Advanced drug delivery nanosystems for shikonin: A calorimetric and

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Advanced drug delivery nanosystems for shikonin: A calorimetric and EPR study Konstantinos Kontogiannopoulos, Athanasia Dasargyri, Maria Francesca Ottaviani, Michela Cangiotti, Dimitrios Fessas, Vassilios Peter Papageorgiou, and Andreana N. Assimopoulou Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00751 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Advanced drug delivery nanosystems for shikonin: A calorimetric and EPR study

Konstantinos N. Kontogiannopoulos1,, Athanasia Dasargyri1, Maria F. Ottaviani2, Michela Cangiotti2, Dimitrios Fessas3, Vassilios P. Papageorgiou1, Andreana N. Assimopoulou1,*

1

Organic Chemistry Laboratory, School of Chemical Engineering, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.

2

Department of Pure and Applied Sciences, Scientific Campus E. Mattei, University of Urbino, 61029 Urbino, Italy. 3

Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy.

Keywords: Alkannin, Shikonin, Quinones, Liposomes, Hyperbranched Polymers, Calorimetry, EPR, Drug Delivery Systems, micro-DSC.

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ABSTRACT

Drug delivery is considered a mature scientific and technological platform for producing innovative medicines with nanosystems composed of intelligent bio-materials that carry active pharmaceutical ingredients forming advanced drug delivery systems (aDDnSs). Shikonin and its enantiomer alkannin are natural products that have been extensively studied in vitro and in vivo for, among others, their antitumor activity and various efforts have been made to prepare shikonin-loaded drug delivery systems. This study is focused on chimeric aDDnSs and specifically on liposomal formulations combining three lipids (egg-phosphatidylcholine, EPC; dipalmitoyl phosphatidylcholine, DPPC; and distearoyl phosphatidylcholine, DSPC) and a hyperbranched polymer (PFH-64-OH). Furthermore, PEGylated liposomal formulations of all samples were also prepared. Calorimetric techniques and electron paramagnetic resonance (EPR) were used to explore and evaluate the interactions and stability of the liposomal formulations, showing that the presence of hyperbranched polymers promote the overall stability of the chimeric aDDnSs based on the drug release profile enhancement. Furthermore, results showed that polyethylene glycol (PEG) enhances drug stabilization inside the liposomes, forming a stable and promising carrier for shikonin with improved characteristics.

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INTRODUCTION Liposomes are considered one of the most extensively investigated drug delivery system (DDS), exhibiting high therapeutic efficiency, no immunogenicity and the capability to minimize toxic side effects due to targeted delivery. Unfortunately, conventional liposomes (without PEG) still present some disadvantages. The main one is the rapid recognition and uptake by reticuloendothelial system, reducing their plasma half-life, as well as degradation by lysosomal enzymes, after their endocytosis. These facts reduce the amount of the drug received by the cell and alter the desired pharmacokinetic profile. In order to overcome these problems, PEGylated or Stealth® liposomes have been developed, as well as more sophisticated liposomal formulations, composing of two different biomaterials (liposomes and polymers/dendrimers). These formulations have been categorized as advanced Drug Delivery nano Systems (aDDnSs).1-5 A recently introduced type of dendritic polymers, called hyperbranched polymers (HBPs), has attracted much attention as promising candidates for the development of novel DDS.6-9 In this concept, HBPs have been successfully utilized to form DDS for shikonin, either as complexes or as advanced liposomal formulations (aDDnSs) by our group.3, 9 Studying the interactions of various additives with model lipid bilayers4-5,

10-11

, it is quite

obvious that the incorporation of molecules like copolymers and dendrimers, into lipid bilayers could cause changes on their thermotropic behavior, as well as on their conformation and interacting properties. Several techniques have been used to study the interactions among various bioactive compounds and model lipid bilayers. Among them, DSC is a valuable and sensitive tool, measuring thermal changes on lipid bilayers, for the exploration of the thermodynamic lipid phase transition.12-14 Changes on thermotropic properties of lipid bilayers, Page 3 of 43


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such as transition enthalpy (∆Η); transition temperature (Τm); half-height width (∆T1/2) and cooperativity index etc., have been used for designing liposomal formulations and for evaluating the percentage of bioactive compounds eventually incorporated into them.14-16 Electron paramagnetic resonance (EPR) technique has proven very useful for the characterization of dendrimer/liposome or vesicle complexes, providing information about their structure and the types and strengths of interactions in these systems.17-19 Furthermore, it allows differentiating the internal/external interacting sites of the dendrimers and their flexibility in respect to their availability for ion complexation and trapping.20-21 Alkannin and shikonin (A/S; Figures 1a and 1b) are optical antipodes of plant origin, biosynthesized in the roots of at least 150 species belonging mainly to the Boraginaceae plant family. Intense investigations over the past 40 years have shown a wide spectrum of biological activity of these compounds, such as strong wound healing, antimicrobial, antioxidant, antiinflammatory, tissue regenerative, and most prominently antitumor ones.22-24 Extensive scientific studies (in vitro, in vivo and clinical trials), conducted in the past few years (more than 140), have established the effectiveness of A/S on several tumors and approximated the mechanism(s) of their antitumor action.25-26 Recently, shikonin has been proposed as a novel dietary agent with great potential as a breast-cancer preventative.27-29 Our group has successfully developed and characterized liposomal DDS of A/S in terms of physicochemical characteristics, pharmacokinetics and stability.3,

30-32

However, mechanisms

and interactions behind these systems are not completely clarified yet. In the present study, interactions between shikonin, HBPs and various phospholipids were assessed, as well as it was defined how these interactions affect the incorporation of the drug and the stability of the system, aiming to exploit and improve the design and development of shikonin-loaded aDDnSs. Page 4 of 43


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For this purpose, various drug delivery formulations with and without shikonin were prepared (e.g. conventional and PEGylated liposomes, HBPs complexes and chimeric advanced Drug Delivery nano Systems (chi-aDDnSs)) and characterized by two different techniques: EPR and micro-DSC. This is the first report on the application of electron paramagnetic resonance (EPR) together with calorimetric techniques for the exploitation and evaluation of shikonin-loaded liposomal formulation’s stability. More specifically, the goals of this study were: (i) to characterize shikonin-loaded liposomes (conventional and PEGylated) and to assess the interactions of shikonin with various phospholipids,

such

as

egg

phosphatidylcholine

(EPC

-

Figure

1c),

dipalmitoyl

phosphatidylcholine (DPPC – Figure 1d) and distearoyl phosphatidylcholine (DSPC - Figure 1e); (ii) to characterize shikonin-HBPs complexes and assess shikonin–HBPs interactions; and finally, (iii) to characterize both conventional and PEGylated shikonin–aDDnSs (formed with liposomes and HBPs), to evaluate the interactions established between shikonin, HBPs and phospholipids at different lipid compositions, and to define how these interactions affect the incorporation of the drug and the stability of all final formulations.

EXPERIMENTAL SECTION Materials Shikonin (S) purchased from Ikeda Corporation (Tokyo, Japan) was further purified via silica gel column chromatography followed by recrystallization (n-hexane), according to Assimopoulou et al. 33 aiming to incorporate a purified drug.

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Dipalmitoyl phosphatidylcholine (DPPC; MW 734.04), egg phosphatidylcholine (EPC; MW 770.12)

and

Ν-(carbonyl-methoxypolyethyleneglycol

2000)-1,2

distearoyl-en-glycero-3-

phosphoethanolamine (DSPE-mPEG2000 – Figure 1f; MW 2806.0) were purchased from Genzyme Pharmaceuticals (Cambridge, USA). Distearoyl phosphatidylcholine (DSPC; MW 790.15) was generously donated by Lipoid GmbH (Ludwigshafen, Germany). Hyperbranched polymer (PFH-64-OH– Figure 2; MW 7323.3) was obtained from Perstorp (Sweden). Cholesterol (Chol), phosphate buffer saline pH 7.4 (PBS) and all organic solvents (analytical grade) were purchased from Sigma–Aldrich (St. Louis, USA). Water used in all experiments was of HPLC grade.

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Figure 1. Chemical structures of (a) Alkannin; (b) Shikonin; (c) EPC; (d) DPPC; (e) DSPC; and (f) DSPE-mPEG2000. Page 7 of 43


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Figure 2. Chemical structure of the hyperbranched polymer PFH-64-OH.

Preparation of conventional and PEGylated liposomes Conventional (samples 1,3 and 5) and pegylated (samples 2, 4 and 6) liposomes (with and without shikonin) were prepared according to our previous study.31 Briefly, lipids, cholesterol and shikonin were dissolved in CHCl3/MeOH 2:1 (v/v). All samples were prepared using the same molar ratios, i.e. lipid/Chol (4.5:1), lipid/shikonin (30:1) and lipid/DPSE-mPEG2000 (13:1). Table 1 depicts the exact amount of the components in each formulation. . All formulations were prepared in a round–bottom flask in order to form a thin lipid film, after the removal of Page 8 of 43


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organic solvent in a rotary evaporator (EYELA N-N Series, Tokyo, Japan). Subsequently, the flask was left overnight under vacuum to remove any traces of organic solvent. The hydration of the lipid film was performed using 10 mL PBS (pH 7.4) for 1 h in a water bath, and the temperature was set above the main phase transition temperature (Tm) of each lipid (45oC for EPC lipids, 51oC for DPPC lipids and 67oC for DSPC lipids). The prepared multilamellar vesicles (MLVs) were vortexed at 1500 rpm using one IKA MS2 Minishaker (IKA Works, Inc, Wilmington, USA) for 10 min. Sonication was applied to obtain small unilamellar vesicles (SUVs) using a probe sonicator from Heat Systems–Ultrasonics Inc (W–375 Cell Disruptors). Sonication conditions were set to amplitude 0.7 pulser 50%, performing two 5 min periods interrupted by a 5 min resting period (in ice bath). In order to anneal any structural defects, the resultant vesicles were left for 30 min. Afterwards, all drug-loaded samples were passed through a size exclusion Sephadex G75 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden), in order to remove any nonencapsulated shikonin. For the preparation of conventional shikonin-loaded liposomes, the same procedure was followed, without the use of DPSE-mPEG2000 in the initial lipid mixture. Table 1 presents the composition of all formulations prepared.

Preparation of shikonin/HBP complexes Shikonin and HBP were diluted in methanol at a constant molar ratio (shikonin:HBP 0.1:1) in a round–bottom flask and the solution was stirred for 22 h. Methanol was evaporated to dryness at 35oC (under vacuum) in a rotary evaporator (Rotavapor R-114, Buchi, Switzerland) and the flask was left under vacuum overnight. The dry residue was hydrated with PBS (final HBP concentration 10 mg/mL) and left under stirring for 24 h at 37oC. Non-entrapped shikonin was Page 9 of 43


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removed through centrifugation (5000 rpm for 15 min, using a SORVAL T-880 centrifuge with fixed angle rotor).3

Preparation of shikonin-loaded chi-aDDnSs (conventional and PEGylated) Shikonin-loaded chi-aDDnSs (samples 8-12) were prepared according to Kontogiannopoulos et al.3 All samples were prepared using the same molar ratios, i.e. shikonin/HBP (0.1:1), lipid/Chol (4.5:1), and lipid/DPSE-mPEG2000 (13:1). Table 1 depicts the exact amount of the components in each formulation. In brief, shikonin and HBP (diluted in methanol) were mixed and stirred for 22 h. Subsequently, the chloroform solution of lipids and cholesterol was added and stirred for 1 h. The organic solvent was evaporated using rotary evaporator and thence the experimental procedure is the same as described in section “Preparation of conventional and PEGylated liposomes”, in order to obtain small unilamellar vesicles (SUVs). Three set of experiments were designed and conducted. The first set contains conventional and PEGylated liposomes using three different lipids, with and without shikonin: DSPC lipid (samples 1-2), EPC lipid (samples 3-4) and DPPC lipid (sample 5-6). The second set uses HBP complexes with (sample 7) and without shikonin (sample 7e). Finally, the third set concerns conventional and PEGylated aDDnSs prepared with three different lipids, with and without shikonin: DSPC lipid (samples 8 and 9, respectively), EPC lipid (samples 10 and 11, respectively), and DPPC lipid (sample 12).

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Table 1. Composition of all liposomal formulations prepared.

Chol

Shikonin

(mg)

DSPE-mPEG2000 HBP (mg) (mg)

(mg)

(mg)

1

DSPC (30)

-

-

3.25

0.365

1e

DSPC (30)

-

-

3.25

-

2

DSPC (30)

8.35

-

3.25

0.39

2e

DSPC (30)

8.35

-

3.25

-

3

EPC (30)

-

-

3.36

0.37

3e

EPC (30)

-

-

3.36

-

4

EPC (30)

8.6

-

3.36

0.4

4e

EPC (30)

8.6

-

3.36

-

5

DPPC (30)

-

-

3.51

0.39

5e

DPPC (30)

-

-

3.51

-

6

DPPC (30)

9

-

3.51

0.39

6e

DPPC (30)

9

-

3.51

-

7

-

-

20

-

0.08

7e

-

-

20

-

-

8

DSPC (62)

-

20

6.74

0.08

8e

DSPC (62)

-

20

6.74

-

9

DSPC (62)

17

20

6.74

0.08

Lipid Sample

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9e

DSPC (62)

17

20

6.74

-

10

EPC (60)

-

20

6.72

0.08

10e

EPC (60)

-

20

6.72

-

11

EPC (60)

17

20

6.72

0.08

11e

EPC (60)

17

20

6.72

-

12

DPPC (58)

-

20

6.79

0.05

12e

DPPC (58)

-

20

6.79

-

e: stands for formulations without drug (empty).

Light-scattering characterization of free and shikonin-loaded liposomes The mean hydrodynamic diameter and ζ-potential of all liposomal formulations were determined by light scattering immediately after their preparation. Measurements were performed using a Malvern ZetaSizer Nano ΖS (Instruments, Malvern, UK) at 25oC, after the appropriate dilution of the samples (50µL of sample, diluted 60-times in PBS, pH 7.4). All measurements were repeated three times.

EPR Analysis Insertion of the spin probe Different samples were studied by means of EPR, inserting the spin probe 5-doxylstearic acid (5-DSA) into the systems and then analyzing the spectral variations from one to another sample.

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We selected the 5-DSA probe because it already showed to insert well into the liposomes and then monitor their structural variations upon interactions with dendrimers in solution.17-18 Initially, the 5-DSA probe was dissolved in a stock solution (2.5×10-3M). An appropriate amount of the probe solution was placed in vials, followed by solvent evaporation before the addition of the prepared samples into the vials. All EPR studies were performed using a low concentration of 1×10-4 M probe in the examined systems. Previous studies indicated that this low concentration does not perturb the nanostructures. After addition of the sample solution to the vials containing 5-DSA, the vials were closed and left equilibrating for 24 h. The equilibration of the samples was controlled by checking the invariance of the EPR spectra for longer waiting times. The EPR spectra were therefore recorded 24 h after sample preparation.

EPR Instrumentation and method An EMX-Bruker spectrometer was utilized to record all EPR spectra, operating at X band (9.5 GHz), controlled by a PC with a software from Bruker to handle and record all spectra. Temperature control was attained through a Bruker ST3000 variable-temperature assembly cooled using liquid nitrogen. Each experiment was repeated three different times (at least) to ensure reproducibility. In order to compare all the samples under the same experimental conditions, all EPR spectra were performed at 298 K using the same experimental setup, as follows: modulation amplitude 0.1 mT; microwave frequency 9.87 GHz; center magnetic field 352 mT; sweep width 10 mT; resolution 1024; time constant 40.96 ms; and conversion time 81.92 ms. Furthermore, pyrex capillary tubes of the same inner diameter (~1 mm) were used as sample containers.

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Computation of the EPR spectra Useful information regarding micropolarity, microviscosity and the order of the lipid aggregates arise from the EPR study (by means of the 5-DSA spin probe). Due to its amphiphilic characteristic, the probe could localize between surfactant molecules reporting on the nearby environment of different structures. The EPR spectra of 5-DSA in solution are constituted by three hyperfine lines (due to the hyperfine coupling between the unpaired electron spin, S = 1/2, and the N14 nitrogen nuclear spin, I=1) at almost the same intensities. The insertion of the probe into a viscous environment results in a shift of the spectral line shape provoking the need for a computational procedure. This procedure is performed taking into account the relaxation process and the different interactions of the magnetic components. The sanctioned procedure of Budil et al. was used to compute spectra.34 Constant gii values (measuring the coupling between the electron spin and the magnetic field) were assumed in order to follow the behavior of the system (i.e. gii=2.009, 2.006, 2.003). The Aii values (measuring the coupling between the electron spin and the nitrogen nuclear spin) of 5-DSA into the phospholipid bilayers were assumed as Aii= 6 G, 6 G, 33 G ( = 1/3 (Axx + Ayy + Azz) = 15 G), which indicates a medium-low polarity environment of the probe. Of course these parameters will change if the spin probe is no more embedded into the phospholipid bilayer, but we assumed Axx=Ayy=6 G in all cases to limit the number of variables, and therefore only Azz was changed. When the spectrum is narrow, the accuracy in the Azz or parameter is high, being the error below ± 0.01 G. This error increases for the spectra constituted by broader lines, up to ± 0.1 G. During the fitting procedure, the following parameters were altered: (i) the correlation time for the diffusion rotational motion of the probe, τ, which measures the microviscosity and therefore corresponds to the interactions occurring Page 14 of 43


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between the nitroxide group and the environmental molecules; (ii) the order parameter, S, which reports on the order of the lipid layer where the probe is embedded. S may change from 0 (no order) to 1 (maximum order); (iii) the Heisenberg exchange frequency, Wex, which measures the high local concentration of probes colliding in a restricted space. The percentage of probes distributed in two or even more environments may be evaluated if the correspondent spectral contributions superimpose as resolved and recognizable components in the overall EPR spectrum. By adding the computed components at the proper relative percentages, the experimental signal was fitted. The reproducibility of the EPR spectra in the same experimental conditions also allowed us to evaluate, by double integration of the EPR spectra, the absolute intensity of the spectra which is considered significant only in a comparative way. A decrease of the absolute intensity provides a rough evaluation of strong spin-spin interactions due to radical self-aggregation or eventual phase separation: in both cases the radicals undergoing to such effects “disappear” from the EPR spectra and the intensity diminishes. Simulation was utilized to determine the accuracy of these parameters (2%). Parameters with variation larger than 2% resulted in a perceivable change of the simulated line shape, leading to a worse fitting between experimental and simulated spectra. Several computations were performed in order to ensure that the reported parameters are the most reliable for the physical meaning of the systems (although other sets parameter-sets could also provide equal or even better fittings).

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Thermal analysis measurements The thermodynamic stability of the prepared liposomal formulations was assessed using thermal analysis, with specific reference to lipid phase’s transitions. Micro-DSC was selected as the most suitable14 technique to study the thermotropic changes of the liposomes induced by the addition of HΒP and/or PEG and shikonin. All measurements were performed with a Setaram micro DSCIII using hermetically closed pans (1 mL) and a scanning rate of 0.5oC/min. The samples investigated were DPPC/Chol and DSPC/Chol liposomes; empty as well as in the presence of HΒP and/or PEG and shikonin, in PBS buffer, pH = 7.4, with a 3 mM concentration for the overall lipid content. A special dedicated software called "THESEUS" was employed to analyze raw data35. Lipid's excess specific heat C Pex (T ) / kJ K-1 mol-1 (compared to the low temperature lipid state) was obtained by finally scaling the apparent specific heat trace C Papp with respect to the base-line. As a result of this transformation, the relevant transitions enthalpy of the lipid phase is calculated by the area beneath the recorded peaks. Each sample was submitted into two heatingcooling cycles.. The observed parameters of the thermotropic transitions were evaluated using the second heating curves. Three replicates (at least) were performed to assess any errors.

Statistical analysis All statistical analyses were performed using SPSS version 22.0 (SPSS Inc., Chicago, Illinois, USA). One-way ANOVA and Student’s t-test were employed to perform multiple comparisons. P values of less than 0.05 were considered statistically significant. Results are shown as mean value ± standard deviation (S.D.) of three independent experiments.

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RESULTS AND DISCUSSION This study focuses on the assessment of the interactions between shikonin and various aDDnSs combining HBPs and different phospholipids, as long as on defining how these interactions affect the stability of shikonin-loaded drug delivery systems.

Characterization of the prepared formulations The prepared formulations were characterized for their particle size distribution, polydispersity index (PDI) as a particle size heterogeneity index and ζ-potential as a physical stability indicator. Polydispersity index expresses the uniformity of molecule or particle sizes in a mixture. Larger PDI values correspond to a wider size distribution in the particle sample. Results are shown in Table 2.

Particle size Lipid composition seems to influence the size of the prepared liposomes to some extent. This could be attributed to variations in lipid tail length, molecular shape and membrane fluidity.38 As shown in Table 2, the mean particle size of the conventional drug-loaded liposomal formulations (samples 1, 3 and 5) varied from 156.6 nm to 238.2 nm, whilst the size of the corresponding PEGylated liposomes (samples 2, 4 and 6) was found from 96.3 nm to 130.0 nm. Furthermore, Table 2 depicts that the use of different type of lipids produced formulations with considerable variations in their mean particle size. More precisely, drug-loaded formulations prepared with EPC had the smaller mean particle size and lower polydispersity index (concerning both conventional and PEGylated liposomes). Moreover, the use of DSPC and Page 17 of 43


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DPPC lipids formed liposomes with larger mean particle sized and slightly higher PDI. This observation is in accordance with our previous study.31 The same trend was observed for the respective empty liposomes (both conventional and PEGylated) regarding their mean particle size and polydispersity index. The further addition of the PEGylated lipid enhances the lateral repulsive properties of the surface of lipid bilayers by extensive hydration around the head group. In order to reduce the degree of repulsion, the liposome size decrease so as to increase the curvature of the grafting surface.39 In agreement with this fact, all PEGylated liposomes displayed lower particle size values compared to the corresponding conventional. Specifically, in case of DPPC formulations (samples 5 and 6), size decrease reached 53%. Additionally, the PDI of the stealth liposomes was considerably lower (10% - 60% on average) in comparison with conventional ones. Thus, this particle size reduction could be attributed to bilayer’s curvature in order to restrict the intensity of lateral repulsion, provoked by the addition of PEG in the lipid bilayer. An increase of the interlamellar repulsion could be attributed to the PEGylated lipid, causing a reduction in lamellarity, as confirmed by numerous studies with different bioactive molecules.40-42

ζ- Potential ζ-potential could be described as particle’s surface electrostatic charge, acting acts as a repulsive energy barrier regulating the stability of dispersion and resisting the aggregation of liposomes in buffer solution.43 For the shikonin-loaded PEGylated liposomes (samples 2, 4 and 6), their ζ-potential ranged from -18.58 mV to -21.87 mV (as shown in Table 2), whilst the values of the corresponding conventional sample (1, 3 and 5) varied from -7.84 mV to -15.87 mV. Page 18 of 43


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Liposomes charge (negative, positive or neutral) is affected by the type of lipid used during their

preparation.44

In

this

study,

formulations

were

prepared

using

different

phosphatidylcholines, which are neutral lipids; nevertheless, they possessed a slight negative charge. These findings are in accordance with the outcomes obtained by Garcia-Manyes & Sanz (2005), who stated that phosphatidylcholine bilayer may acquire negative ζ-potential values due to hydration layers shaped around the surface and to the orientation of lipid headgroups.45 As depicted in Table 2, drug-loaded liposomes formed with DSPC and EPC (sample 1 and 3) shown higher ζ-potential (-21.87 mV and -19.87 mV respectively) compared to the ones prepared with DPPC (sample 5; -18.58 mV). Liposomal formulations without drug, shown similar results. This observation is a clear indicator that the type of lipid has a significant effect on liposome’s ζ-potential. Furthermore, comparing PEGylated and conventional formulations, the former shown higher ζ-potential values in all cases, which could be interpreted as an indicator of higher stability for these samples, since ζ-potential increase the repulsive interactions, reducing the frequency of liposome agglomeration and precipitation.46 Regarding the prepared aDDnSs, all samples shown ζ-potential values slightly lower compared to the corresponding liposomes without HBP (Table 2), which is in accordance to a previous study where the addition of the same HBP caused a slight reduction of the ζ-potential values.8 Thus, it could be assumed that the addition of HBP has a negative effect on the ζpotential of the prepared liposomes. Finally, it should be noted that all drug-free liposomes presented lower ζ-potential values compared with the drug-loaded ones, which is in accordance with the results of our previous study.31 This observation could be ascribed to the fact that drug incorporation causes notable alterations in the liposome surface structure and the orientation of the phosphatidylcholine head group.47 Page 19 of 43


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Table 2. Physicochemical characteristics of the prepared liposomal formulations.

Sample

Mean Particle Polydispersity Size (nm) (PDI)

index

1

236.8±19.2

0.53

-8.01±0.87

1e

203.5±14.2

0.52

-4.11±0.54

2

130.0±18.4

0.19

-21.87±4.58

2e

124.3±6.7

0.29

-18.78±2.04

3

156.6±4.2

0.26

-15.87±3.21

3e

139.5±8.5

0.34

-9.58±2.57

4

96.3±3.8

0.24

-19.87±2.58

4e

95.1±6.7

0.31

-15.34±2.01

5

238.2±21.5

0.43

-7.84±3.58

5e

222.6±19.6

0.54

-4.01±1.02

6

110.8±9.4

0.25

-18.58±1.87

6e

100.2±7.2

0.21

-16.88±1.46

8

128.1±12.1

0.37

-12.37±1.96

8e

116.6±14.3

0.42

-10.52±2.41

9

148.3±21.3

0.19

-9.87±1.67

9e

137.8±20.8

0.35

-6.88±2.51

10

173.2±20.4

0.39

2.47±1.12

10e

165.9±17.2

0.49

0.85±1.45

11

252.9±18.3

0.35

1.12±2.63

11e

230.7±16.2

0.25

0.52±3.41

ζ-Potential (mV)

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12

204.5±16.2

0.28

-8.87±2.44

12e

197.1±17.8

0.33

-7.09±1.86

EPR analysis Figure 3 shows selected examples of experimental and computed EPR spectra for 5-DSA in PBS or in solutions with the various components at the defined molar ratios: (a) PBS or DSPC+Chol+S (DSPC/S=30; DSPC/Chol=4.5); (b) HBP and HBP+S (HBP/S=0.1); (c) DSPC+HBP; (d) DPPC+Chol (DPPC/Chol=4.5); and (e) PC+PEG+S+HBP (PC/PEG=13; S/HBP=0.2).

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Figure 3. Examples of experimental and computed EPR spectra for 5-DSA in PBS or in solutions with the various components at the defined molar ratios: (a) PBS or DSPC+Chol+S (DSPC/S=30; DSPC/Chol=4.5); (b) HBP and HBP+S (HBP/S=0.1); (c) DSPC+Chol+HBP; (d) DPPC+Chol (DPPC/Chol=4.5); and (e) PC+Chol+PEG+S+HBP (PC/PEG=13; S/HBP=0.2) Page 22 of 43


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The main parameters used for computing the spectra in Figure 3, namely, the relative percentages of the spectral components; the hyperfine coupling constant, , measuring the polarity; the correlation time for the rotational diffusional motion, τ, measuring the strength of interactions; the order parameter, S; and the Heisenberg exchange frequency, Wex, measuring the local concentration of probes, are shown in Table 3.

Table 3. Main parameters used for computation of the spectra in Figure 3: the relative percentages of the spectral components; the hyperfine coupling constant, , measuring the polarity; the correlation time for the rotational diffusional motion, τ, measuring the strength of interactions; the order parameter, S; and the Heisenberg exchange frequency, Wex, measuring the local concentration of probes. The molar ratios are reported in the experimental section. 5-DSA Sample

% components

/G τ /ns

S

Wex/x108 s-1

PBS

100

17

0.02

0

0

DSPC+Chol+S (Sample 1)

100

17

0.02

0

0

HBP (Sample 7e)

64

15.7

0.13

0

0

36

15.7

0.13

0

3.2

80

15.7

0.13

0

0

20

15.7

0.13

0

3.2

DSPC+Chol+HBP (Sample 8e)

100

15

15

0.7

0

DPPC+Chol (Sample 5e)

54

15

15

0.7

0

46

15.7

0.2

0

0

15

2.65

0.56

0

HBP+S (Sample 7)

PC+Chol+PEG+S+HBP (Sample 11) 100

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First, the spectrum in Figure 3a is the one for 5-DSA in PBS, but it is the same recorded for 5DSA in DSPC+Chol+S (DSPC/S=30; DSPC/Chol=4.5). Therefore, in the latter sample, the probe was almost completely extruded from the DSPC liposomes due to the presence of both Chol and shikonin (S). The analysis of the spectrum of 5-DSA in HBP solutions in the absence and presence of shikonin (Figure 3b) indicated that HBP generated a slower rotational mobility (higher τ) and a lower polarity (lower ) in the probe environment if compared to pure-PBS environment. But, the spectrum in HBP solution was constituted by two spectral components: a broad signal, indicated with an arrow in Figure 3b, superimposed to the narrow three-lines signal; this broad signal came from unsolved-self aggregated 5-DSA probes. We noted that the relative percentage of this broad signal decreases by adding shikonin to HBP. This means that shikonin improves the solubility of the spin probes into the PBS+HBP medium. The spectrum of 5-DSA + HBP embedded into the DSPC liposomes is completely different with respect to those described for Figures 3a and 3b, as shown in Figure 3c. Τhe microviscosity (τ) and the order (S) strongly increased when the spin probes inserted into the ordered phospholipid structure, while the polarity (measured by ) significantly decreased, as expected for a low-polar liposome-internal environment. This spectrum, characterized by high τ (slow mobility) and S (high order), is present as a component in the spectrum in Figure 3d for 5DSA in DPPC+Chol, but it superimposes to a "fast" component (low τ and S values). Indeed, this fast component arises from fast moving 5-DSA radicals, as those originating the spectra in Figures 3a and 3b. However, the computation parameters of the fast component in Figure 3d (Table 3) showed a decreased mobility (higher τ) and polarity (higher ) with respect to those measured for the spectrum in Figure 3a. Moreover, as obtained from calculation (Table 3), in this DPPC+Chol system, the spin probes distributed in two different environments: 54 % of Page 24 of 43


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probes were embedded into the well packed and ordered phospholipid bilayer, while 46 % of probes were in a rather fluid area at middle polarity, that is probably the liposome/PBS interface. Finally, the spin probes were all embedded into a PC liposome, showing the same polarity, but lower microviscosity and order with respect to DSPC and DPPC liposomes. This is revealed in Figure 3e for the system PC+Chol+PEG+S+HBP (PC/PEG=13; S/HBP=0.2), chosen as example. The parameters of computation are also shown in Table 3. Since significant variations of parameters occurred between the PC systems and DPPC or DSPC systems, the results obtained in these two cases are henceforth separately discussed. In each of these two cases, the main parameters of computation changed from one to another system (for instance, in the absence and presence of shikonin or PEG). Sometimes these variations were quite small, at the limit of the accuracy in the calculation, but the trends were well reproducible and trustable.

DSPC and DPPC liposomes Figure 4 shows in form of histograms the variations of (a) the absolute intensity of the slow component, (b) the order parameter of the slow component, (c) the relative percentage of the fast component, and (d) the polarity of the fast component, for the various samples based on DSPC and DPPC.

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Figure 4. Variations of (a) the absolute intensity of the slow component; (b) the order parameter of the slow component; (c) the relative percentage of fast component, and (d) the polarity of the fast component for the various samples based on DSPC and DPPC (Chol was also added in all samples; the molar ratios are reported in the experimental section).

The variation of the parameters showed that PEG decreased the packing of the phospholipids towards liposomes’ formation. The lower packing also allowed the probe head to approach and interact with the polar external surface of the liposomes.

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Similarly, the variation of the EPR parameter showed that shikonin partially de-structured the DSPC liposomes, while DPPC showed a better capability to incorporate the drug into the ordered bilayer structure. PEG helped to stabilize the drug inside the liposomes. The results were in line with the higher fluidity of the DPPC liposome with respect to the DSPC one. The higher fluidity promoted the internalization of the surfactant probes into the less ordered DPPC liposome. The more disordered DPPC structure favored the formation of microaggregates of 5-DSA inside the liposomes. In respect to HBP effect, the EPR results showed that HBP favored the formation of more ordered and packed DSPC liposomes, also favoring the insertion of the surfactant probes into the bilayer. Probably the polymer interacts with the surface of the packed bilayer.

PC liposomes In the PC case, the spectra were always constituted by one component, corresponding to 5DSA probes all embedded into the liposomes in slow motion and ordered conditions (100% slow component). The unsaturated chains in PC (C16 saturated+C18 unsaturated) rendered the bilayer more fluid (lower τ) and less ordered (lower S) with respect to DPPC (C16-C16 saturated) and DSPC (C18-C18 saturated). This also improved 5-DSA solubility and consequently enhanced the absolute intensity measured for 5-DSA in PC liposome with respect to DPPC and DSPC liposomes. Figure 5 depicts in form of histograms the variations of (a) the absolute intensity and (b) the order parameter, for the various samples based on PC.

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Figure 5. Variations of (a) the absolute intensity and (b) the order parameter, for the various samples based on PC (Chol is present in all samples; the molar ratios are reported in the experimental section).

The effects of PEG and drug addition were in large part similar to those found for DPPC and DSPC. However, the addition of PEG in the absence of the drug increased the order of PC liposomes, while the opposite was observed for DPPC and DSPC. This means that, in PC liposomes, PEG partially avoided the disordering effect of the unsaturated chain, probably because of PEG - PC binding. The radical probes indicated interactions between Shikonin and PC (similarly to DPPC) liposomes. However, the PC liposome structure was partially destabilized by the drug. Unlike DSPC, HBP decreased the order of PC liposomes. Probably the polymer is able to partially enter the PC liposome structure. The increase in order in the presence of both HBP and Shikonin indicates that the drug impedes the HBP penetration into the liposome structure.

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Thermal analysis (micro-DSC) In order to highlight the thermodynamic features i.e. the enthalpy and/or entropy contribution on the overall thermodynamic stability of liposomes added with the drug and/or HBPS, formulations with different lipids were investigated through micro-DSC.

Figure 6. Micro-DSC thermograms (second heating) of selected formulations (with and without drug).

Figure 6 reports the micro-DSC signal of “empty” liposomes (without drug) prepared with DPPC and DSPC lipids. These traces correspond to the well-known gel-liquid transition of lipid membranes i.e. the change from a gel to a liquid crystal state.

14

As a first comment, it can be

stated that the signals are very broad, shouldered and as depicted do not preceded by a pretransition. Contiguous observations were acquired form studies with SUVs and LUVs.14, 48-49 As Page 29 of 43


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presented in other studies, the cooperativity of the phase transition is different in case of MLBs (multi lamellar bilayers) than in liposomes50 and depends on the geometry of the membrane i.e. decreases as the vesicle curvature increases.48 This low cooperativity indicates a dispersion of lipid regions with varying stability and cooperativity parameters. The presence of cholesterol as well as the addition of shikonin and/or HBPs would further affect the aforementioned regions. In this case, despite this cooperativity differences, transition enthalpy (33±2, 45±2 kJ mol-1 for DPPC/Chol and DSPC/Chol respectively) and Tm were very close to those of MLBs (main transition enthalpy: 34±2, 46±2 kJ mol-1, Tm: 40.8±0.3, 54.0±0.3 °C for DPPC/Chol and DSPC/Chol respectively) indicating entropic driven processes.50 Both the transition enthalpy and Tm indicate that DSPC/Chol membranes are more stable compared to DPPC/Chol. The main effect of shikonin to the micro-DSC traces, in both types of liposomes (samples 1 and 5, Figure 6) was the clear appearance of a shoulder which suggests that the presence of shikonin enhanced the fluidity of the membrane. Furthermore, the presence of shikonin promoted phase separation i.e. the formation of domains within the lipidic envelope with distinct order parameters and stability. It should be noted, that the presence of a phase separation might result in an apparent decrease of the overall order (as overall order index is considered the half height width ∆T1/2 of the DSC curve; the smaller the width the higher the order and cooperativity). This suggests that, during the preparation procedure of the liposomes, a fraction of shikonin initial load might be partially misplaced in the lipid phase of the vesicle membrane. The overall transition enthalpies dο not seem to be affected by the drug presence. The above DSC observations were in line with the EPR data. Results from EPR analysis showed that the presence of the drug increased the fluidity and decreased the order of the membranes, promoting the insertion of the surfactant probes inside the bilayer in an Page 30 of 43


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inhomogeneous organization that is coherent with phase separation revealed by DSC. All these effects were more evident in DPPC liposomes compared to DSPC ones (see Figure 6), confirming that the intrinsic stability of DSPC bilayer prevented major variations. The high packing of DSPC bilayers impeded the EPR probe solubilization inside the lipid phase of the pure liposome promoted by the drug and HBP binding at the interphase. In case of aDDnSs (samples 8 and 12, Figure 6) the more stable phase seems to be the favourite. The overall transition enthalpies dο not seem to be affected, indicating again, entropic driven interactions. These observations are in line with previous studies,14 i.e. a phase separation within the lipid bilayer may be triggered by polymers with sufficient molecular size and branching degree, promoting in this way, the liposome-HBP-drug system toward a more stable thermodynamic state, as also suggested by the EPR data. In agreement with the DSC results, the EPR analysis indicated the existence of two different environments of the EPR probes, a more fluid one at the interface, and packed and ordered regions inside the DPPC liposomes. It should be noted here that, due to shikonin’s hydrophobicity, the drug does not exhibit such a strong effect on the overall stabilization of the membrane in the final system (aDDnSs formulations) in comparison with those observed in similar studies with hydrophilic drugs.50 Finally, the presence of PEG had rather peculiar and system/sample dependent effects, without a clear overall view. In case of liposomal formulations with DPPC, the presence of PEG did not compromise the general trend of thermodynamic stabilization. Furthermore, in case of DSPC formulations, the effect seems almost negligible and/or slightly negative, also coherently with the EPR data, which indicated a small perturbing effect of PEG on the packing of the liposomes.

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CONCLUSION Chimeric advanced Drug Delivery nano Systems

is a new trend for designing more

sophisticated liposomal formulations combining different biomaterials (liposomes and polymers/dendrimers) aiming to enhance the desired pharmacokinetic profile of the drug. The interaction and incorporation of these biomaterials, as well as of the drug into lipid bilayers could cause alterations on their thermotropic behavior and conformation altering the properties of the final formulation. In this study differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) were deployed in order to assess the interactions between shikonin, hyperbranched polymers (HBPs) and various phospholipids, as long as to define how these interactions affect the stability of various shikonin-loaded aDDnSs. The EPR results showed that PEG decreased the order of DPPC and DSPC liposomes due to interactions with the phospholipid heads, while, in PC liposomes, PEG partially avoided the disordering effect of the unsaturated chains. Shikonin partially de-structured the liposomes. However, a more fluid liposome structure showed a better capability to incorporate the drug into ordered bilayers. PEG helped to stabilize the drug inside the liposomes. HBP in the absence of the drug was able to partially enter the fluid PC liposome structure, but the drug avoids this effect. Conversely, a packed and ordered DSPC bilayer was formed due to HBP-liposome surface interactions. Concerning micro-DSC analysis, results from both transition enthalpy and Tm indicated that DSPC/Chol membranes were thermodynamically more stable compared to DPPC/Chol ones. Furthermore, the presence of shikonin during the preparation procedure promoted phase separation and a fraction of shikonin initial load, might be fully or partially misplaced in the Page 32 of 43


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lipid phase of the vesicle membrane. In case of aDDnSs, the more stable phase seems to be the favorite, i.e. polymers with sufficient molecular size may trigger a phase separation within the lipid bilayer, promoting the liposome-HBPs-drug system toward a more stable thermodynamic state, as also suggested by EPR data. Finally, the presence of PEG did not seem to compromise the general trend of stabilization when used with DPPC. In case of DPSC, the addition of PEG seemed almost negligible. In conclusion, it should be noted that the incorporation of HBPs and PEG into lipid membranes changes the thermal and physicochemical characteristics of both bilayers and liposomes, as shown from both EPR and micro-DSC analyses. Thus, these techniques could efficiently be utilized for studying and analyzing the behavior of artificial biological membranes and as a tool in our effort to rationally design stable, safe and efficient liposomal formulations.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected],

ORCID Konstantinos N. Kontogiannopoulos: 0000-0001-8728-0233 Athanasia Dasargyri: Maria F. Ottaviani: 0000-0002-4681-4718 Michela Cangiotti: 0000-0002-5713-7740 Page 33 of 43


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Dimitrios Fessas: 0000-0003-4998-308X Vassilios P. Papageorgiou: 0000-0001-9202-1420 Andreana N. Assimopoulou: 0000-0002-2833-2694

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors are grateful to Lipoid Company (Lipoid GmbH, Ludwigshafen, Germany) for generously donating distearoyl phosphatidylcholine (DSPC). COST Action TD0802 “Dendrimers in Biomedical Applications” is acknowledged for supporting networking.

ABBREVIATIONS 5-DSA, 5-doxylstearic acid; A, Alkannin; aDDnSs, advanced Drug Delivery nano Systems; chiaDDnSs, chimeric advanced Drug Delivery nano Systems; Chol, Cholesterol; DPPC, Dipalmitoyl phosphatidylcholine; DSC, Differential Scanning Calorimetry; DSPC, Distearoyl phosphatidylcholine; DSPE-mPEG2000, Ν-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 dipalmitoyl-en-glycero-3-phosphoethanolamine; EPC, Egg phosphatidylcholine; EPR, Electron Paramagnetic Resonance; HBPs, HyperBranched Polymers; PC, Phosphatidylcholine; PBS,

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Phosphate buffer saline; PEG, Polyethylene glycol; RES, Reticuloendothelial system; S, Shikonin.

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Chemical structures of (a) Alkannin; (b) Shikonin; (c) EPC; (d) DPPC; (e) DSPC; and (f) DSPE-mPEG2000. 210x319mm (300 x 300 DPI)


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Chemical structure of the hyperbranched polymer PFH-64-OH. 105x82mm (150 x 150 DPI)


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Examples of experimental and computed EPR spectra for 5-DSA in PBS or in solutions with the various components at the defined molar ratios: (a) PBS or DSPC+Chol+S (DSPC/S=30; DSPC/Chol=4.5); (b) HBP and HBP+S (HBP/S=0.1); (c) DSPC+Chol+HBP; (d) DPPC+Chol (DPPC/Chol=4.5); and (e) PC+Chol+PEG+S+HBP (PC/PEG=13; S/HBP=0.2) 260x320mm (300 x 300 DPI)


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Variations of (a) the absolute intensity of the slow component; (b) the order parameter of the slow component; (c) the relative percentage of fast component, and (d) the polarity of the fast component for the various samples based on DSPC and DPPC (Chol was also added in all samples; the molar ratios are reported in the experimental section). 169x137mm (300 x 300 DPI)


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Variations of (a) the absolute intensity and (b) the order parameter, for the various samples based on PC (Chol is present in all samples; the molar ratios are reported in the experimental section). 84x33mm (300 x 300 DPI)


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Micro-DSC thermograms (second heating) of selected formulations (with and without drug). 114x79mm (300 x 300 DPI)


Advanced drug delivery nanosystems for shikonin: A calorimetric and

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