent in place of the Pluronic F127. However, lower intensity characteristic peaks for the internal H2 phase with smaller lattice parameter at about 5.79 nm were depicted on a top of diffuse scattering, indicating the coexistence of hexosomes with a higher fraction of vesicles and micelles (Figure 3B) as compared to Pluronic F127-stabilized hexosomes. The occurrence of coexisting vesicles and normal micelles is expected due to the tendency of TPGS-PEG2000 to form normal micelles and stabilize vesicles in excess water.58

Synchrotron

small-angle

X-ray

scattering

(SAXS)

measurements

on

drug-loaded

nanodispersions. Figures 3C and 3D show the SAXS patterns of cisplatin-loaded DPPS-containing nanodispersions stabilized by Pluronic F127 and TPGS-PEG 2000, respectively. In all lipid ratios, the characteristic scattering peaks of the internal H2 phase was inferior due to the presence of a broad peak, demonstrating a possible co-existence of hexosomes with vesicles (Figure 3C). As presented in Table 1, encapsulation of cisplatin in a concentration dependent manner has a significant influence on the internal non-lamellar LC phases of the investigated nanodispersions. For instance, the internal H2 phase of hexosomes was enlarged on solubilizing cisplatin (in comparison with the structural features of the control drug-free hexosomes [see Table 1]) in three PHYT/DPPS/Vit E nanodispersions prepared at the following lipid weight ratios: 6.5:1.5:2, 6:2:2 and 5.5:2.5:2. The lattice parameters of the internal H2 phase were increased to 4.95, 5.19 to 5.25


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nm, respectively. These results indicate that a considerable amount of cisplatin has been successfully localized in the internal H2 nanostructure leading to an enlargement of its hydrophilic nanochannels. At relatively higher concentrations of solubilized cisplatin, more complicated structural features including the appearance of newly formed coexisting phases were detected (Figure 3C). At 46 % EE, the SAXS pattern for the nanodispersion based on PHYT/DPPS/Vit E at a lipid weight ratio of 5:3:2 indicated the formation of non-lamellar LC nanoparticles enveloping internal H2 phase and bicontinuous cubic phase of the symmetry Im3m (primitive, QIIP) with lattice parameters of about 5.46 and 17.58 nm, respectively. Moreover, loading cisplatin was associated with the appearance of a more diffuse dominating SAXS scattering pattern indicating the presence of a higher fraction of vesicles. SAXS result however, was intricate at higher DPPS content (PHYT/DPPS/Vit E at a lipid weight ratio of 3:4:3) due to considerably higher EE (53 %). The effect of cisplatin loading on TPGS-PEG2000-stabilized formulation (Figure 3D) was also found to induce the formation of relatively large fraction of co-existing vesicles, thus making the SAXS analysis and identification of coexisting phases rather difficult. Presumably, these mesophases as illustrated in Figure 5B, are consisted of multi-phase nanostructures. These involve the formation of a high fraction of vesicles in coexistence with nanoparticles enveloping highly disorder H2 phase (identified form the q value approximately at 1.1 nm-1) and, highly disorder cubic Im3m phase (ambiguously detected at q value at 0.6 nm-1). The assignment of the possible formation of coexisting H2 and cubic Im3m phases was based on the SAXS data analysis of the aforementioned Pluronic F127-stabilized nanodispersions loaded with cisplatin and prepared at different lipid compositions (Figure 3C). Furthermore, a complete transition from non-lamellar LC phases to vesicles was detected by SAXS and cryo-TEM for DPPS-containing nanodispersions at the highest cisplatin loading (81 and 98 %) (Figure 6).


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Similar phase transition order was revealed for cisplatin-loaded DSPG-containing formulations (Figure 4). However, the internal coexisting cubic Im3m phase was clearly detected due to ineffective swelling of the hydrophilic nanochannels (lower capability of these formulations to encapsulate cisplatin (Table 1), particularly for drug-loaded nanodispersions at PHYT/DSPG/Vit E ratios of 7.8:0.2:2 and 6.5:1.5:2. At these compositions, the EE was only 15 and 19% and the calculated lattice parameters of the cubic Im3m phase were 19.19 and 19.82 nm, and 5.10 and 5.27 nm for the coexisting H2 phase, respectively. Accordingly, at slightly higher EE of 21 % (6:2:2) and 25 % (5.5:2.5:2), the cubic Im3m phase started to obscure due to the presence of a higher fraction of vesicles, while the H2 phase was shifted to lower q value indicating cisplatin-triggered enlargement of its hydrophilic nanochannels. Taken together, the influence of lipid composition and EE of cisplatin on the structural features of the investigated nanoparticles, the SAXS findings further support a structural nonlamellar-lamellar transition mechanism, in which the encapsulation of cisplatin and its strong interactions with the hydrophilic headgroups of the anionic phospholipid (DSPG or DPPS) induce significant enlargement of the internal H2 phase of hexosomes with a possible re-organization of the embedded lipid molecules at the water-lipid interfacial area. This induces the formation of domains rich with cisplatin-phospholipid complexes leading in a concentration dependent manner to destabilization of the internal swollen H2 phase and promotes transitions to nanoparticles enveloping self-assembled nanostructures with less spontaneous curvatures. This explains the occurrence of internal cubic Im3m and Lα phases that are built up from curved and flattened bilayers, respectively, with increasing the encapsulated drug concentration. In other words, the experimental findings indicate that encapsulation of cisplatin triggers in a concentration dependent manner a colloidal transformation in the following order: hexosomes → cubosomes coexisting with hexosomes → MLVs. However, the role of water and cisplatin diffusion to the surrounding continuous aqueous


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medium, and the influence of the stabilizer on modulating the structural features during the structural transition events remain unclear. In combination with the loaded cisplatin, a possible penetration of Pluronic F127 to the hydrophilic domains of the non-lamellar (H2 and cubic Im3m) and lamellar (Lα) phases during the detected phase transitions that involve significant topological changes could facilitate the observed nonlamellar-lamellar transitions. In this context, it was reported that the temperature-induced cubic Im3m-Lα phase transition in monoelaidin nanodispersion is associated with the accommodation of a considerable amount of Pluronic F127 in the nanoparticles’ self-assembled interiors.59 A similar concentration-dependent structural transition from non-lamellar liquid crystalline phases to Lα phase and normal micelles was recently detected on the solubilization of the poorly water soluble antimicrobial peptide LL-37 to cubosomes and oleic acid nanodispersions.60,61

Morphological characterization of the drug-free and drug-loaded nanostructural aqueous Nanodispersions.

Figures 7A and 7B show representative cryo-TEM images of F127-stabilized PHYT/DSPG/Vit E nanodispersion prepared at PHYT/DSPG/Vit E weight ratio of 6.5:1.5:2. Near spherical and faceted nanoparticles of hexagonal shape with an internal nanostructure and size in the range of 100-200 nm were detected. The coexistence of these nanoparticles with vesicles as shown in Figure 7B is consistent with previous reports on the typical detection of coexisting vesicles with cubosomes and hexosomes.1-3,62,63 We emphasize that the size characteristics of the observed nanoparticles is consistent with the mean nanodispersion size (diameter) as determined by DLS. The structural periodicity of an internal H2 phase (hexosomes) was further verified by Fast Fourier transforms (FFT) analysis (Figure 7C) and supported by SAXS (Figure 4) as discussed above. Hexosomes enveloping an internal H2 phase were also detected in the drug-free DPPS-containing


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nanodispersion that was stabilized by F127 and prepared at PHYT/DPPS/Vit E weight ratio of 5.5:2.5:2 (Figures 7E and 7F). For this sample, the internal H2 phase was also verified by FFT (Figure 7D) and was consistent with SAXS analysis (Figure 3A). A cryo-TEM image at a lower magnification (Figure 7F) further revealed the presence of coexisting vesicles with hexosomes in this nanodispersion. An interesting observation was the sensitivity of the morphological and structural features of the investigated nanodispersions (Figures 7 and 8) to cisplatin encapsulation. In good agreement with SAXS analysis (Figures 3A and 4), significant structural and morphological alterations including the occurrence of nanoparticles enveloping di- and multiphasic nanostructures coexisting with uniand oligo-lamellar vesicles were detected in representative cryo-TEM images presented in Figure 8A-D. For instance, the di-compartment nanoparticles (core-shell nanoparticles) presented in Figure 8D were apparently consisted of an internal vesicular structure (a vesicular core) with thick dense shell (surface phase) of non-lamellar liquid crystalline nanostructure. In line with SAXS experiments on the significant cisplatin-triggered structural alterations, the formation of these unique nanoparticles could be attributed to uneven distribution of the embedded lipids at the interfacial area as discussed above. Thus, DPPS (or DSPG) are most likely concentrated in the aqueous core of the vesicular structure due to their strong interactions with cisplatin; whereas PHYT is localized at the outer surface of nanoparticles to form such thick shell of surface phase. In contrast to previous reports on the formation of a thin “surface phase” of vesicular or sponge (L3 phase) on the outer surfaces of cubosomes, hexosomes and other related nanodispersions45,64-66, we observed unique colloidal entities with opposite organized compartments of vesicular core surrounded by a surface non-lamellar liquid crystalline phase. Similar nanoparticles enveloping three internal coexisting phases (lamellar and bicontinuous cubic Pn3m and Im3m phases) have also been observed in monoolein/eicosapentaenoic acid nanoparticles loaded with relatively high


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concentration of human recombinant brain-derived neurotrophic factor (BDNF).67 However, in contrast to the presently reported cisplatin-triggered nonlamellar-lamellar structural alterations, they reported that BDNF solubilization induces a transition in the opposite order: vesicles to three-phase liquid crystalline nanoparticles.

Conclusion We showed the potential of non-lamellar LC nanostructures to accommodate cisplatin in their wellorder interiors that were characterized using complementary techniques including SEC-UV, SAXS, DLS, and cryo-TEM. Depending on the type of the negatively charged phospholipid (DSPG or DPPS), EE varied, where the highest loading was observed for DPPS-based nanodispersions prepared at pH 7.4 and low chloride ion concentration. However, accommodation of cisplatin in the internal nanostructures of non-lamellar LC nanodispersions led to significant structural and morphological changes including the formation of unique di- and multi-compartment nanoparticles. These observations show the complexity surrounding drug encapsulation in non-lamellar LC nanodispersions, which in turn may affect the formulation performance in biological microenvironment. Earlier, we demonstrated the effect of human serum and plasma on modulating the internal nanostructure of various hexosome and cubosome preparations.45,63,64 Since, cisplatin encapsulation generates different multi-compartmental nanostructures, a further modulatory role of biological fluids (e.g., plasma, lymph, tumour interstitial fluid) on these nanostructures as well as cisplatin release profile warrants further investigation. Improved understating of multifaceted physicochemical and biological processes modulating the internal nanostructure and stability of non-lamellar LC nanodispersion could lead to development of improved clinical formulations with pharmaceutical attributes.


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ACKNOWLEDGMENTS Financial support by the Danish Council for Independent Research | Technology and Production Sciences, reference 1335-00150b (to AY and SMM) is gratefully acknowledged. AY further acknowledges financial support from the Danish Natural Sciences Research Council (DanScatt) for SAXS experiments. IDMA is a recipient of a PhD Scholarship Award from the Ministry of Higher Education of Malaysia (MOHE) and Universiti Putra Malaysia (UPM).


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Figure 1. Molecular structures of (a) cisplatin; (b) Pluronic F127; (c) phytantriol (PHYT); (d) alpha-tocopherol (vitamin E); (e) 1,2-Dipalmitoyl-rac-glycero-3-phospho-L-serine sodium salt (DPPS); and (f) 1,2-Distearoyl-sn-glycero-3-phosphoglycerol sodium salt (DSPG).


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Figure 2. Representative chromatograms of SEC-UV on separation of free- and encapsulated cisplatin measured at 50 min. (a) Baseline; injection of 20 µL PBS buffer of 150 mM NaCl (pH 7.4). The presence of negative peak (or “vacancy” peak) in A is presumably due to the difference in UV absorbance of solute of the injected sample (PBS) than that of mobile phase (Tris-HCl) or due to salting-out effect, which commonly observed with PBS at higher chloride concentration. Cisplatin-loaded PHYT/DPPS/Vit E at lipid weight ratio of 3:4:3 stabilized by (b) TPGS-PEG 2000 (using PBS buffer containing 50 mM NaCl at pH 7.4) and (c) Pluronic F127 (using PBS buffer containing 150 mM NaCl at pH 5.6) resulted in 98 and 53% EE, respectively. The observed peak at ~43 min represents the un-loaded drugs. (d) Cisplatin standard solution at a concentration of 3 mg L-1. The newly formed compounds (marked with asterisk) are consistent with the hydrolysis products of cisplatin.68


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Figure 3. SAXS patterns of drug free (A) and drug loaded (C) DPPS-based hexosomal nanodispersions stabilized with 1 wt% Pluronic F127 at different ratios of PHYT/DPPS/Vit E; where each ratio in panel A and B corresponds to each color of the scattering line (red: 6.5:1.5:2, green: 6:2:2, blue: 5.5:2.5:2, yellow: 5:3:2 and black: 3:4:3). In panel C, the Bragg reflections marked with star, asterisk, and arrow are given to lamellar, cubic Im3m, and hexagonal (H2) phases, respectively. SAXS patterns display drug-free (B) and drug-loaded (D) DPPS-based hexosomal nanodispersions at PHYT/DPPS/Vit E (3:4:3) stabilized with 2 wt% of TPGS-PEG2000. All SAXS experiments were performed at 37 °C.


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Figure 4. SAXS patterns of drug free and drug loaded DSPG-based hexosomal nanodispersions at different PHYT/DSPG/Vit E weight ratios. Reflections indicating the coexistence of cubosomes with internally cubic phase of the symmetry Im3m and hexosomes of an inverted-type hexagonal (H2) phase were marked with asterisks and arrows, respectively. All SAXS experiments were performed at 37 °C.


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Figure 5. Schematic illustrations for the structural effects of inclusion DPPS or DSPG on cisplatin-free nano-self-assemblies of the binary PHYT/Vit E mixture (A), and the solubilization of cisplatin on the nanoself-assemblies of the ternary PHYT/DPPS (or DSPG)/Vit E mixture (B). In absence of cisplatin (A), the inclusion of the negatively charged phospholipid (DPPS or DSPG) to PHYT/Vit E hexosomes is associated with a significant enlargement of the hydrophilic nanochannels of the internal H2 phase and enhances the formation of coexisting vesicles. Cisplatin loading to these DPPS- or DSPG-containing nano-self-assemblies leads to significant structural and morphological alterations including the formation of dicompartments that are most likely comprised from vesicular core rich with the anionic phospholipid (DSPG or DPPS) and encapsulated cisplatin and surface H2 phase rich with PHYT or binary PHYT /Vit E mixture (B).


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Figure 6. Representative (A) SAXS pattern and (B) cryo-TEM image prepared at 3:4:3 (PHYT/DPPS/Vit E) stabilized by TPGS-PEG 2000; where a transition to uni- and oligo-lamellar vesicles was evolved on higher cisplatin encapsulation efficiency (81 %). Red arrows show a presence of most likely small region of non-lamellar phases covering the vesicles. Yellow arrows demonstrate partially collapsed lipid bilayer resulting from high loading capacity of cisplatin into vesicles.


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Figure 7. Cryo-TEM images of drug-free nanodispersions; where A & B represent nanodispersion at PHYT/DSPG/Vit E ratio of 6.5:1.5:2, while E & F display nanodispersion at PHYT/DPPS/Vit E ratio of 5.5:2.5:2. Also shown in C & D are the identified H2 phase using Fast Fourier transform (FFT) analysis for DSPG- and DPPS-based nanodispersions, respectively.


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Figure 8. Cryo-TEM images of F127-stabilized drug-loaded nanodispersion (A & D) prepared at PHYT/DSPG/Vit E ratio of 6.5:1.5:2 and 19 % EE of cisplatin; where the images display surface non-lamellar liquid crystalline phases enveloping most likely a vesicular core. Representative cryo-TEM images taken from cisplatin-loaded nanodispersions prepared at PHYT/DPPS/Vit E weight ratio of 5.5:2.5:2 at 42 % EE of cisplatin are given in B & C and the formation of a thick surface non-lamellar liquid crystalline phase (see the marked green areas in the images). For instance, the image in D displays a surface H2 phase.


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REFERENCES (1) Azmi, I. D. M.; Moghimi, S. M.; Yaghmur, A. Cubosomes and Hexosomes as Versatile Platforms for Drug Delivery. Ther. Delivery 2015, 6, 1347-1364. (2) Fong, W. K.; Negrini, R.; Vallooran, J. J.; Mezzenga, R.; Boyd, B. J. Responsive Self-Assembled Nanostructured Lipid Systems for Drug Delivery and Diagnostics. J. Colloid Interface Sci. 2016, 484, 320-339. (3) Angelova, A.; Garamus, V. M.; Angelov, B.; Tian, Z.; Li, Y.; Zou, A. Advances in Structural Design of Lipid-Based Nanoparticle Carriers for Delivery of Macromolecular Drugs, Phytochemicals and AntiTumor Agents. Adv. Colloid Interface Sci. 2018, 249, 331-345. (4) Meli, V.; Caltagirone, C.; Falchi, A. M.; Hyde, S. T.; Lippolis, V.; Monduzzi, M.; Obiols-Rabasa, M.; Rosa, A.; Schmidt, J.; Talmon, Y.; Murgia, S. Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics. Langmuir 2015, 31, 9566-9575.

(5) Koynova, R.; Tenchov, B. Recent Patents on Nonlamellar Liquid Crystalline Lipid Phases in Drug Delivery. Recent Pat. Drug Delivery Formulation 2013, 7, 165-173. (6) Nilsson, C.; Barrios-Lopez, B.; Kallinen, A.; Laurinmaki, P.; Butcher, S. J.; Raki, M.; Weisell, J.; Bergstrom, K.; Larsen, S. W.; Ostergaard, J.; Larsen, C.; Urtti, A.; Airaksinen, A. J.; Yaghmur, A. SPECT/CT Imaging of Radiolabeled Cubosomes and Hexosomes for Potential Theranostic Applications. Biomaterials 2013, 34, 8491-8503. (7) Tran, N.; Bye, N.; Moffat, B. A.; Wright, D. K.; Cuddihy, A.; Hinton, T. M.; Hawley, A. M.; Reynolds,

N. P.; Waddington, L. J.; Mulet, X.; Turnley, A. M.; Morganti-Kossmann, M. C.; Muir, B. W. DualModality NIRF-MRI Cubosomes and Hexosomes: High Throughput Formulation and in Vivo Biodistribution. Mater. Sci. Eng. C. Mater. Biol. Appl. 2017, 71, 584-593.

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Abstract Graphic


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Cisplatin encapsulation in the internal nanostructures of non-lamellar liquid crystalline nanodispersions generates multi-compartment nanoparticles with unique structural features 65x47mm (150 x 150 DPI)


Cisplatin Encapsulation Generates Morphologically Different

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