STERICALLY STABILIZED LIPOSOMES AS A POTENT CARRIER

FOR SHIKONIN

Konstantinos N. Kontogiannopoulos 1, Andreana N. Assimopoulou

1, Vassilios P.

Papageorgiou 1,*

1 Organic Chemistry Laboratory, Chemical Engineering Department, Aristotle

University of Thessaloniki, 54124 Thessaloniki, Greece.

* Corresponding author. Tel: +30 2310996241, Fax: +30 2310996252,

e–mail address: vaspap@eng.auth.gr

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Abstract

Asdasdas

Keywords: Alkannin; Naphthoquinone; Pegylated Liposomes; Sterically Stabilized

Liposomes; cancer

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Abbreviations

Hp Hyperbranched polymers

Chi-aDDnSs Chimeric advanced Drug Delivery nano Systems

A Alkannin

S Shikonin

MCRnSs Modulatory Controlled Release nano Systems

EPC Egg phosphatidylcholine

DPPC Dipalmitoyl phosphatidylcholine

DSPC Distearoyl phosphatidylcholine

DSPE-mPEG2000 Ν-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2

dipalmitoyl-en-glycero-3-phosphoethanolamine

CHOL Cholesterol

PBS Phosphate buffer saline pH 7.4

SLS Sodium lauryl sulfate

PDI Polydispersity index

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1. Introduction

Over the past few decades, increasing attention has been given to drug targeting

in order to reduce side-effects and improve therapeutic efficacy by preventing

undesired drug localization in healthy tissue sites and decreasing rapid degradation or

elimination of drugs (Han et al., 2007; Vyas et al., 2006; Yousefi et al., 2009). Among

a variety of targeted drug carrier systems, liposomes have been studied extensively

because of their capability to accommodate a large variety of drugs, alongside their

good biocompatibility, low toxicity and lack of immune system activation or

suppression. In general, an optimized system is consisted of nanoliposomes, which

possess a long circulation lifetime. Such liposomes will circulate sufficiently long to

accumulate at sites of disease, such as tumors, as a result of the leaky vasculature and

reduced blood flow exhibited by the diseased tissue (Drummond et al., 2008; Fenske

& Cullis, 2005; Yang et al., 2007).

Liposomes were one of the first nanoparticulate drug delivery systems to show

increased delivery of low molecular weight anticancer agents to solid tumors.

Liposomes with diameters in the range of 100 nm can accumulate in solid tumors via

the enhanced permeability and retention (EPR) effect (Maeda et al., 2000), which

occurs when nanoparticulates extravasate from the circulation into tumors through

gaps in the vasculature endothelium (Ishida et al., 2009; Jain, 2001). The ability of

liposomes to localize in solid tumors via the EPR effect partly depends on their long

circulating properties, which can be achieved by grafting polyethylene glycol (PEG)

to the surface of the liposomes (pegylated liposomes or sterically stabilized

liposomes-SSL) (Papahadjopoulos et al., 1991). Anticancer agents encapsulated in

SSL have shown increased efficiency and lower toxicity in treatment of solid tumors

by achieving higher accumulation in tumor tissue but limited accumulation in healthy

organs (Gabizon et al., 1994; Vaage et al., 1993). Doxorubicin-containing SSL (DXR-

SSL), Doxil/Caelyx, has been approved for clinical use (Engels et al., 2007).

Alkannin and Shikonin (A/S; Figure 1) are chiral–pair of naturally occurring

isohexenylnaphthazarins. They are found in the external layer of the roots of at least a

hundred and fifty species that belong mainly to the genera Alkanna, Lithospermum,

Echium, Onosma and Anchusa of the Boraginaceae family (Papageorgiou et al., 1999;

Papageorgiou et al., 2006).

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Figure 1: The chiral pair alkannin and shikonin that possess major biological activity.

Alkannin, Shikonin and their derivatives were originally introduced and

established as wound healing agents by Prof. Papageorgiou. A wound healing

pharmaceutical ointment is already commercially available under the trademark

HELIXDERM®

and the medical devices HELIXFILM, HELIXGEL and

HELIXSPRAY (wound healing collagen film, gel and spray respectively) are under

development. Further biological investigations over the last 35 years have shown that

A/S are potent pharmaceutical substances with a well–established wide spectrum of

antimicrobial, anti–inflammatory and antioxidant activity (Papageorgiou, 1980;

Papageorgiou et al., 1999; Papageorgiou et al., 2006; Papageorgiou et al., 2008).

Extensive scientific research has been conducted the last years on cancer

chemotherapy, focusing on A/S effectiveness on several tumors and mechanism(s) of

antitumor action (Chen et al., 2002; Komi et al., 2009; Lee et al., 2008; Papageorgiou

et al., 1999; Papageorgiou et al., 2006; Yang et al., 2009; Yao & Zhou, 2010; Zeng et

al., 2009).

The scarce aqueous solubility of A/S (0.00002M) (He, 2009) is a barrier for their

oral and internal administration, since they cannot be easily dissolved and further

absorbed from the receptor. A/S are also oxidized (Cheng et al., 1995), polymerized

(Assimopoulou & Papageorgiou, 2004a, b; Papageorgiou et al., 2002) and internally

metabolized (Meselhy et al., 1994a; Meselhy et al., 1994b). Regarding the toxicity of

the active compounds, alkannin was found to bear a LD50 of 3 g/kg in mice and less

than 1 g/kg in rats, when administered orally in a feeding study (Majlathova, 1971).

Shikonin, on the other hand, was found to be rather more toxic to mice by

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intraperitoneal administration, with a LD50 of 20±5 mg/kg (Hayashi, 1977). In

addition, during in vivo testing (mice, intraperitoneal administration), shikonin

showed toxicity at dosages higher than 15 mg/kg/day (Sarcoma–180) and at 10x5

mg/kg/day (L-1210) (Sankawa et al., 1977). Both the solubility and instability matters

could be overcome by delivering A/S through a drug delivery nano-system, such as a

liposomal formulation, which could furthermore enhance their antitumor activity

through toxicity decrease and targeted delivery.

The purpose of this work was to prepare and characterize shikonin–loaded

liposomes as a new drug delivery system for shikonin, in order to reduce side effects

of the free drug, to enhance selectivity against cancer cells and to protect shikonin

from internal biotransformations (Meselhy et al., 1994b; Meselhy et al., 1994a). In

this context, three new pegylated liposomal formulations of shikonin were prepared

and characterised in terms of their physicochemical characteristics, pharmacokinetics

and stability and also compared to the corresponding conventional liposomes.

Characterization of all liposomal formulations prepared (both conventional and

pegylated) was performed in terms of particle size distribution, ζ–potential,

entrapment efficiency and release profile of the entrapped drug. Finally, a stability

study was performed at 4oC for a 28 days period in order to examine the

physicochemical and pharmacological stability of the prepared formulations (both

conventional and pegylated).

This research is a continuation study of the authors on exploiting the biological

properties of A/S and other naphthoquinones through the preparation of DDSs, such

as microcapsules (Assimopoulou et al., 2003; Assimopoulou & Papageorgiou,

2004d), cyclodextrins (Assimopoulou & Papageorgiou, 2004c), liposomes

(Kontogiannopoulos et al., 2011a), chimeric advanced drug delivery nanosystems

(combining dendritic and liposomal technology) (Kontogiannopoulos et al., 2011b)

and electrospun fiber mats (Kontogiannopoulos et al., 2011c).

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2. Materials and Methods

2.1 Materials

Shikonin was used after purification from a commercial sample (Ikeda

Corporation, Tokyo, Japan), by silica gel column chromatography (gradient mixtures

of n-hexane: dichloromethane: chloroform) followed by recrystallization (n-hexane),

according to the procedure proposed by Prof. Papageorgiou (Assimopoulou et al.,

2008) (purity obtained 100%).

Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (EPC),

distearoyl phosphatidylcholine (DSPC) and Ν-(Carbonyl-methoxypolyethyleneglycol

2000)-1,2 dipalmitoyl-en-glycero-3-phosphoethanolamine (DSPE-mPEG2000) were

purchased from Genzyme Pharmaceuticals (Cambridge, USA). Cholesterol (CHOL),

phosphate buffer saline pH 7.4 (PBS), sodium lauryl sulfate (SLS), dialysis sacks

(molecular weight cut off 13000) and Sephadex G75 were purchased from Sigma–

Aldrich (St. Louis, USA). All organic solvents were of analytical grade and were

purchased from Sigma–Aldrich (St. Louis, USA). Water used in all experiments was

of HPLC grade.

2.2 Preparation of shikonin-loaded pegylated liposomes

Initially, the lipids together with the cholesterol were dissolved in

chloroform:methanol 2:1 (v/v) at constant molar ratios 13:1 lipid/DPSE-mPEG2000

(mol/mol) and 4.5:1 lipid/CHOL (mol/mol). Shikonin was diluted in the same organic

solvent (in a different flask) and added to the above mixture under stirring in a 50 mL

round–bottom flask at 30:1 lipid/drug molar ratio (mol/mol). The organic solvent was

slowly removed under reduced pressure using a rotary evaporator (EYELA Rotary

Vacuum Evaporator N-N Series, Digital Water bath SB–651, Tokyo, Japan), forming

a thin film of the lipid on the inner side of the flask. The flask containing the lipid

film was left overnight under vacuum, for the removal of organic solvent traces. The

lipid film was then hydrated with 10 mL PBS (pH 7.4) for 1 h, in water bath above

the main phase transition temperature (Tm) of the lipids (45oC for EPC lipids, 51

oC

for DPPC lipids and 65oC for DSPC lipids) in order to prepare multilamellar vesicles

8

(MLVs). The system was vortexed at 1500 rpm using an IKA MS2 Minishaker (IKA

Works, Inc, Wilmington, USA) for 10 min.

Small unilamellar vesicles (SUVs) were prepared from the resultant liposomal

suspension (MLVs), after sonication for two 5 min periods interrupted by a 5 min

resting period, using a probe sonicator (amplitude 0.7; pulser 50%; Heat Systems–

Ultrasonics Inc., Sonicator W–375 Cell Disruptors). The resultant vesicles were

allowed for 30 min to anneal any structural defects. Non-encapsulated shikonin was

removed by passing the liposomal suspensions through a Sephadex G75 column that

was swollen with water overnight.

2.3 Characterization of shikonin-loaded pegylated liposomes

2.3.1 Particle size measurement and ζ-potential

Size and ζ-potential of liposomes are crucial parameters that indicate their

physical stability. The hydrodynamic diameter of all liposomal formulations was

measured by light scattering. 50 μL of each liposomal formulation were 60-fold

diluted in PBS (pH 7.4) immediately after preparation and z-average mean and ζ-

potential were measured. Measurements were made at 25oC and at a 90° angle in a

photon correlation spectrometer (Malvern ZetaSizer Nano S, Malvern Instruments,

Malvern, UK) and analyzed by the CONTIN method (MALVERN software).

2.3.2 Determination of entrapment efficiency

To remove the non–encapsulated shikonin, liposomal suspensions were passed

through a Sephadex G75 column prior to the determination of the entrapment

efficiency. The percentage of shikonin incorporated into liposomes was estimated by

UV–vis spectrophotometry (UV-Vis Hitachi U1900, Hitachi High-Technologies

Corporation, Tokyo, Japan) at the characteristic wavelength of shikonin (516 nm). 0.5

mL of each liposomal formulation in PBS were suspended in 2.5 mL of methanol to

destroy the liposomal structure, releasing the drug into the organic phase. Absorbance

of the organic phase was measured and shikonin concentration was determined using

the following shikonin calibration curve in methanol:

Drug Concentration (mg/mL) = 0.0485 x Absorbance - 0.00009 ; (R2=0.99992) (1)

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The entrapment efficiency was calculated using the following equation:

Entrapment Efficiency (%) = (Fi/Ft) x 100 (2)

where Fi is the amount of shikonin incorporated into shikonin-Hp complexes and Ft is

the initially added amount of shikonin.

2.3.3 In vitro drug release

The release profile of shikonin from all liposomal formulations was studied in

(PBS+1% SLS) at 37oC. 3 mL of each sample were placed in dialysis sacks

(molecular weight cut off 13,000; Sigma-Aldrich). Dialysis sacks were inserted in 20

mL (PBS+1% SLS) in shaking water bath (Selecta) set at 37oC. Aliquots of samples

(3 ml) were taken from the external solution at specific time intervals and that volume

was replaced with fresh release medium in order to maintain sink conditions. The

amount of shikonin released at various times, up to 72 h, was determined using UV–

vis spectrometer at λmax=518 nm with the aid of the following calibration curve of

shikonin in the release medium:

Drug Concentration (mg/mL) = 0.04837 x Absorbance - 0.00004 ; (R2=0.99998) (3)

The cumulative percentage of drug release was calculated and plotted versus time

using the equation:

% Cumulative Drug Releasedt = Drug Releasedt / Total Entrapped Shikonin x 100 (4)

2.3.4 Stability studies

All liposomal formulations (both conventional and pegylated) were tested for

their stability by means of drug leakage, mean particle size, polydispersity index

(PDI), and ζ–potential. Specifically, immediately after preparation liposomes

formulations were placed in glass vials and stored at 4oC for 28 days. Aliquots of

samples were taken at specific time intervals and mean particle size, ζ–potential and

entrapment efficiency were measured as described earlier.

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2.4 Statistical analysis

Results are shown as mean value ± standard deviation (S.D.) of three independent

experiments. Statistical analysis was performed using Student’s t-test and multiple

comparisons were done using one-way ANOVA. P values <0.05 were considered

statistically significant. All statistical analyses were performed using “SPSS 14.0”.

3. Results and Discussion

The distinct features of pegylated liposomes such as reduced uptake by the

reticulo-endothelial system, favourable pharmacokinetics (long circulating time, slow

clearance rate, small volume of distribution), reduced accumulation in healthy tissues

and, most importantly, preferential tumour uptake owing to their ability to extravasate

through the hyperpermeable tumour vasculature are best illustrated by PEGylated

liposomal doxorubicin (Caelyx, Doxil, Myocet) (Engels et al., 2007).

In the present study pegylated (or sterically stabilized liposomes) are used for the

first time, as drug delivery system for shikonin, using three types of lipids (EPC,

DPPC and DSPC).

3.1 Particle size measurement and ζ-potential

Fdsfsdf

Fsdfa

3.2 Entrapment efficiency

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Dsfasdf

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3.3 In vitro drug release

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Dsfasdf

3.4 Stability studies

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